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Sample records for cell free system

  1. A Continuous-Exchange Cell-Free Protein Synthesis System Based on Extracts from Cultured Insect Cells

    Science.gov (United States)

    Stech, Marlitt; Quast, Robert B.; Sachse, Rita; Schulze, Corina; Wüstenhagen, Doreen A.; Kubick, Stefan

    2014-01-01

    In this study, we present a novel technique for the synthesis of complex prokaryotic and eukaryotic proteins by using a continuous-exchange cell-free (CECF) protein synthesis system based on extracts from cultured insect cells. Our approach consists of two basic elements: First, protein synthesis is performed in insect cell lysates which harbor endogenous microsomal vesicles, enabling a translocation of de novo synthesized target proteins into the lumen of the insect vesicles or, in the case of membrane proteins, their embedding into a natural membrane scaffold. Second, cell-free reactions are performed in a two chamber dialysis device for 48 h. The combination of the eukaryotic cell-free translation system based on insect cell extracts and the CECF translation system results in significantly prolonged reaction life times and increased protein yields compared to conventional batch reactions. In this context, we demonstrate the synthesis of various representative model proteins, among them cytosolic proteins, pharmacological relevant membrane proteins and glycosylated proteins in an endotoxin-free environment. Furthermore, the cell-free system used in this study is well-suited for the synthesis of biologically active tissue-type-plasminogen activator, a complex eukaryotic protein harboring multiple disulfide bonds. PMID:24804975

  2. Spore coat protein synthesis in cell-free systems from sporulating cells of Bacillus subtilis.

    Science.gov (United States)

    Nakayama, T; Munoz, L E; Sadaie, Y; Doi, R H

    1978-09-01

    Cell-free systems for protein synthesis were prepared from Bacillus subtilis 168 cells at several stages of sporulation. Immunological methods were used to determine whether spore coat protein could be synthesized in the cell-free systems prepared from sporulating cells. Spore coat protein synthesis first occurred in extracts from stage t2 cells. The proportion of spore coat protein to total proteins synthesized in the cell-free systems was 2.4 and 3.9% at stages t2 and t4, respectively. The sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis patterns of immunoprecipitates from the cell-free systems showed the complete synthesis of an apparent spore coat protein precursor (molecular weight, 25,000). A polypeptide of this weight was previously identified in studies in vivo (L.E. Munoz, Y. Sadaie, and R.H. Doi, J. Biol. Chem., in press). The synthesis in vitro of polysome-associated nascent spore coat polypeptides with varying molecular weights up to 23,000 was also detected. These results indicate that the spore coat protein may be synthesized as a precursor protein. The removal of proteases in the crude extracts by treatment with hemoglobin-Sepharose affinity techniques may be preventing the conversion of the large 25,000-dalton precursor to the 12,500-dalton mature spore coat protein.

  3. Synthetic Biology Outside the Cell: Linking Computational Tools to Cell-Free Systems

    International Nuclear Information System (INIS)

    Lewis, Daniel D.; Villarreal, Fernando D.; Wu, Fan; Tan, Cheemeng

    2014-01-01

    As mathematical models become more commonly integrated into the study of biology, a common language for describing biological processes is manifesting. Many tools have emerged for the simulation of in vivo synthetic biological systems, with only a few examples of prominent work done on predicting the dynamics of cell-free synthetic systems. At the same time, experimental biologists have begun to study dynamics of in vitro systems encapsulated by amphiphilic molecules, opening the door for the development of a new generation of biomimetic systems. In this review, we explore both in vivo and in vitro models of biochemical networks with a special focus on tools that could be applied to the construction of cell-free expression systems. We believe that quantitative studies of complex cellular mechanisms and pathways in synthetic systems can yield important insights into what makes cells different from conventional chemical systems.

  4. Synthetic Biology Outside the Cell: Linking Computational Tools to Cell-Free Systems

    Energy Technology Data Exchange (ETDEWEB)

    Lewis, Daniel D. [Integrative Genetics and Genomics, University of California Davis, Davis, CA (United States); Department of Biomedical Engineering, University of California Davis, Davis, CA (United States); Villarreal, Fernando D.; Wu, Fan; Tan, Cheemeng, E-mail: cmtan@ucdavis.edu [Department of Biomedical Engineering, University of California Davis, Davis, CA (United States)

    2014-12-09

    As mathematical models become more commonly integrated into the study of biology, a common language for describing biological processes is manifesting. Many tools have emerged for the simulation of in vivo synthetic biological systems, with only a few examples of prominent work done on predicting the dynamics of cell-free synthetic systems. At the same time, experimental biologists have begun to study dynamics of in vitro systems encapsulated by amphiphilic molecules, opening the door for the development of a new generation of biomimetic systems. In this review, we explore both in vivo and in vitro models of biochemical networks with a special focus on tools that could be applied to the construction of cell-free expression systems. We believe that quantitative studies of complex cellular mechanisms and pathways in synthetic systems can yield important insights into what makes cells different from conventional chemical systems.

  5. Synthetic Biology Outside the Cell: Linking Computational Tools to Cell-Free Systems

    Directory of Open Access Journals (Sweden)

    Daniel eLewis

    2014-12-01

    Full Text Available As mathematical models become more commonly integrated into the study of biology, a common language for describing biological processes is manifesting. Many tools have emerged for the simulation of in vivo systems, with only a few examples of prominent work done on predicting the dynamics of cell-free systems. At the same time, experimental biologists have begun to study dynamics of in vitro systems encapsulated by amphiphilic molecules, opening the door for the development of a new generation of biomimetic systems. In this review, we explore both in vivo and in vitro models of biochemical networks with a special focus on tools that could be applied to the construction of cell-free expression systems. We believe that quantitative studies of complex cellular mechanisms and pathways in synthetic systems can yield important insights into what makes cells different from conventional chemical systems.

  6. Label-free single-cell separation and imaging of cancer cells using an integrated microfluidic system

    DEFF Research Database (Denmark)

    Antfolk, Maria; Kim, Soo Hyeon; Koizumi, Saori

    2017-01-01

    , an integrated system is presented that efficiently eliminates this risk by integrating label-free separation with single cell arraying of the target cell population, enabling direct on-chip tumor cell identification and enumeration. Prostate cancer cells (DU145) spiked into a sample with whole blood...... a fully integrated system for rapid label-free separation and on-chip phenotypic characterization of circulating tumor cells from peripheral venous blood in clinical practice....

  7. A single-cell and feeder-free culture system for monkey embryonic stem cells.

    Science.gov (United States)

    Ono, Takashi; Suzuki, Yutaka; Kato, Yosuke; Fujita, Risako; Araki, Toshihiro; Yamashita, Tomoko; Kato, Hidemasa; Torii, Ryuzo; Sato, Naoya

    2014-01-01

    Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.

  8. Cell-free synthetic biology: thinking outside the cell.

    Science.gov (United States)

    Hodgman, C Eric; Jewett, Michael C

    2012-05-01

    Cell-free synthetic biology is emerging as a powerful approach aimed to understand, harness, and expand the capabilities of natural biological systems without using intact cells. Cell-free systems bypass cell walls and remove genetic regulation to enable direct access to the inner workings of the cell. The unprecedented level of control and freedom of design, relative to in vivo systems, has inspired the rapid development of engineering foundations for cell-free systems in recent years. These efforts have led to programmed circuits, spatially organized pathways, co-activated catalytic ensembles, rational optimization of synthetic multi-enzyme pathways, and linear scalability from the micro-liter to the 100-liter scale. It is now clear that cell-free systems offer a versatile test-bed for understanding why nature's designs work the way they do and also for enabling biosynthetic routes to novel chemicals, sustainable fuels, and new classes of tunable materials. While challenges remain, the emergence of cell-free systems is poised to open the way to novel products that until now have been impractical, if not impossible, to produce by other means. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. Preparation of ubiquitin-conjugated proteins using an insect cell-free protein synthesis system.

    Science.gov (United States)

    Suzuki, Takashi; Ezure, Toru; Ando, Eiji; Nishimura, Osamu; Utsumi, Toshihiko; Tsunasawa, Susumu

    2010-01-01

    Ubiquitination is one of the most significant posttranslational modifications (PTMs). To evaluate the ability of an insect cell-free protein synthesis system to carry out ubiquitin (Ub) conjugation to in vitro translated proteins, poly-Ub chain formation was studied in an insect cell-free protein synthesis system. Poly-Ub was generated in the presence of Ub aldehyde (UA), a de-ubiquitinating enzyme inhibitor. In vitro ubiquitination of the p53 tumor suppressor protein was also analyzed, and p53 was poly-ubiquitinated when Ub, UA, and Mdm2, an E3 Ub ligase (E3) for p53, were added to the in vitro reaction mixture. These results suggest that the insect cell-free protein synthesis system contains enzymatic activities capable of carrying out ubiquitination. CBB-detectable ubiquitinated p53 was easily purified from the insect cell-free protein synthesis system, allowing analysis of the Ub-conjugated proteins by mass spectrometry (MS). Lys 305 of p53 was identified as one of the Ub acceptor sites using this strategy. Thus, we conclude that the insect cell-free protein synthesis system is a powerful tool for studying various PTMs of eukaryotic proteins including ubiqutination presented here.

  10. Sodium nitrite induces acute central nervous system toxicity in guinea pigs exposed to systemic cell-free hemoglobin

    International Nuclear Information System (INIS)

    Buehler, Paul W.; Butt, Omer I.; D'Agnillo, Felice

    2011-01-01

    Highlights: → Toxicological implications associated with the use of NaNO 2 therapy to treat systemic cell-free Hb exposure are not well-defined. → Systemic Hb exposure followed by NaNO 2 infusion induces acute CNS toxicities in guinea pigs. → These CNS effects were not reproduced by the infusion of cell-free Hb or NaNO 2 alone. → NaNO 2 -mediated oxidation of cell-free Hb may play a causative role in the observed CNS changes. -- Abstract: Systemic cell-free hemoglobin (Hb) released via hemolysis disrupts vascular homeostasis, in part, through the scavenging of nitric oxide (NO). Sodium nitrite (NaNO 2 ) therapy can attenuate the hypertensive effects of Hb. However, the chemical reactivity of NaNO 2 with Hb may enhance heme- or iron-mediated toxicities. Here, we investigate the effect of NaNO 2 on the central nervous system (CNS) in guinea pigs exposed to systemic cell-free Hb. Intravascular infusion of NaNO 2 , at doses sufficient to alleviate Hb-mediated blood pressure changes, reduced the expression of occludin, but not zona occludens-1 (ZO-1) or claudin-5, in cerebral tight junctions 4 h after Hb infusion. This was accompanied by increased perivascular heme oxygenase-1 expression, neuronal iron deposition, increased astrocyte and microglial activation, and reduced expression of neuron-specific nuclear protein (NeuN). These CNS changes were not observed in animals treated with Hb or NaNO 2 alone. Taken together, these findings suggest that the use of nitrite salts to treat systemic Hb exposure may promote acute CNS toxicity.

  11. Sodium nitrite induces acute central nervous system toxicity in guinea pigs exposed to systemic cell-free hemoglobin

    Energy Technology Data Exchange (ETDEWEB)

    Buehler, Paul W.; Butt, Omer I. [Laboratory of Biochemistry and Vascular Biology, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD (United States); D' Agnillo, Felice, E-mail: felice.dagnillo@fda.hhs.gov [Laboratory of Biochemistry and Vascular Biology, Division of Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD (United States)

    2011-06-10

    Highlights: {yields} Toxicological implications associated with the use of NaNO{sub 2} therapy to treat systemic cell-free Hb exposure are not well-defined. {yields} Systemic Hb exposure followed by NaNO{sub 2} infusion induces acute CNS toxicities in guinea pigs. {yields} These CNS effects were not reproduced by the infusion of cell-free Hb or NaNO{sub 2} alone. {yields} NaNO{sub 2}-mediated oxidation of cell-free Hb may play a causative role in the observed CNS changes. -- Abstract: Systemic cell-free hemoglobin (Hb) released via hemolysis disrupts vascular homeostasis, in part, through the scavenging of nitric oxide (NO). Sodium nitrite (NaNO{sub 2}) therapy can attenuate the hypertensive effects of Hb. However, the chemical reactivity of NaNO{sub 2} with Hb may enhance heme- or iron-mediated toxicities. Here, we investigate the effect of NaNO{sub 2} on the central nervous system (CNS) in guinea pigs exposed to systemic cell-free Hb. Intravascular infusion of NaNO{sub 2}, at doses sufficient to alleviate Hb-mediated blood pressure changes, reduced the expression of occludin, but not zona occludens-1 (ZO-1) or claudin-5, in cerebral tight junctions 4 h after Hb infusion. This was accompanied by increased perivascular heme oxygenase-1 expression, neuronal iron deposition, increased astrocyte and microglial activation, and reduced expression of neuron-specific nuclear protein (NeuN). These CNS changes were not observed in animals treated with Hb or NaNO{sub 2} alone. Taken together, these findings suggest that the use of nitrite salts to treat systemic Hb exposure may promote acute CNS toxicity.

  12. Translation in cell-free systems

    International Nuclear Information System (INIS)

    Jagus, R.

    1987-01-01

    The simplest, unambiguous identification of a particular mRNA is the identification of its protein product. This can be established by translation of the mRNA of interest in a cell-free protein-synthesizing system. Messenger RNA protein product identification is important in the isolation of a particular mRNA species for cDNA cloning and in the identification of positive cDNA clones. The two high-activity translation systems in common use are those prepared from rabbit reticulocytes and from wheat germ. Both systems are easy to prepare, and both are available commercially. Each has advantages and disadvantages over the other and a choice between the two will depend on the type of mRNAs to be translated, the prejudices of experience, and availability. The main disadvantage of the reticulocyte system is that it requires removal of endogenous mRNA. However, this is a relatively simple procedure. The wheat germ system does not require removal of endogenous mRNA and may translate weakly initiating mRNAs more efficiently. However, ionic optima for translation in the wheat germ system are more sensitive to the nature and concentration of mRNA and may need to be determined for each template. The biggest problem with the use of the wheat germ system is its tendency to produce incomplete translation products due to premature termination

  13. Controlling cell-free metabolism through physiochemical perturbations.

    Science.gov (United States)

    Karim, Ashty S; Heggestad, Jacob T; Crowe, Samantha A; Jewett, Michael C

    2018-01-01

    Building biosynthetic pathways and engineering metabolic reactions in cells can be time-consuming due to complexities in cellular metabolism. These complexities often convolute the combinatorial testing of biosynthetic pathway designs needed to define an optimal biosynthetic system. To simplify the optimization of biosynthetic systems, we recently reported a new cell-free framework for pathway construction and testing. In this framework, multiple crude-cell extracts are selectively enriched with individual pathway enzymes, which are then mixed to construct full biosynthetic pathways on the time scale of a day. This rapid approach to building pathways aids in the study of metabolic pathway performance by providing a unique freedom of design to modify and control biological systems for both fundamental and applied biotechnology. The goal of this work was to demonstrate the ability to probe biosynthetic pathway performance in our cell-free framework by perturbing physiochemical conditions, using n-butanol synthesis as a model. We carried out three unique case studies. First, we demonstrated the power of our cell-free approach to maximize biosynthesis yields by mapping physiochemical landscapes using a robotic liquid-handler. This allowed us to determine that NAD and CoA are the most important factors that govern cell-free n-butanol metabolism. Second, we compared metabolic profile differences between two different approaches for building pathways from enriched lysates, heterologous expression and cell-free protein synthesis. We discover that phosphate from PEP utilization, along with other physiochemical reagents, during cell-free protein synthesis-coupled, crude-lysate metabolic system operation inhibits optimal cell-free n-butanol metabolism. Third, we show that non-phosphorylated secondary energy substrates can be used to fuel cell-free protein synthesis and n-butanol biosynthesis. Taken together, our work highlights the ease of using cell-free systems to explore

  14. Absence of regulation of tumor cholesterogenesis in cell-free synthesizing systems

    International Nuclear Information System (INIS)

    Azrolan, N.; Coleman, P.S.

    1986-01-01

    In tumors, cholesterol synthesis de novo is deregulated relative to normal tissues. But no previous study has demonstrated the decontrol of tumor cholesterogenesis with cell-free cytosolic systems. They have utilized a lipid synthesizing, post-mitochondrial supernatant system (PMS), with 14 C-citrate as substrate, to characterize the cholesterogenic pathway in Morris Hepatoma 3924A and normal rat liver. The rate of cholesterogenesis in the hepatoma PMS was 6-fold higher than that in the liver system on a per cell basis. The ratio of sterol-to-fatty acid synthesis was also significantly greater in the tumor versus the liver PMS. The authors determined the steady-state carbon flux through the early intermediates of the lipogenic pathways. Whereas the liver system displayed a metabolic crossover point at the HMG-CoA reductase reaction, the hepatoma system showed no evidence of control at this rate-limiting site of sterol synthesis. Furthermore, acetyl-CoA formation from added citrate (via ATP-citrate lyase) exhibited rates of 42% and 88% in excess of that required for lipidogenesis by liver and tumor PMS systems, respectively. Clearly, a cell-free PMS system from tumor tissue displays the property of deregulated lipidogenesis, especially cholesterol biosynthesis. The authors suggest that deregulated and continuously operating cholesterogenesis would provide for an increased level of a mevalonate-derived sterol pathway intermediate proposed as a trigger for DNA synthesis and cell proliferation in tumors

  15. Cell-Free and In Vivo Characterization of Lux, Las, and Rpa Quorum Activation Systems in E. coli.

    Science.gov (United States)

    Halleran, Andrew D; Murray, Richard M

    2018-02-16

    Synthetic biologists have turned toward quorum systems as a path for building sophisticated microbial consortia that exhibit group decision making. Currently, however, even the most complex consortium circuits rely on only one or two quorum sensing systems, greatly restricting the available design space. High-throughput characterization of available quorum sensing systems is useful for finding compatible sets of systems that are suitable for a defined circuit architecture. Recently, cell-free systems have gained popularity as a test-bed for rapid prototyping of genetic circuitry. We take advantage of the transcription-translation cell-free system to characterize three commonly used Lux-type quorum activators, Lux, Las, and Rpa. We then compare the cell-free characterization to results obtained in vivo. We find significant genetic crosstalk in both the Las and Rpa systems and substantial signal crosstalk in Lux activation. We show that cell-free characterization predicts crosstalk observed in vivo.

  16. In vitro Fab display: a cell-free system for IgG discovery

    Science.gov (United States)

    Stafford, Ryan L.; Matsumoto, Marissa L.; Yin, Gang; Cai, Qi; Fung, Juan Jose; Stephenson, Heather; Gill, Avinash; You, Monica; Lin, Shwu-Hwa; Wang, Willie D.; Masikat, Mary Rose; Li, Xiaofan; Penta, Kalyani; Steiner, Alex R.; Baliga, Ramesh; Murray, Christopher J.; Thanos, Christopher D.; Hallam, Trevor J.; Sato, Aaron K.

    2014-01-01

    Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF. PMID:24586053

  17. Cell-free protein synthesis: applications in proteomics and biotechnology.

    Science.gov (United States)

    He, Mingyue

    2008-01-01

    Protein production is one of the key steps in biotechnology and functional proteomics. Expression of proteins in heterologous hosts (such as in E. coli) is generally lengthy and costly. Cell-free protein synthesis is thus emerging as an attractive alternative. In addition to the simplicity and speed for protein production, cell-free expression allows generation of functional proteins that are difficult to produce by in vivo systems. Recent exploitation of cell-free systems enables novel development of technologies for rapid discovery of proteins with desirable properties from very large libraries. This article reviews the recent development in cell-free systems and their application in the large scale protein analysis.

  18. Reconstituted AIM2 inflammasome in cell-free system.

    Science.gov (United States)

    Kaneko, Naoe; Ito, Yuki; Iwasaki, Tomoyuki; Takeda, Hiroyuki; Sawasaki, Tatsuya; Migita, Kiyoshi; Agematsu, Kazunaga; Kawakami, Atsushi; Morikawa, Shinnosuke; Mokuda, Sho; Kurata, Mie; Masumoto, Junya

    2015-11-01

    Absent in melanoma 2 (AIM2) is an intracellular pattern-recognition receptor, which is a member of the PYHIN protein family, consisting of a PYD domain and an IFN-inducible nuclear localization (HIN) domain. AIM2 is reported to oligomerize with adaptor protein ASC upon sensing bacterial and viral cytosolic DNA in order to form the AIM2 inflammasome, which activates caspase-1 leading to IL-1β secretion. Dysregulation of AIM2 inflammasome is supposed to result in autoinflammatory and autoimmune diseases. Thus, the development of new targeted drugs against AIM2 inflammasome would be important for the treatment of these diseases. However, since AIM2 inflammasome is an intracellular receptor, enforced internalization of both ligands and candidate molecules is necessary for the screening of AIM2-inflammasome-targeted molecules. We developed a reconstituted AIM2 inflammasome in a cell-free system with amplified luminescent proximity homogeneous assay (Alpha). Strong Alpha signal was detected upon incubation with poly-deoxyadenylic-deoxythymidylic acid, poly(dA:dT), whereas no Alpha signal was detected upon incubation with muramyl dipeptide, one of the NLR ligands of Nod2 ligand. The interaction between AIM2 and ASC was disrupted by an anti-human ASC monoclonal antibody, CRID3, a class of diarylsulfonylurea-containing compounds, and glycyrrhizin, a substance found in liquorice root. Thus, the reconstituted AIM2 inflammasome in a cell-free system is useful for screening AIM2-inflammasome-targeted therapeutic molecules. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Cell-free unnatural amino acid incorporation with alternative energy systems and linear expression templates.

    Science.gov (United States)

    Shrestha, Prashanta; Smith, Mark Thomas; Bundy, Bradley Charles

    2014-01-25

    Site-specific incorporation of unnatural amino acids (uAAs) during protein synthesis expands the proteomic code through the addition of unique residue chemistry. This field provides a unique tool to improve pharmacokinetics, cancer treatments, vaccine development, proteomics and protein engineering. The limited ability to predict the characteristics of proteins with uAA-incorporation creates a need for a low-cost system with the potential for rapid screening. Escherichia coli-based cell-free protein synthesis is a compelling platform for uAA incorporation due to the open and accessible nature of the reaction environment. However, typical cell-free systems can be expensive due to the high cost of energizing reagents. By employing alternative energy sources, we reduce the cost of uAA-incorporation in CFPS by 55%. While alternative energy systems reduce cost, the time investment to develop gene libraries can remain cumbersome. Cell-free systems allow the direct use of PCR products known as linear expression templates, thus alleviating tedious plasmid library preparations steps. We report the specific costs of CFPS with uAA incorporation, demonstrate that LETs are suitable expression templates with uAA-incorporation, and consider the substantial reduction in labor intensity using LET-based expression for CFPS uAA incorporation. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Production of functional bacteriorhodopsin by an Escherichia coli cell-free protein synthesis system supplemented with steroid detergent and lipid

    OpenAIRE

    Shimono, Kazumi; Goto, Mie; Kikukawa, Takashi; Miyauchi, Seiji; Shirouzu, Mikako; Kamo, Naoki; Yokoyama, Shigeyuki

    2009-01-01

    Cell-free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis-based Escherichia coli cell-free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light-driven proton pump bacteriorhodopsin, consisting of seven transmembrane α-helices. The cell-free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lip...

  1. A Robust, Cell-free Production System for On-Demand Protein Synthesis in Space

    Data.gov (United States)

    National Aeronautics and Space Administration — We will develop a new cell-free expression system that functions after rehydrating from a freeze-dried condition. Freeze-dried powder that can be stored or...

  2. Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of "Difficult-to-Express" Proteins and Future Perspectives.

    Directory of Open Access Journals (Sweden)

    Lena Thoring

    Full Text Available Nowadays, biotechnological processes play a pivotal role in target protein production. In this context, Chinese Hamster Ovary (CHO cells are one of the most prominent cell lines for the expression of recombinant proteins and revealed as a safe host for nearly 40 years. Nevertheless, the major bottleneck of common in vivo protein expression platforms becomes obvious when looking at the production of so called "difficult-to-express" proteins. This class of proteins comprises in particular several ion channels and multipass membrane proteins as well as cytotoxic proteins. To enhance the production of "difficult-to-express" proteins, alternative technologies were developed, mainly based on translationally active cell lysates. These so called "cell-free" protein synthesis systems enable an efficient production of different classes of proteins. Eukaryotic cell-free systems harboring endogenous microsomal structures for the synthesis of functional membrane proteins and posttranslationally modified proteins are of particular interest for future applications. Therefore, we present current developments in cell-free protein synthesis based on translationally active CHO cell extracts, underlining the high potential of this platform. We present novel results highlighting the optimization of protein yields, the synthesis of various "difficult-to-express" proteins and the cotranslational incorporation of non-standard amino acids, which was exemplarily demonstrated by residue specific labeling of the glycoprotein Erythropoietin and the multimeric membrane protein KCSA.

  3. A versatile coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates.

    Science.gov (United States)

    Buntru, Matthias; Vogel, Simon; Stoff, Katrin; Spiegel, Holger; Schillberg, Stefan

    2015-05-01

    Cell-free protein synthesis is a powerful method for the high-throughput production of recombinant proteins, especially proteins that are difficult to express in living cells. Here we describe a coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates (BYLs). Using a combination of fractional factorial designs and response surface models, we developed a cap-independent system that produces more than 250 μg/mL of functional enhanced yellow fluorescent protein (eYFP) and about 270 μg/mL of firefly luciferase using plasmid templates, and up to 180 μg/mL eYFP using linear templates (PCR products) in 18 h batch reactions. The BYL contains actively-translocating microsomal vesicles derived from the endoplasmic reticulum, promoting the formation of disulfide bonds, glycosylation and the cotranslational integration of membrane proteins. This was demonstrated by expressing a functional full-size antibody (∼ 150 μg/mL), the model enzyme glucose oxidase (GOx) (∼ 7.3 U/mL), and a transmembrane growth factor (∼ 25 μg/mL). Subsequent in vitro treatment of GOx with peptide-N-glycosidase F confirmed the presence of N-glycans. Our results show that the BYL can be used as a high-throughput expression and screening platform that is particularly suitable for complex and cytotoxic proteins. © 2014 Wiley Periodicals, Inc.

  4. Establishment of feeder-free culture system for human induced pluripotent stem cell on DAS nanocrystalline graphene

    Science.gov (United States)

    Lee, Hyunah; Nam, Donggyu; Choi, Jae-Kyung; Araúzo-Bravo, Marcos J.; Kwon, Soon-Yong; Zaehres, Holm; Lee, Taehee; Park, Chan Young; Kang, Hyun-Wook; Schöler, Hans R.; Kim, Jeong Beom

    2016-02-01

    The maintenance of undifferentiated human pluripotent stem cells (hPSC) under xeno-free condition requires the use of human feeder cells or extracellular matrix (ECM) coating. However, human-derived sources may cause human pathogen contamination by viral or non-viral agents to the patients. Here we demonstrate feeder-free and xeno-free culture system for hPSC expansion using diffusion assisted synthesis-grown nanocrystalline graphene (DAS-NG), a synthetic non-biological nanomaterial which completely rule out the concern of human pathogen contamination. DAS-NG exhibited advanced biocompatibilities including surface nanoroughness, oxygen containing functional groups and hydrophilicity. hPSC cultured on DAS-NG could maintain pluripotency in vitro and in vivo, and especially cell adhesion-related gene expression profile was comparable to those of cultured on feeders, while hPSC cultured without DAS-NG differentiated spontaneously with high expression of somatic cell-enriched adhesion genes. This feeder-free and xeno-free culture method using DAS-NG will facilitate the generation of clinical-grade hPSC.

  5. Cell-Free Protein Synthesis Enhancement from Real-Time NMR Metabolite Kinetics: Redirecting Energy Fluxes in Hybrid RRL Systems.

    Science.gov (United States)

    Panthu, Baptiste; Ohlmann, Théophile; Perrier, Johan; Schlattner, Uwe; Jalinot, Pierre; Elena-Herrmann, Bénédicte; Rautureau, Gilles J P

    2018-01-19

    A counterintuitive cell-free protein synthesis (CFPS) strategy, based on reducing the ribosomal fraction in rabbit reticulocyte lysate (RRL), triggers the development of hybrid systems composed of RRL ribosome-free supernatant complemented with ribosomes from different mammalian cell-types. Hybrid RRL systems maintain translational properties of the original ribosome cell types, and deliver protein expression levels similar to RRL. Here, we show that persistent ribosome-associated metabolic activity consuming ATP is a major obstacle for maximal protein yield. We provide a detailed picture of hybrid CFPS systems energetic metabolism based on real-time nuclear magnetic resonance (NMR) investigation of metabolites kinetics. We demonstrate that protein synthesis capacity has an upper limit at native ribosome concentration and that lower amounts of the ribosomal fraction optimize energy fluxes toward protein translation, consequently increasing CFPS yield. These results provide a rationalized strategy for further mammalian CFPS developments and reveal the potential of real-time NMR metabolism phenotyping for optimization of cell-free protein expression systems.

  6. Gateway-compatible vectors for high-throughput protein expression in pro- and eukaryotic cell-free systems.

    Science.gov (United States)

    Gagoski, Dejan; Mureev, Sergey; Giles, Nichole; Johnston, Wayne; Dahmer-Heath, Mareike; Škalamera, Dubravka; Gonda, Thomas J; Alexandrov, Kirill

    2015-02-10

    Although numerous techniques for protein expression and production are available the pace of genome sequencing outstrips our ability to analyze the encoded proteins. To address this bottleneck, we have established a system for parallelized cloning, DNA production and cell-free expression of large numbers of proteins. This system is based on a suite of pCellFree Gateway destination vectors that utilize a Species Independent Translation Initiation Sequence (SITS) that mediates recombinant protein expression in any in vitro translation system. These vectors introduce C or N terminal EGFP and mCherry fluorescent and affinity tags, enabling direct analysis and purification of the expressed proteins. To maximize throughput and minimize the cost of protein production we combined Gateway cloning with Rolling Circle DNA Amplification. We demonstrate that as little as 0.1 ng of plasmid DNA is sufficient for template amplification and production of recombinant human protein in Leishmania tarentolae and Escherichia coli cell-free expression systems. Our experiments indicate that this approach can be applied to large gene libraries as it can be reliably performed in multi-well plates. The resulting protein expression pipeline provides a valuable new tool for applications of the post genomic era. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. A stromal cell free culture system generates mouse pro-T cells that can reconstitute T-cell compartments in vivo.

    Science.gov (United States)

    Gehre, Nadine; Nusser, Anja; von Muenchow, Lilly; Tussiwand, Roxane; Engdahl, Corinne; Capoferri, Giuseppina; Bosco, Nabil; Ceredig, Rhodri; Rolink, Antonius G

    2015-03-01

    T-cell lymphopenia following BM transplantation or diseases such as AIDS result in immunodeficiency. Novel approaches to ameliorate this situation are urgently required. Herein, we describe a novel stromal cell free culture system in which Lineage(-) Sca1(+)c-kit(+) BM hematopoietic progenitors very efficiently differentiate into pro-T cells. This culture system consists of plate-bound Delta-like 4 Notch ligand and the cytokines SCF and IL-7. The pro-T cells developing in these cultures express CD25, CD117, and partially CD44; express cytoplasmic CD3ε; and have their TCRβ locus partially D-J rearranged. They could be expanded for over 3 months and used to reconstitute the T-cell compartments of sublethally irradiated T-cell-deficient CD3ε(-/-) mice or lethally irradiated WT mice. Pro-T cells generated in this system could partially correct the T-cell lymphopenia of pre-Tα(-/-) mice. However, reconstituted CD3ε(-/-) mice suffered from a wasting disease that was prevented by co-injection of purified CD4(+) CD25(high) WT Treg cells. In a T-cell-sufficient or T-lymphopenic setting, the development of disease was not observed. Thus, this in vitro culture system represents a powerful tool to generate large numbers of pro-T cells for transplantation and possibly with clinical applications. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Cell-free synthetic biology for environmental sensing and remediation.

    Science.gov (United States)

    Karig, David K

    2017-06-01

    The fields of biosensing and bioremediation leverage the phenomenal array of sensing and metabolic capabilities offered by natural microbes. Synthetic biology provides tools for transforming these fields through complex integration of natural and novel biological components to achieve sophisticated sensing, regulation, and metabolic function. However, the majority of synthetic biology efforts are conducted in living cells, and concerns over releasing genetically modified organisms constitute a key barrier to environmental applications. Cell-free protein expression systems offer a path towards leveraging synthetic biology, while preventing the spread of engineered organisms in nature. Recent efforts in the areas of cell-free approaches for sensing, regulation, and metabolic pathway implementation, as well as for preserving and deploying cell-free expression components, embody key steps towards realizing the potential of cell-free systems for environmental sensing and remediation. Copyright © 2017 The Author. Published by Elsevier Ltd.. All rights reserved.

  9. The emerging age of cell-free synthetic biology.

    Science.gov (United States)

    Smith, Mark Thomas; Wilding, Kristen M; Hunt, Jeremy M; Bennett, Anthony M; Bundy, Bradley C

    2014-08-25

    The engineering of and mastery over biological parts has catalyzed the emergence of synthetic biology. This field has grown exponentially in the past decade. As increasingly more applications of synthetic biology are pursued, more challenges are encountered, such as delivering genetic material into cells and optimizing genetic circuits in vivo. An in vitro or cell-free approach to synthetic biology simplifies and avoids many of the pitfalls of in vivo synthetic biology. In this review, we describe some of the innate features that make cell-free systems compelling platforms for synthetic biology and discuss emerging improvements of cell-free technologies. We also select and highlight recent and emerging applications of cell-free synthetic biology. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  10. The circulating cell-free microrna profile in systemic sclerosis is distinct from both healthy controls and Systemic Lupus Erythematosus

    DEFF Research Database (Denmark)

    Steen, S. O.; Iversen, L. V.; Carlsen, A. L.

    2015-01-01

    Objective. To evaluate the expression profile of cell-free circulating microRNA (miRNA) in systemic sclerosis (SSc), healthy controls (HC), and systemic lupus erythematosus (SLE). Methods. Total RNA was purified from plasma and 45 different, mature miRNA were measured using quantitative PCR assays...

  11. Nonhomologous DNA End Joining in Cell-Free Extracts

    Directory of Open Access Journals (Sweden)

    Sheetal Sharma

    2010-01-01

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

  12. Effect of intravenous administration of d-lysergic acid diethylamide on subsequent protein synthesis in a cell-free system derived from brain.

    Science.gov (United States)

    Cosgrove, J W; Clark, B D; Brown, I R

    1981-03-01

    An initiating cell-free protein synthesis system derived from brain was utilized to demonstrate that the intravenous injection of d-lysergic acid diethylamide (LSD) to rabbits induced a transient inhibition of translation following a brief stimulatory period. Subfractionation of the brain cell-free system into postribosomal supernatant (PRS) and microsome fractions demonstrated that LSD in vivo induced alterations in both of these fractions. In addition to the overall inhibition of translation in the cell-free system, differential effects were noted, i.e., greater than average relative decreases in in vitro labeling of certain brain proteins and relative increases in others. The brain proteins of molecular weights 75K and 95K, which were increased in relative labeling under conditions of LSD-induced hyperthermia, are similar in molecular weight to two of the major "heat shock" proteins reported in tissue culture systems. Injection of LSD to rabbits at 4 degrees C prevented LSD-induced hyperthermia but behavioral effects of the drug were still apparent. The overall decrease in cell-free translation was still observed but the differential labeling effects were not. LSD appeared to influence cell-free translation in the brain at two dissociable levels: (a) an overall decrease in translation that was observed even in the absence of LSD-induced hyperthermia and (b) differential labeling effects on particular proteins that were dependent on LSD-induced hyperthermia.

  13. Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system.

    Science.gov (United States)

    Buntru, Matthias; Vogel, Simon; Spiegel, Holger; Schillberg, Stefan

    2014-05-03

    Cell-free protein synthesis is a rapid and efficient method for the production of recombinant proteins. Usage of prokaryotic cell-free extracts often leads to non-functional proteins. Eukaryotic counterparts such as wheat germ extract (WGE) and rabbit reticulocyte lysate (RLL) may improve solubility and promote the correct folding of eukaryotic multi-domain proteins that are difficult to express in bacteria. However, the preparation of WGEs is complex and time-consuming, whereas RLLs suffer from low yields. Here we report the development of a novel cell-free system based on tobacco Bright Yellow 2 (BY-2) cells harvested in the exponential growth phase. The highly-productive BY-2 lysate (BYL) can be prepared quickly within 4-5 h, compared to 4-5 d for WGE. The efficiency of the BYL was tested using three model proteins: enhanced yellow fluorescent protein (eYFP) and two versions of luciferase. The added mRNA was optimized by testing different 5' and 3' untranslated regions (UTRs). The protein yield in batch and dialysis reactions using BYL was much higher than that of a commercial Promega WGE preparation, achieving a maximum yield of 80 μg/mL of eYFP and 100 μg/mL of luciferase, compared to only 45 μg/mL of eYFP and 35 μg/mL of luciferase in WGEs. In dialysis reactions, the BYL yielded about 400 μg/mL eYFP, representing up to 50% more of the target protein than the Promega WGE, and equivalent to the amount using 5Prime WGE system. Due to the high yield and the short preparation time the BYL represents a remarkable improvement over current eukaryotic cell-free systems.

  14. Probing the biology of cell boundary conditions through confinement of Xenopus cell-free cytoplasmic extracts.

    Science.gov (United States)

    Bermudez, Jessica G; Chen, Hui; Einstein, Lily C; Good, Matthew C

    2017-01-01

    Cell-free cytoplasmic extracts prepared from Xenopus eggs and embryos have for decades provided a biochemical system with which to interrogate complex cell biological processes in vitro. Recently, the application of microfabrication and microfluidic strategies in biology has narrowed the gap between in vitro and in vivo studies by enabling formation of cell-size compartments containing functional cytoplasm. These approaches provide numerous advantages over traditional biochemical experiments performed in a test tube. Most notably, the cell-free cytoplasm is confined using a two- or three-dimensional boundary, which mimics the natural configuration of a cell. This strategy enables characterization of the spatial organization of a cell, and the role that boundaries play in regulating intracellular assembly and function. In this review, we describe the marriage of Xenopus cell-free cytoplasm and confinement technologies to generate synthetic cell-like systems, the recent biological insights they have enabled, and the promise they hold for future scientific discovery. © 2017 Wiley Periodicals, Inc.

  15. Arraying proteins by cell-free synthesis.

    Science.gov (United States)

    He, Mingyue; Wang, Ming-Wei

    2007-10-01

    Recent advances in life science have led to great motivation for the development of protein arrays to study functions of genome-encoded proteins. While traditional cell-based methods have been commonly used for generating protein arrays, they are usually a time-consuming process with a number of technical challenges. Cell-free protein synthesis offers an attractive system for making protein arrays, not only does it rapidly converts the genetic information into functional proteins without the need for DNA cloning, but also presents a flexible environment amenable to production of folded proteins or proteins with defined modifications. Recent advancements have made it possible to rapidly generate protein arrays from PCR DNA templates through parallel on-chip protein synthesis. This article reviews current cell-free protein array technologies and their proteomic applications.

  16. Production of functional bacteriorhodopsin by an Escherichia coli cell-free protein synthesis system supplemented with steroid detergent and lipid.

    Science.gov (United States)

    Shimono, Kazumi; Goto, Mie; Kikukawa, Takashi; Miyauchi, Seiji; Shirouzu, Mikako; Kamo, Naoki; Yokoyama, Shigeyuki

    2009-10-01

    Cell-free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis-based Escherichia coli cell-free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light-driven proton pump bacteriorhodopsin, consisting of seven transmembrane alpha-helices. The cell-free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lipid, phosphatidylcholine from egg yolk, in only the reaction mixture. By examining a variety of detergents, we found that the combination of a steroid detergent (digitonin, cholate, or CHAPS) and egg phosphatidylcholine yielded a large amount (0.3-0.7 mg/mL reaction mixture) of the fully functional bacteriorhodopsin. We also analyzed the process of functional expression in our system. The synthesized polypeptide was well protected from aggregation by the detergent-lipid mixed micelles and/or lipid disks, and was integrated into liposomes upon detergent removal by dialysis. This approach might be useful for the high yield production of functional membrane proteins.

  17. Insights to the effects of free cells on community structure of attached cells and chalcopyrite bioleaching during different stages.

    Science.gov (United States)

    Feng, Shoushuai; Yang, Hailin; Wang, Wu

    2016-01-01

    The effects of free cells on community structure of attached cells and chalcopyrite bioleaching by Acidithiobacillus sp. during different stages were investigated. The attached cells of Acidithiobacillus thiooxidans owned the community advantage from 14thd to the end of bioprocess in the normal system. The community structure of attached cells was greatly influenced in the free cells-deficient systems. Compared to A. thiooxidans, the attached cells community of Acidithiobacillus ferrooxidans had a higher dependence on its free cells. Meanwhile, the analysis of key biochemical parameters revealed that the effects of free cells on chalcopyrite bioleaching in different stages were diverse, ranging from 32.8% to 64.3%. The bioleaching contribution of free cells of A. ferrooxidans in the stationary stage (8-14thd) was higher than those of A. thiooxidans, while the situation was gradually reversed in the jarosite passivation inhibited stage (26-40thd). These results may be useful in guiding chalcopyrite bioleaching. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Free energy analysis of cell spreading.

    Science.gov (United States)

    McEvoy, Eóin; Deshpande, Vikram S; McGarry, Patrick

    2017-10-01

    In this study we present a steady-state adaptation of the thermodynamically motivated stress fiber (SF) model of Vigliotti et al. (2015). We implement this steady-state formulation in a non-local finite element setting where we also consider global conservation of the total number of cytoskeletal proteins within the cell, global conservation of the number of binding integrins on the cell membrane, and adhesion limiting ligand density on the substrate surface. We present a number of simulations of cell spreading in which we consider a limited subset of the possible deformed spread-states assumed by the cell in order to examine the hypothesis that free energy minimization drives the process of cell spreading. Simulations suggest that cell spreading can be viewed as a competition between (i) decreasing cytoskeletal free energy due to strain induced assembly of cytoskeletal proteins into contractile SFs, and (ii) increasing elastic free energy due to stretching of the mechanically passive components of the cell. The computed minimum free energy spread area is shown to be lower for a cell on a compliant substrate than on a rigid substrate. Furthermore, a low substrate ligand density is found to limit cell spreading. The predicted dependence of cell spread area on substrate stiffness and ligand density is in agreement with the experiments of Engler et al. (2003). We also simulate the experiments of Théry et al. (2006), whereby initially circular cells deform and adhere to "V-shaped" and "Y-shaped" ligand patches. Analysis of a number of different spread states reveals that deformed configurations with the lowest free energy exhibit a SF distribution that corresponds to experimental observations, i.e. a high concentration of highly aligned SFs occurs along free edges, with lower SF concentrations in the interior of the cell. In summary, the results of this study suggest that cell spreading is driven by free energy minimization based on a competition between decreasing

  19. Differentiation of oligodendrocyte progenitor cells from dissociated monolayer and feeder-free cultured pluripotent stem cells.

    Science.gov (United States)

    Yamashita, Tomoko; Miyamoto, Yuki; Bando, Yoshio; Ono, Takashi; Kobayashi, Sakurako; Doi, Ayano; Araki, Toshihiro; Kato, Yosuke; Shirakawa, Takayuki; Suzuki, Yutaka; Yamauchi, Junji; Yoshida, Shigetaka; Sato, Naoya

    2017-01-01

    Oligodendrocytes myelinate axons and form myelin sheaths in the central nervous system. The development of therapies for demyelinating diseases, including multiple sclerosis and leukodystrophies, is a challenge because the pathogenic mechanisms of disease remain poorly understood. Primate pluripotent stem cell-derived oligodendrocytes are expected to help elucidate the molecular pathogenesis of these diseases. Oligodendrocytes have been successfully differentiated from human pluripotent stem cells. However, it is challenging to prepare large amounts of oligodendrocytes over a short amount of time because of manipulation difficulties under conventional primate pluripotent stem cell culture methods. We developed a proprietary dissociated monolayer and feeder-free culture system to handle pluripotent stem cell cultures. Because the dissociated monolayer and feeder-free culture system improves the quality and growth of primate pluripotent stem cells, these cells could potentially be differentiated into any desired functional cells and consistently cultured in large-scale conditions. In the current study, oligodendrocyte progenitor cells and mature oligodendrocytes were generated within three months from monkey embryonic stem cells. The embryonic stem cell-derived oligodendrocytes exhibited in vitro myelinogenic potency with rat dorsal root ganglion neurons. Additionally, the transplanted oligodendrocyte progenitor cells differentiated into myelin basic protein-positive mature oligodendrocytes in the mouse corpus callosum. This preparative method was used for human induced pluripotent stem cells, which were also successfully differentiated into oligodendrocyte progenitor cells and mature oligodendrocytes that were capable of myelinating rat dorsal root ganglion neurons. Moreover, it was possible to freeze, thaw, and successfully re-culture the differentiating cells. These results showed that embryonic stem cells and human induced pluripotent stem cells maintained in a

  20. Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70

    Directory of Open Access Journals (Sweden)

    Noireaux Vincent

    2010-06-01

    Full Text Available Abstract Background Escherichia coli cell-free expression systems use bacteriophage RNA polymerases, such as T7, to synthesize large amounts of recombinant proteins. These systems are used for many applications in biotechnology, such as proteomics. Recently, informational processes have been reconstituted in vitro with cell-free systems. These synthetic approaches, however, have been seriously limited by a lack of transcription modularity. The current available cell-free systems have been optimized to work with bacteriophage RNA polymerases, which put significant restrictions to engineer processes related to biological information. The development of efficient cell-free systems with broader transcription capabilities is required to study complex informational processes in vitro. Results In this work, an efficient cell-free expression system that uses the endogenous E. coli RNA polymerase only and sigma factor 70 for transcription was prepared. Approximately 0.75 mg/ml of Firefly luciferase and enhanced green fluorescent protein were produced in batch mode. A plasmid was optimized with different regulatory parts to increase the expression. In addition, a new eGFP was engineered that is more translatable in cell-free systems than the original eGFP. The protein production was characterized with three different adenosine triphosphate (ATP regeneration systems: creatine phosphate (CP, phosphoenolpyruvate (PEP, and 3-phosphoglyceric acid (3-PGA. The maximum protein production was obtained with 3-PGA. Preparation of the crude extract was streamlined to a simple routine procedure that takes 12 hours including cell culture. Conclusions Although it uses the endogenous E. coli transcription machinery, this cell-free system can produce active proteins in quantities comparable to bacteriophage systems. The E. coli transcription provides much more possibilities to engineer informational processes in vitro. Many E. coli promoters/operators specific to sigma

  1. Cell-free synthetic biology for in vitro prototype engineering.

    Science.gov (United States)

    Moore, Simon J; MacDonald, James T; Freemont, Paul S

    2017-06-15

    Cell-free transcription-translation is an expanding field in synthetic biology as a rapid prototyping platform for blueprinting the design of synthetic biological devices. Exemplar efforts include translation of prototype designs into medical test kits for on-site identification of viruses (Zika and Ebola), while gene circuit cascades can be tested, debugged and re-designed within rapid turnover times. Coupled with mathematical modelling, this discipline lends itself towards the precision engineering of new synthetic life. The next stages of cell-free look set to unlock new microbial hosts that remain slow to engineer and unsuited to rapid iterative design cycles. It is hoped that the development of such systems will provide new tools to aid the transition from cell-free prototype designs to functioning synthetic genetic circuits and engineered natural product pathways in living cells. © 2017 The Author(s).

  2. An amino acid depleted cell-free protein synthesis system for the incorporation of non-canonical amino acid analogs into proteins.

    Science.gov (United States)

    Singh-Blom, Amrita; Hughes, Randall A; Ellington, Andrew D

    2014-05-20

    Residue-specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an efficient amino acid depleted cell-free protein synthesis system that can be used to study residue-specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines a simple methodology and high protein expression titers with a high-efficiency analog substitution into a target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein-wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo methodologies. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Dynamic Modeling of Cell-Free Biochemical Networks Using Effective Kinetic Models

    Directory of Open Access Journals (Sweden)

    Joseph A. Wayman

    2015-03-01

    Full Text Available Cell-free systems offer many advantages for the study, manipulation and modeling of metabolism compared to in vivo processes. Many of the challenges confronting genome-scale kinetic modeling can potentially be overcome in a cell-free system. For example, there is no complex transcriptional regulation to consider, transient metabolic measurements are easier to obtain, and we no longer have to consider cell growth. Thus, cell-free operation holds several significant advantages for model development, identification and validation. Theoretically, genome-scale cell-free kinetic models may be possible for industrially important organisms, such as E. coli, if a simple, tractable framework for integrating allosteric regulation with enzyme kinetics can be formulated. Toward this unmet need, we present an effective biochemical network modeling framework for building dynamic cell-free metabolic models. The key innovation of our approach is the integration of simple effective rules encoding complex allosteric regulation with traditional kinetic pathway modeling. We tested our approach by modeling the time evolution of several hypothetical cell-free metabolic networks. We found that simple effective rules, when integrated with traditional enzyme kinetic expressions, captured complex allosteric patterns such as ultrasensitivity or non-competitive inhibition in the absence of mechanistic information. Second, when integrated into network models, these rules captured classic regulatory patterns such as product-induced feedback inhibition. Lastly, we showed, at least for the network architectures considered here, that we could simultaneously estimate kinetic parameters and allosteric connectivity from synthetic data starting from an unbiased collection of possible allosteric structures using particle swarm optimization. However, when starting with an initial population that was heavily enriched with incorrect structures, our particle swarm approach could converge

  4. Applications of cell-free protein synthesis in synthetic biology: Interfacing bio-machinery with synthetic environments.

    Science.gov (United States)

    Lee, Kyung-Ho; Kim, Dong-Myung

    2013-11-01

    Synthetic biology is built on the synthesis, engineering, and assembly of biological parts. Proteins are the first components considered for the construction of systems with designed biological functions because proteins carry out most of the biological functions and chemical reactions inside cells. Protein synthesis is considered to comprise the most basic levels of the hierarchical structure of synthetic biology. Cell-free protein synthesis has emerged as a powerful technology that can potentially transform the concept of bioprocesses. With the ability to harness the synthetic power of biology without many of the constraints of cell-based systems, cell-free protein synthesis enables the rapid creation of protein molecules from diverse sources of genetic information. Cell-free protein synthesis is virtually free from the intrinsic constraints of cell-based methods and offers greater flexibility in system design and manipulability of biological synthetic machinery. Among its potential applications, cell-free protein synthesis can be combined with various man-made devices for rapid functional analysis of genomic sequences. This review covers recent efforts to integrate cell-free protein synthesis with various reaction devices and analytical platforms. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Fuel cell system with interconnect

    Science.gov (United States)

    Goettler, Richard; Liu, Zhien

    2017-12-12

    The present invention includes a fuel cell system having a plurality of adjacent electrochemical cells formed of an anode layer, a cathode layer spaced apart from the anode layer, and an electrolyte layer disposed between the anode layer and the cathode layer. The fuel cell system also includes at least one interconnect, the interconnect being structured to conduct free electrons between adjacent electrochemical cells. Each interconnect includes a primary conductor embedded within the electrolyte layer and structured to conduct the free electrons.

  6. Efficient production and purification of functional bacteriorhodopsin with a wheat-germ cell-free system and a combination of Fos-choline and CHAPS detergents.

    Science.gov (United States)

    Genji, Takahisa; Nozawa, Akira; Tozawa, Yuzuru

    2010-10-01

    Cell-free translation is one potential approach to the production of functional transmembrane proteins. We have now examined various detergents as supplements to a wheat-germ cell-free system in order to optimize the production and subsequent purification of a functional model transmembrane protein, bacteriorhodopsin. We found that Fos-choline and CHAPS detergents counteracted each other's inhibitory effects on cell-free translation activity and thereby allowed the efficient production and subsequent purification of functional bacteriorhodopsin in high yield. Copyright © 2010 Elsevier Inc. All rights reserved.

  7. Rapid acquisition and model-based analysis of cell-free transcription–translation reactions from nonmodel bacteria

    Science.gov (United States)

    Wienecke, Sarah; Ishwarbhai, Alka; Tsipa, Argyro; Aw, Rochelle; Kylilis, Nicolas; Bell, David J.; McClymont, David W.; Jensen, Kirsten; Biedendieck, Rebekka

    2018-01-01

    Native cell-free transcription–translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, Bacillus megaterium, is a giant Gram-positive bacterium with potential biotechnology applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to B. megaterium to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quantitative models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple experimental conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription–translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition experiment. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based analysis of cell-free transcription–translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biology and biotechnology applications. PMID:29666238

  8. Differential control of the cholesterol biosynthetic pathway in tumor versus liver: evidence for decontrolled tumor cholesterogenesis in a cell-free system

    International Nuclear Information System (INIS)

    Azrolan, N.

    1987-01-01

    Cholesterol biosynthesis was characterized in cell-free post-mitochondrial supernatant (PMS) systems prepared from both normal rat liver and Morris hepatoma 3924A. Per cell, the rate of cholesterol synthesis from either 14 C-citrate of 14 -acetate in the hepatoma system was 9-fold greater than that observed in the liver system. Furthermore, the ratio of sterol-to-fatty acid synthesis rates from 14 C-citrate was more than 3-fold greater in the tumor than in the normal liver system. Incubations using radiolabeled acetate and mevalonate have demonstrated the loss of a normally rate-limiting control site within the early portion of the cholesterol biosynthetic pathway in the tumor system. Upon analysis of the steady-state levels of early lipogenic intermediates, the specific site of decontrol in the tumor was identified as the 3-hydroxy-3-methylglutaryl-CoA → mevalonate site of this pathway. In contrast, this reaction appeared to retain its rate-limiting properties in the cell-free system from normal liver

  9. Effect of intravenous administration of D-lysergic acid diethylamide on initiation of protein synthesis in a cell-free system derived from brain.

    Science.gov (United States)

    Cosgrove, J W; Brown, I R

    1984-05-01

    An initiating cell-free protein synthesis system derived from brain was utilized to demonstrate that the intravenous injection of D-lysergic acid diethylamide (LSD) to rabbits resulted in a lesion at the initiation stage of brain protein synthesis. Three inhibitors of initiation, edeine, poly(I), and aurintricarboxylic acid were used to demonstrate a reduction in initiation-dependent amino acid incorporation in the brain cell-free system. One hour after LSD injection, there was also a measurable decrease in the formation of 40S and 80S initiation complexes in vitro, using either [35S]methionine or [35S]Met-tRNAf. Analysis of the methionine pool size after LSD administration indicated there was no change in methionine levels. Analysis of the formation of initiation complexes in the brain cell-free protein synthesis system prepared 6 h after LSD administration indicated that there was a return to control levels at this time. The effects of LSD on steps in the initiation process are thus reversible.

  10. Validation of cell-free culture using scanning electron microscopy (SEM) and gene expression studies.

    Science.gov (United States)

    Yang, R; Elankumaran, Y; Hijjawi, N; Ryan, U

    2015-06-01

    A cell-free culture system for Cryptosporidium parvum was analysed using scanning electron microscopy (SEM) to characterise life cycle stages and compare gene expression in cell-free culture and cell culture using HCT-8 cells. Cryptosporidium parvum samples were harvested at 2 h, 8 h, 14 h, 26 h, 50 h, 74 h, 98 h, 122 h and 170 h, chemically fixed and specimens were observed using a Zeiss 1555 scanning electron microscope. The presence of sporozoites, trophozoites and type I merozoites were identified by SEM. Gene expression in cell culture and cell-free culture was studied using reverse transcriptase quantitative PCR (RT-qPCR) of the sporozoite surface antigen protein (cp15), the glycoprotein 900 (gp900), the Cryptosporidium oocyst wall protein (COWP) and 18S ribosomal RNA (rRNA) genes in both cell free and conventional cell culture. In cell culture, cp15 expression peaked at 74 h, gp900 expression peaked at 74 h and 98 h and COWP expression peaked at 50 h. In cell-free culture, CP15 expression peaked at 98 h, gp900 expression peaked at 74 h and COWP expression peaked at 122 h. The present study is the first to compare gene expression of C. parvum in cell culture and cell-free culture and to characterise life cycle stages of C. parvum in cell-free culture using SEM. Findings from this study showed that gene expression patterns in cell culture and cell-free culture were similar but in cell-free culture, gene expression was delayed for CP15 and COWP in cell free culture compared with the cell culture system and was lower. Although three life cycle stageswere conclusively identified, improvements in SEM methodology should lead to the detection of more life cycle stages. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Scaling Up ITO-free solar cells

    DEFF Research Database (Denmark)

    Galagan, Yulia; Coenen, Erica W. C.; Zimmermann, Birger

    2014-01-01

    Indium-tin-oxide-free (ITO-free) polymer solar cells with composite electrodes containing current-collecting grids and a semitransparent poly(3,4-ethylenedioxythiophene):polystyrenesulfonate) (PEDOT:PSS) conductor are demonstrated. The up-scaling of the length of the solar cell from 1 to 6 cm...... resistances. The performance of ITO-free organic solar cells with different dimensions and different electrode resistances are evaluated for different light intensities. The current generation and electric potential distribution are found to not be uniformly distributed in large-area devices at simulated 1...

  12. Cell-free protein synthesis for structure determination by X-ray crystallography.

    Science.gov (United States)

    Watanabe, Miki; Miyazono, Ken-ichi; Tanokura, Masaru; Sawasaki, Tatsuya; Endo, Yaeta; Kobayashi, Ichizo

    2010-01-01

    Structure determination has been difficult for those proteins that are toxic to the cells and cannot be prepared in a large amount in vivo. These proteins, even when biologically very interesting, tend to be left uncharacterized in the structural genomics projects. Their cell-free synthesis can bypass the toxicity problem. Among the various cell-free systems, the wheat-germ-based system is of special interest due to the following points: (1) Because the gene is placed under a plant translational signal, its toxic expression in a bacterial host is reduced. (2) It has only little codon preference and, especially, little discrimination between methionine and selenomethionine (SeMet), which allows easy preparation of selenomethionylated proteins for crystal structure determination by SAD and MAD methods. (3) Translation is uncoupled from transcription, so that the toxicity of the translation product on DNA and its transcription, if any, can be bypassed. We have shown that the wheat-germ-based cell-free protein synthesis is useful for X-ray crystallography of one of the 4-bp cutter restriction enzymes, which are expected to be very toxic to all forms of cells retaining the genome. Our report on its structure represents the first report of structure determination by X-ray crystallography using protein overexpressed with the wheat-germ-based cell-free protein expression system. This will be a method of choice for cytotoxic proteins when its cost is not a problem. Its use will become popular when the crystal structure determination technology has evolved to require only a tiny amount of protein.

  13. Community dynamics of attached and free cells and the effects of attached cells on chalcopyrite bioleaching by Acidithiobacillus sp.

    Science.gov (United States)

    Yang, Hailin; Feng, Shoushuai; Xin, Yu; Wang, Wu

    2014-02-01

    The community dynamics of attached and free cells of Acidithiobacillus sp. were investigated and compared during chalcopyrite bioleaching process. In the mixed strains system, Acidithiobacillus ferrooxidans was the dominant species at the early stage while Acidithiobacillus thiooxidans owned competitive advantage from the middle stage to the end of bioprocess. Meanwhile, compared to A. ferrooxidans, more significant effects of attached cells on free biomass with A. thiooxidans were shown in either the pure or mixed strains systems. Moreover, the effects of attached cells on key chemical parameters were also studied in different adsorption-deficient systems. Consistently, the greatest reduction of key chemical ion was shown with A. thiooxidans and the loss of bioleaching efficiency was high to 50.5%. These results all demonstrated the bioleaching function of attached cells was more efficient than the free cells, especially with A. thiooxidans. These notable results would help us to further understand the chalcopyrite bioleaching. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Pathophysiological consequences of hemolysis. Role of cell-free hemoglobin

    Directory of Open Access Journals (Sweden)

    Tomasz Misztal

    2011-09-01

    Full Text Available Abundant hemolysis is associated with a number of inherent and acquired diseases including sickle-cell disease (SCD, polycythemia, paroxysmal nocturnal hemoglobinuria (PNH and drug-induced hemolytic anemia. Despite different etiopathology of hemolytic diseases, many concomitant symptoms are comparable and include e.g. hypertension, hemoglobinuria and hypercoagulation state. Studies in the last years have shown a growing list of mechanisms lying at the basis of those symptoms, in particular irreversible reaction between cell-free hemoglobin (Hb and nitric oxide (NO – endogenous vasorelaxant and anti-thrombotic agent. Saturation of protective physiological cell-free Hb-scavenging mechanisms results in accumulation of Hb in plasma and hemoglobinemia. Extensive hemoglobinemia subsequently leads to hemoglobinuria, which may cause kidney damage and development of Fanconi syndrome. A severe problem in patients with SCD and PNH is pulmonary and systemic hypertension. It may lead to circulation failure, including stroke, and it is related to abolition of NO bioavailability for vascular smooth muscle cells. Thrombotic events are the major cause of death in SCD and PNH. It ensues from lack of platelet inhibition evoked by Hb-mediated NO scavenging. A serious complication that affects patients with excessive hemolysis is erectile dysfunction. Also direct cytotoxic, prooxidant and proinflammatory effects of cell-free hemoglobin and heme compose the clinical picture of hemolytic diseases. The pathophysiological role of plasma Hb, mechanisms of its elimination, and direct and indirect (via NO scavenging deleterious effects of cell-free Hb are presented in detail in this review. Understanding the critical role of hemolysis and cell-free Hb is important in the perspective of treating patients with hemolytic diseases and to design new effective therapies in future.

  15. Scaling up ITO-Free solar cells

    NARCIS (Netherlands)

    Galagan, Y.O.; Coenen, E.W.C.; Zimmermann, B.; Slooff, L.H.; Verhees, W.J.H.; Veenstra, S.C.; Kroon, J.M.; Jørgensen, M.; Krebs, F.C.; Andriessen, H.A.J.M.

    2014-01-01

    Indium-tin-oxide-free (ITO-free) polymer solar cells with composite electrodes containing current-collecting grids and a semitransparent poly(3,4-ethylenedioxythiophene):polystyrenesulfonate) (PEDOT:PSS) conductor are demonstrated. The up-scaling of the length of the solar cell from 1 to 6 cm and

  16. Cell-free expression and stable isotope labelling strategies for membrane proteins

    International Nuclear Information System (INIS)

    Sobhanifar, Solmaz; Reckel, Sina; Junge, Friederike; Schwarz, Daniel; Kai, Lei; Karbyshev, Mikhail; Loehr, Frank; Bernhard, Frank; Doetsch, Volker

    2010-01-01

    Membrane proteins are highly underrepresented in the structural data-base and remain one of the most challenging targets for functional and structural elucidation. Their roles in transport and cellular communication, furthermore, often make over-expression toxic to their host, and their hydrophobicity and structural complexity make isolation and reconstitution a complicated task, especially in cases where proteins are targeted to inclusion bodies. The development of cell-free expression systems provides a very interesting alternative to cell-based systems, since it circumvents many problems such as toxicity or necessity for the transportation of the synthesized protein to the membrane, and constitutes the only system that allows for direct production of membrane proteins in membrane-mimetic environments which may be suitable for liquid state NMR measurements. The unique advantages of the cell-free expression system, including strong expression yields as well as the direct incorporation of almost any combination of amino acids with very little metabolic scrambling, has allowed for the development of a wide-array of isotope labelling techniques which facilitate structural investigations of proteins whose spectral congestion and broad line-widths may have earlier rendered them beyond the scope of NMR. Here we explore various labelling strategies in conjunction with cell-free developments, with a particular focus on α-helical transmembrane proteins which benefit most from such methods.

  17. Dynamic imaging of cell-free and cell-associated viral capture in mature dendritic cells.

    Science.gov (United States)

    Izquierdo-Useros, Nuria; Esteban, Olga; Rodriguez-Plata, Maria T; Erkizia, Itziar; Prado, Julia G; Blanco, Julià; García-Parajo, Maria F; Martinez-Picado, Javier

    2011-12-01

    Dendritic cells (DCs) capture human immunodeficiency virus (HIV) through a non-fusogenic mechanism that enables viral transmission to CD4(+) T cells, contributing to in vivo viral dissemination. Although previous studies have provided important clues to cell-free viral capture by mature DCs (mDCs), dynamic and kinetic insight on this process is still missing. Here, we used three-dimensional video microscopy and single-particle tracking approaches to dynamically dissect both cell-free and cell-associated viral capture by living mDCs. We show that cell-free virus capture by mDCs operates through three sequential phases: virus binding through specific determinants expressed in the viral particle, polarized or directional movements toward concrete regions of the cell membrane and virus accumulation in a sac-like structure where trapped viral particles display a hindered diffusive behavior. Moreover, real-time imaging of cell-associated viral transfer to mDCs showed a similar dynamics to that exhibited by cell-free virus endocytosis leading to viral accumulation in compartments. However, cell-associated HIV type 1 transfer to mDCs was the most effective pathway, boosted throughout enhanced cellular contacts with infected CD4(+) T cells. Our results suggest that in lymphoid tissues, mDC viral uptake could occur either by encountering cell-free or cell-associated virus produced by infected cells generating the perfect scenario to promote HIV pathogenesis and impact disease progression. © 2011 John Wiley & Sons A/S.

  18. Development of serum-free media for lepidopteran insect cell lines.

    Science.gov (United States)

    Agathos, Spiros N

    2007-01-01

    Lepidopteran insect cell culture technology has progressed to the point of becoming an essential part of one of the most successful eukaryotic expression systems and is increasingly used industrially on a large scale. Therefore, there is a constant need for convenient and low-cost culture media capable of supporting good insect cell growth and ensuring high yield of baculovirus as well as the strong expression of recombinant proteins. Vertebrate sera or invertebrate hemolymph were essential supplements in first-generation insect cell media. These supplements, however, are cumbersome and expensive for routine large-scale culture; thus, their use is now circumvented by substituting the essential growth factors present in these supplements with serum-free substances. Such non-serum supplements are typically of non-animal origin and include protein hydrolysates, lipid emulsions, and specialized substances (e.g., surfactants and shear damage protecting chemicals). These supplements need to complement the defined, synthetic basal medium to ensure that the fundamental nutritional needs of the cells are satisfied. Although there is a significant number of proprietary serum-free and low-protein or protein-free media on the market, the lack of information concerning their detailed composition is a drawback in their adoption for different applications, including their adaptation to the metabolic and kinetic analysis and monitoring of a given insect cell based bioprocess. Hence, there is wide appeal for formulating serum-free media based on a rational assessment of the metabolic requirements of the lepidopteran cells during both the growth and the production phases. Techniques such as statistical experimental design and genetic algorithms adapted to the cellular behavior and the bioreactor operation mode (batch, fed-batch, or perfusion) permit the formulation of versatile serum- and protein-free media. These techniques are illustrated with recent developments of serum-free

  19. Dry storage systems with free convection air cooling

    International Nuclear Information System (INIS)

    Kioes, S.R.

    1980-01-01

    Several design principles to remove heat from the spent fuel by free air convection are illustrated and described. The key safety considerations were felt to be: loss of coolant is impossible as the passive system uses air as a coolant; overheating is precluded because as the temperatures of the containers rises the coolant flow rate increases; mass of the storage building provides a large heat sink and therefore a rapid temperature rise is impossible; and lack of any active external support requirements makes the cooling process less likely to equipment or operator failures. An example of this type of storage already exists. The German HTGR is operated with spherical graphite fuel elements which are stored in canister and in storage cells. The concept is a double cooling system with free convection inside the cells and heat exchange via two side walls of the cell to the ambient air in the cooling ducts. Technical description of the TN 1300 cask is also presented

  20. Defined Essential 8™ Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems

    Science.gov (United States)

    Badenes, Sara M.; Fernandes, Tiago G.; Cordeiro, Cláudia S. M.; Boucher, Shayne; Kuninger, David; Vemuri, Mohan C.; Diogo, Maria Margarida; Cabral, Joaquim M. S.

    2016-01-01

    Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL spinner flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation speed, and c) to maximize cell yield in spinner flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm2 of microcarrier surface area and using 44 rpm, which generates a cell density of 1.4x106 cells/mL after 10 days of culture. After dynamic culture, hiPS cells maintained their typical morphology upon re-plating, exhibited pluripotency-associated marker expression as well as tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid body formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, robust and cost-effective xeno-free culture system was successfully developed and implemented for the scale-up production of hiPS cells. PMID:26999816

  1. Free-electron laser system with Raman amplifier outcoupling

    Energy Technology Data Exchange (ETDEWEB)

    Linford, G.J.

    1988-05-03

    A free-electron laser system is described comprising: a free-electron laser pump beam generator producing a high-power optical output beam in a vacuum environement; a Raman amplifier cell located in the path of the output beam from the pump beam generator; means for generating and introducing a Stokes seed beam into the Raman amplifier cell, a pair of gaseous windows through which the output beam enters and leaves the Raman amplifier cell, each window having a stream of gas moving continuously in a direction generally perpendicular to the beam; and a mirror positioned in the path of the output beam from the Raman amplifier, the mirror functioning to reflect and further direct the output beam, but not the unwanted spectral components.

  2. Oral epithelial cells are susceptible to cell-free and cell-associated HIV-1 infection in vitro

    International Nuclear Information System (INIS)

    Moore, Jennifer S.; Rahemtulla, Firoz; Kent, Leigh W.; Hall, Stacy D.; Ikizler, Mine R.; Wright, Peter F.; Nguyen, Huan H.; Jackson, Susan

    2003-01-01

    Epithelial cells lining the oral cavity are exposed to HIV-1 through breast-feeding and oral-genital contact. Genital secretions and breast milk of HIV-1-infected subjects contain both cell-free and cell-associated virus. To determine if oral epithelial cells can be infected with HIV-1 we exposed gingival keratinocytes and adenoid epithelial cells to cell-free virus and HIV-1-infected peripheral blood mononuclear cells and monocytes. Using primary isolates we determined that gingival keratinocytes are susceptible to HIV-1 infection via cell-free CD4-independent infection only. R5 but not X4 viral strains were capable of infecting the keratinocytes. Further, infected cells were able to release infectious virus. In addition, primary epithelial cells isolated from adenoids were also susceptible to infection; both cell-free and cell-associated virus infected these cells. These data have potential implications in the transmission of HIV-1 in the oral cavity

  3. Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Minh Vu Chuong, E-mail: mvchuong@yahoo.fr [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France); Zhang, Leilei [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France); Lhomme, Stanislas; Mouz, Nicolas [PX' Therapeutics, MINATEC/Batiment de Haute Technologie, Grenoble (France); Lenormand, Jean-Luc [HumProTher Laboratory, TheReX/TIMC-IMAG UMR 5525 CNRS UJF, Universite Joseph Fourier, UFR de Medecine, Domaine de la Merci, 38706 La Tronche (France); Lardy, Bernard; Morel, Francoise [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France)

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer A comparison of two bacterial cell and cell-free protein expression systems is presented. Black-Right-Pointing-Pointer Soluble and active truncated Nox4 proteins are produced. Black-Right-Pointing-Pointer Nox4 has a constitutive diaphorase activity which is independent of cytosolic factors. Black-Right-Pointing-Pointer Isoform Nox4B is unable to initiate the first electronic transfer step. Black-Right-Pointing-Pointer Findings contribute to the understanding of the mechanism of Nox4 oxidase activity. -- Abstract: The membrane protein NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 constitutively generates reactive oxygen species differing from other NADPH oxidases activity, particularly in Nox2 which needs a stimulus to be active. Although the precise mechanism of production of reactive oxygen species by Nox2 is well characterized, the electronic transfer throughout Nox4 remains unclear. Our study aims to investigate the initial electronic transfer step (diaphorase activity) of the cytosolic tail of Nox4. For this purpose, we developed two different approaches to produce soluble and active truncated Nox4 proteins. We synthesized soluble recombinant proteins either by in vitro translation or by bacteria induction. While proteins obtained by bacteria induction demonstrate an activity of 4.4 {+-} 1.7 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 20.5 {+-} 2.8 nmol/min/nmol with cytochrome c, the soluble proteins produced by cell-free expression system exhibit a diaphorase activity with a turn-over of 26 {+-} 2.6 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 48 {+-} 20.2 nmol/min/nmol with cytochrome c. Furthermore, the activity of the soluble proteins is constitutive and does not need any stimulus. We also show that the cytosolic tail of the isoform Nox4B lacking the first NADPH binding site is unable to demonstrate any diaphorase activity pointing out the

  4. On Orbit Immuno-Based, Label-Free, White Blood Cell Counting System with MicroElectroMechanical Sensor (MEMS) Technology (OILWBCS-MEMS), Phase II

    Data.gov (United States)

    National Aeronautics and Space Administration — Aurora Flight Sciences Corporation and partner, Draper Laboratory, propose to develop an on-orbit immuno-based label-free white blood cell counting system using MEMS...

  5. On Orbit Immuno-Based, Label-Free, White Blood Cell Counting System with MicroElectroMechanical Sensor (MEMS) Technology (OILWBCS-MEMS), Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — Aurora Flight Sciences Corporation and our partner, Draper Laboratory, propose to develop an on orbit immuno-based, label-free, white blood cell counting system for...

  6. Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis.

    Science.gov (United States)

    Gan, Qinglei; Fan, Chenguang

    2017-11-01

    Cell-free protein synthesis provides a robust platform for co-translational incorporation of noncanonical amino acid (ncAA) into proteins to facilitate biological studies and biotechnological applications. Recently, eliminating the activity of release factor 1 has been shown to increase ncAA incorporation in response to amber codons. However, this approach could promote mis-incorporation of canonical amino acids by near cognate suppression. We performed a facile protocol to remove near cognate tRNA isoacceptors of the amber codon from total tRNAs, and used the phosphoserine (Sep) incorporation system as validation. By manipulating codon usage of target genes and tRNA species introduced into the cell-free protein synthesis system, we increased the fidelity of Sep incorporation at a specific position. By removing three near cognate tRNA isoacceptors of the amber stop codon [tRNA Lys , tRNA Tyr , and tRNA Gln (CUG)] from the total tRNA, the near cognate suppression decreased by 5-fold without impairing normal protein synthesis in the cell-free protein synthesis system. Mass spectrometry analyses indicated that the fidelity of ncAA incorporation was improved. Removal of near cognate tRNA isoacceptors of the amber codon could increase ncAA incorporation fidelity towards the amber stop codon in release factor deficiency systems. We provide a general strategy to improve fidelity of ncAA incorporation towards stop, quadruplet and sense codons in cell-free protein synthesis systems. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Microbial fuel cell as a free-radical scavenging tool

    International Nuclear Information System (INIS)

    Koleva, Ralitza; Yemendzhiev, Hyusein; Nenov, Valentin

    2017-01-01

    Microbial fuel cells (MFCs) are known for their capability to directly convert organic substrates into electricity by the biochemical activity of specific microorganisms. Availability of a proper terminal electron acceptor is crucial for this process. Free radicals, with their one or more unpaired electrons, are extremely reducible and could be considered as electron acceptors in terms of cathodic processes in MFC. During this reduction, free radicals could be transformed in the same manner as they are transformed by antioxidants. The present study investigated this opportunity by utilization of 2,2-diphenyl-1-picrylhydrazyl (150 mmol/dm"3 methanol solution) as a free-radical molecule. During the studied process, over 90% radical neutralization was observed in less than 16 hours. The results obtained demonstrate for the first time the potential of MFC type bioelectrochemical systems to serve as a free-radical scavenging tool and to provide antioxidant and anti-radical activity. In this way, this study opens a completely new field of research and application of bio-electrochemical systems

  8. Radio-modification by caffeine alone and in combination with phosphorothioates: in vivo and cell-free studies

    International Nuclear Information System (INIS)

    Swenberg, C.E.; Landauer, M.R.; Weiss, J.F.

    1997-01-01

    Caffeine is generally considered to result in radiosensitization by affecting the cell cycle. Data from in vivo studies, however, do not suggest sensitization; caffeine administration did not adversely affect survival of mice irradiated at doses causing hematopoietic injury, or gastrointestinal injury, or when administered in combination with phosphorothioates. For example, caffeine administration (20 mg/kg IP) in combination with the radioprotector WR-151327, S-2-(3-methyl-amino-propyl-amino)propyl-phosphoro-thioic acid. (200 mg/kg IP) resulted in a dose modification factor of 1.54 in comparison to 1.51 for WR-151327 treatment alone. In a cell-free system, the active metabolites of phosphorothiotates, i.e. free thiols and disulfides, appear to mimic polyamines and modulate enzymes involves in DNA structure and synthesis. The free thiol of WR-151327 (WR-151326) actively enhanced topoisomerase I-mediated unwinding of supercoiled plB130 DNA and super-coiling of DNA mediated by DNA gyrase (topoisomerase II). Caffeine, in general, had opposite effects on potoisomerase activities compared to WR-151326. When caffeine was added to the cell-free system together with WR-151326, the stimulatory effects of WR-151326 were suppressed. Further studies are needed in cell-free systems, cells, and animals to elucidate the potential utility of caffeine administration in combination with radiation and other therapeutic agents. (authors)

  9. Radio-modification by caffeine alone and in combination with phosphorothioates: in vivo and cell-free studies

    Energy Technology Data Exchange (ETDEWEB)

    Swenberg, C.E.; Landauer, M.R. [Armed Forces Radiobiology Research Institute, Bethesda (United States); Weiss, J.F. [Office of International Health Programs, Department of Energy, Germantown (United States)

    1997-03-01

    Caffeine is generally considered to result in radiosensitization by affecting the cell cycle. Data from in vivo studies, however, do not suggest sensitization; caffeine administration did not adversely affect survival of mice irradiated at doses causing hematopoietic injury, or gastrointestinal injury, or when administered in combination with phosphorothioates. For example, caffeine administration (20 mg/kg IP) in combination with the radioprotector WR-151327, S-2-(3-methyl-amino-propyl-amino)propyl-phosphoro-thioic acid. (200 mg/kg IP) resulted in a dose modification factor of 1.54 in comparison to 1.51 for WR-151327 treatment alone. In a cell-free system, the active metabolites of phosphorothiotates, i.e. free thiols and disulfides, appear to mimic polyamines and modulate enzymes involves in DNA structure and synthesis. The free thiol of WR-151327 (WR-151326) actively enhanced topoisomerase I-mediated unwinding of supercoiled plB130 DNA and super-coiling of DNA mediated by DNA gyrase (topoisomerase II). Caffeine, in general, had opposite effects on potoisomerase activities compared to WR-151326. When caffeine was added to the cell-free system together with WR-151326, the stimulatory effects of WR-151326 were suppressed. Further studies are needed in cell-free systems, cells, and animals to elucidate the potential utility of caffeine administration in combination with radiation and other therapeutic agents. (authors)

  10. Establishing a high yielding streptomyces-based cell-free protein synthesis system.

    Science.gov (United States)

    Li, Jian; Wang, He; Kwon, Yong-Chan; Jewett, Michael C

    2017-06-01

    Cell-free protein synthesis (CFPS) has emerged as a powerful platform for applied biotechnology and synthetic biology, with a range of applications in synthesizing proteins, evolving proteins, and prototyping genetic circuits. To expand the current CFPS repertoire, we report here the development and optimization of a Streptomyces-based CFPS system for the expression of GC-rich genes. By developing a streamlined crude extract preparation protocol and optimizing reaction conditions, we were able to achieve active enhanced green fluorescent protein (EGFP) yields of greater than 50 μg/mL with batch reactions lasting up to 3 h. By adopting a semi-continuous reaction format, the EGFP yield could be increased to 282 ± 8 μg/mL and the reaction time was extended to 48 h. Notably, our extract preparation procedures were robust to multiple Streptomyces lividans and Streptomyces coelicolor strains, although expression yields varied. We show that our optimized Streptomyces lividans system provides benefits when compared to an Escherichia coli-based CFPS system for increasing percent soluble protein expression for four Streptomyces-originated high GC-content genes that are involved in biosynthesis of the nonribosomal peptides tambromycin and valinomycin. Looking forward, we believe that our Streptomyces-based CFPS system will contribute significantly towards efforts to express complex natural product gene clusters (e.g., nonribosomal peptides and polyketides), providing a new avenue for obtaining and studying natural product biosynthesis pathways. Biotechnol. Bioeng. 2017;114: 1343-1353. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  11. Photovoltaic power generation system free of bypass diodes

    Science.gov (United States)

    Lentine, Anthony L.; Okandan, Murat; Nielson, Gregory N.

    2015-07-28

    A photovoltaic power generation system that includes a solar panel that is free of bypass diodes is described herein. The solar panel includes a plurality of photovoltaic sub-modules, wherein at least two of photovoltaic sub-modules in the plurality of photovoltaic sub-modules are electrically connected in parallel. A photovoltaic sub-module includes a plurality of groups of electrically connected photovoltaic cells, wherein at least two of the groups are electrically connected in series. A photovoltaic group includes a plurality of strings of photovoltaic cells, wherein a string of photovoltaic cells comprises a plurality of photovoltaic cells electrically connected in series. The strings of photovoltaic cells are electrically connected in parallel, and the photovoltaic cells are microsystem-enabled photovoltaic cells.

  12. Comparing handheld and hands-free cell phone usage behaviors while driving.

    Science.gov (United States)

    Soccolich, Susan A; Fitch, Gregory M; Perez, Miguel A; Hanowski, Richard J

    2014-01-01

    The goal of this study was to compare cell phone usage behaviors while driving across 3 types of cell phones: handheld (HH) cell phones, portable hands-free (PHF) cell phones, and integrated hands-free (IHF) cell phones. Naturalistic driving data were used to observe HH, PHF, and IHF usage behaviors in participants' own vehicles without any instructions or manipulations by researchers. In addition to naturalistic driving data, drivers provided their personal cell phone call records. Calls during driving were sampled and observed in naturalistically collected video. Calls were reviewed to identify cell phone type used for, and duration of, cell phone subtasks, non-cell phone secondary tasks, and other use behaviors. Drivers in the study self-identified as HH, PHF, or IHF users if they reported using that cell phone type at least 50% of the time. However, each sampled call was classified as HH, PHF, or IHF if the talking/listening subtask was conducted using that cell phone type, without considering the driver's self-reported group. Drivers with PHF or IHF systems also used HH cell phones (IHF group used HH cell phone in 53.2% of the interactions, PHF group used HH cell phone for 55.5% of interactions). Talking/listening on a PHF phone or an IHF phone was significantly longer than talking/listening on an HH phone (P phone call task for HH phones was significantly longer in duration than the end phone call task for PHF and IHF phones. Of all the non-cell phone-related secondary tasks, eating or drinking was found to occur significantly more often during IHF subtasks (0.58%) than in HH subtasks (0.15%). Drivers observed to reach for their cell phone mostly kept their cell phone in the cup holder (36.3%) or in their seat or lap (29.0% of interactions); however, some observed locations may have required drivers to move out of position. Hands-free cell phone technologies reduce the duration of cell phone visual-manual tasks compared to handheld cell phones. However

  13. Serum-free media formulations are cell line-specific and require optimization for microcarrier culture.

    Science.gov (United States)

    Tan, Kah Yong; Teo, Kim Leng; Lim, Jessica F Y; Chen, Allen K L; Choolani, Mahesh; Reuveny, Shaul; Chan, Jerry; Oh, Steve Kw

    2015-08-01

    Mesenchymal stromal cells (MSCs) are being investigated as potential cell therapies for many different indications. Current methods of production rely on traditional monolayer culture on tissue-culture plastic, usually with the use of serum-supplemented growth media. However, the monolayer culturing system has scale-up limitations and may not meet the projected hundreds of billions to trillions batches of cells needed for therapy. Furthermore, serum-free medium offers several advantages over serum-supplemented medium, which may have supply and contaminant issues, leading to many serum-free medium formulations being developed. We cultured seven MSC lines in six different serum-free media and compared their growth between monolayer and microcarrier culture. We show that (i) expansion levels of MSCs in serum-free monolayer cultures may not correlate with expansion in serum-containing media; (ii) optimal culture conditions (serum-free media for monolayer or microcarrier culture) differ for each cell line; (iii) growth in static microcarrier culture does not correlate with growth in stirred spinner culture; (iv) and that early cell attachment and spreading onto microcarriers does not necessarily predict efficiency of cell expansion in agitated microcarrier culture. Current serum-free media developed for monolayer cultures of MSCs may not support MSC proliferation in microcarrier cultures. Further optimization in medium composition will be required for microcarrier suspension culture for each cell line. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  14. Optimizing Immobilized Enzyme Performance in Cell-Free Environments to Produce Liquid Fuels

    Energy Technology Data Exchange (ETDEWEB)

    Belfort, Georges [Rensselaer Polytechnic Inst., Troy, NY (United States). Dept. of Chemical and Biological Engineering; Grimaldi, Joseph J. [Rensselaer Polytechnic Inst., Troy, NY (United States). Dept. of Chemical and Biological Engineering

    2015-01-27

    Limitations on biofuel production using cell culture (Escherichia coli, Clostridium, Saccharomyces cerevisiae, brown microalgae, blue-green algae and others) include low product (alcohol) concentrations (≤0.2 vol%) due to feedback inhibition, instability of cells, and lack of economical product recovery processes. To overcome these challenges, an alternate simplified biofuel production scheme was tested based on a cell-free immobilized enzyme system. Using this cell free system, we were able to obtain about 2.6 times higher concentrations of iso-butanol using our non-optimized system as compared with live cell systems. This process involved two steps: (i) converts acid to aldehyde using keto-acid decarboxylase (KdcA), and (ii) produces alcohol from aldehyde using alcohol dehydrogenase (ADH) with a cofactor (NADH) conversion from inexpensive formate using a third enzyme, formate dehydrogenase (FDH). To increase stability and conversion efficiency with easy separations, the first two enzymes were immobilized onto methacrylate resin. Fusion proteins of labile KdcA (fKdcA) were expressed to stabilize the covalently immobilized KdcA. Covalently immobilized ADH exhibited long-term stability and efficient conversion of aldehyde to alcohol over multiple batch cycles without fusions. High conversion rates and low protein leaching were achieved by covalent immobilization of enzymes on methacrylate resin. The complete reaction scheme was demonstrated by immobilizing both ADH and fKdcA and using FDH free in solution. The new system without in situ removal of isobutanol achieved a 55% conversion of ketoisovaleric acid to isobutanol at a concentration of 0.5 % (v/v). Further increases in titer will require continuous removal of the isobutanol using our novel brush membrane system that exhibits a 1.5 fold increase in the separation factor of isobutanol from water versus that obtained for commercial silicone rubber membranes. These bio-inspired brush membranes are based on the

  15. Number of infection events per cell during HIV-1 cell-free infection.

    Science.gov (United States)

    Ito, Yusuke; Remion, Azaria; Tauzin, Alexandra; Ejima, Keisuke; Nakaoka, Shinji; Iwasa, Yoh; Iwami, Shingo; Mammano, Fabrizio

    2017-07-26

    HIV-1 accumulates changes in its genome through both recombination and mutation during the course of infection. For recombination to occur, a single cell must be infected by two HIV strains. These coinfection events were experimentally demonstrated to occur more frequently than would be expected for independent infection events and do not follow a random distribution. Previous mathematical modeling approaches demonstrated that differences in target cell susceptibility can explain the non-randomness, both in the context of direct cell-to-cell transmission, and in the context of free virus transmission (Q. Dang et al., Proc. Natl. Acad. Sci. USA 101:632-7, 2004: K. M. Law et al., Cell reports 15:2711-83, 2016). Here, we build on these notions and provide a more detailed and extensive quantitative framework. We developed a novel mathematical model explicitly considering the heterogeneity of target cells and analysed datasets of cell-free HIV-1 single and double infection experiments in cell culture. Particularly, in contrast to the previous studies, we took into account the different susceptibility of the target cells as a continuous distribution. Interestingly, we showed that the number of infection events per cell during cell-free HIV-1 infection follows a negative-binomial distribution, and our model reproduces these datasets.

  16. Cell-Free, De Nova Synthesis of Poliovirus

    Science.gov (United States)

    Molla, Akhteruzzaman; Paul, Aniko V.; Wimmer, Eckard

    1991-12-01

    Cell-free translation of poliovirus RNA in an extract of uninfected human (HeLa) cells yielded viral proteins through proteolysis of the polyprotein. In the extract, newly synthesized proteins catalyzed poliovirus-specific RNA synthesis, and formed infectious poliovirus de novo. Newly formed virions were neutralized by type-specific antiserum, and infection of human cells with them was prevented by poliovirus receptor-specific antibodies. Poliovirus synthesis was increased nearly 70-fold when nucleoside triphosphates were added, but it was abolished in the presence of inhibitors of translation or viral genome replication. The ability to conduct cell-free synthesis of poliovirus will aid in the study of picornavirus proliferation and in the search for the control of picornaviral disease.

  17. Generation of hematopoietic stem cells from human embryonic stem cells using a defined, stepwise, serum-free, and serum replacement-free monolayer culture method.

    Science.gov (United States)

    Kim, So-Jung; Jung, Ji-Won; Ha, Hye-Yeong; Koo, Soo Kyung; Kim, Eung-Gook; Kim, Jung-Hyun

    2017-03-01

    Embryonic stem cells (ESCs) can be expanded infinitely in vitro and have the potential to differentiate into hematopoietic stem cells (HSCs); thus, they are considered a useful source of cells for HSC production. Although several technical in vitro methods for engineering HSCs from pluripotent stem cells have been developed, clinical application of HSCs engineered from pluripotent stem cells is restricted because of the possibility of xenogeneic contamination resulting from the use of murine materials. Human ESCs (CHA-hES15) were cultured on growth factor-reduced Matrigel-coated dishes in the mTeSR1 serum-free medium. When the cells were 70% confluent, we initiated HSC differentiation by three methods involving (1) knockout serum replacement (KSR), cytokines, TGFb1, EPO, and FLT3L; (2) KSR, cytokines, and bFGF; or (3) cytokines and bFGF. Among the three differentiation methods, the minimal number of cytokines without KSR resulted in the greatest production of HSCs. The optimized method resulted in a higher proportion of CD34 + CD43 + hematopoietic progenitor cells (HPCs) and CD34 + CD45 + HPCs compared to the other methods. In addition, the HSCs showed the potential to differentiate into multiple lineages of hematopoietic cells in vitro . In this study, we optimized a two-step, serum-free, animal protein-free, KSR-free, feeder-free, chemically defined monolayer culture method for generation of HSCs and hematopoietic stem and progenitor cells (HSPCs) from human ESCs.

  18. Measuring strand discontinuity-directed mismatch repair in yeast Saccharomyces cerevisiae by cell-free nuclear extracts.

    Science.gov (United States)

    Yuan, Fenghua; Lai, Fangfang; Gu, Liya; Zhou, Wen; El Hokayem, Jimmy; Zhang, Yanbin

    2009-05-01

    Mismatch repair corrects biosynthetic errors generated during DNA replication, whose deficiency causes a mutator phenotype and directly underlies hereditary non-polyposis colorectal cancer and sporadic cancers. Because of remarkably high conservation of the mismatch repair machinery between the budding yeast (Saccharomyces cerevisiae) and humans, the study of mismatch repair in yeast has provided tremendous insights into the mechanisms of this repair pathway in humans. In addition, yeast cells possess an unbeatable advantage over human cells in terms of the easy genetic manipulation, the availability of whole genome deletion strains, and the relatively low cost for setting up the system. Although many components of eukaryotic mismatch repair have been identified, it remains unclear if additional factors, such as DNA helicase(s) and redundant nuclease(s) besides EXO1, participate in eukaryotic mismatch repair. To facilitate the discovery of novel mismatch repair factors, we developed a straightforward in vitro cell-free repair system. Here, we describe the practical protocols for preparation of yeast cell-free nuclear extracts and DNA mismatch substrates, and the in vitro mismatch repair assay. The validity of the cell-free system was confirmed by the mismatch repair deficient yeast strain (Deltamsh2) and the complementation assay with purified yeast MSH2-MSH6.

  19. Temperature and UV light affect the activity of marine cell-free enzymes

    Directory of Open Access Journals (Sweden)

    B. Thomson

    2017-09-01

    Full Text Available Microbial extracellular enzymatic activity (EEA is the rate-limiting step in the degradation of organic matter in the oceans. These extracellular enzymes exist in two forms: cell-bound, which are attached to the microbial cell wall, and cell-free, which are completely free of the cell. Contrary to previous understanding, cell-free extracellular enzymes make up a substantial proportion of the total marine EEA. Little is known about these abundant cell-free enzymes, including what factors control their activity once they are away from their sites (cells. Experiments were run to assess how cell-free enzymes (excluding microbes respond to ultraviolet radiation (UVR and temperature manipulations, previously suggested as potential control factors for these enzymes. The experiments were done with New Zealand coastal waters and the enzymes studied were alkaline phosphatase (APase, β-glucosidase, (BGase, and leucine aminopeptidase (LAPase. Environmentally relevant UVR (i.e. in situ UVR levels measured at our site reduced cell-free enzyme activities by up to 87 % when compared to controls, likely a consequence of photodegradation. This effect of UVR on cell-free enzymes differed depending on the UVR fraction. Ambient levels of UV radiation were shown to reduce the activity of cell-free enzymes for the first time. Elevated temperatures (15 °C increased the activity of cell-free enzymes by up to 53 % when compared to controls (10 °C, likely by enhancing the catalytic activity of the enzymes. Our results suggest the importance of both UVR and temperature as control mechanisms for cell-free enzymes. Given the projected warming ocean environment and the variable UVR light regime, it is possible that there could be major changes in the cell-free EEA and in the enzymes contribution to organic matter remineralization in the future.

  20. Generation of thermostable Moloney murine leukemia virus reverse transcriptase variants using site saturation mutagenesis library and cell-free protein expression system.

    Science.gov (United States)

    Katano, Yuta; Li, Tongyang; Baba, Misato; Nakamura, Miyo; Ito, Masaaki; Kojima, Kenji; Takita, Teisuke; Yasukawa, Kiyoshi

    2017-12-01

    We attempted to increase the thermostability of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT). The eight-site saturation mutagenesis libraries corresponding to Ala70-Arg469 in the whole MMLV RT (Thr24-Leu671), in each of which 1 out of 50 amino acid residues was replaced with other amino acid residue, were constructed. Seven-hundred and sixty eight MMLV RT clones were expressed using a cell-free protein expression system, and their thermostabilities were assessed by the temperature of thermal treatment at which they retained cDNA synthesis activity. One clone D200C was selected as the most thermostable variant. The highest temperature of thermal treatment at which D200C exhibited cDNA synthesis activity was 57ºC, which was higher than for WT (53ºC). Our results suggest that a combination of site saturation mutagenesis library and cell-free protein expression system might be useful for generation of thermostable MMLV RT in a short period of time for expression and selection.

  1. Prenatal Cell-Free DNA Screening

    Science.gov (United States)

    ... poses no physical risks for you or your baby. While prenatal cell-free DNA screening might cause anxiety, it might help you avoid the need for more invasive tests, treatment or monitoring during your pregnancy. Keep in mind, however, that ...

  2. Extracellular Matrix-Dependent Generation of Integration- and Xeno-Free iPS Cells Using a Modified mRNA Transfection Method

    Directory of Open Access Journals (Sweden)

    Kang-In Lee

    2016-01-01

    Full Text Available Human induced pluripotent stem cells (iPS cells hold great promise in the field of regenerative medicine, especially immune-compatible cell therapy. The most important safety-related issues that must be resolved before the clinical use of iPS cells include the generation of “footprint-free” and “xeno-free” iPS cells. In this study, we sought to examine whether an extracellular matrix- (ECM- based xeno-free culture system that we recently established could be used together with a microRNA-enhanced mRNA reprogramming method for the generation of clinically safe iPS cells. The notable features of this method are the use of a xeno-free/feeder-free culture system for the generation and expansion of iPS cells rather than the conventional labor-intensive culture systems using human feeder cells or human feeder-conditioned medium and the enhancement of mRNA-mediated reprogramming via the delivery of microRNAs. Strikingly, we observed the early appearance of iPS cell colonies (~11 days, substantial reprogramming efficiency (~0.2–0.3%, and a high percentage of ESC-like colonies among the total colonies (~87.5%, indicating enhanced kinetics and reprogramming efficiency. Therefore, the combined method established in this study provides a valuable platform for the generation and expansion of clinically safe (i.e., integration- and xeno-free iPS cells, facilitating immune-matched cell therapy in the near future.

  3. Efficient cell-free production of olfactory receptors: detergent optimization, structure, and ligand binding analyses.

    Science.gov (United States)

    Kaiser, Liselotte; Graveland-Bikker, Johanna; Steuerwald, Dirk; Vanberghem, Mélanie; Herlihy, Kara; Zhang, Shuguang

    2008-10-14

    High-level production of membrane proteins, particularly of G protein-coupled receptors (GPCRs) in heterologous cell systems encounters a number of difficulties from their inherent hydrophobicity in their transmembrane domains, which frequently cause protein aggregation and cytotoxicity and thus reduce the protein yield. Recent advances in cell-free protein synthesis circumvent those problems to produce membrane proteins with a yield sometimes exceeding the cell-based approach. Here, we report cell-free production of a human olfactory receptor 17-4 (hOR17-4) using the wheat germ extract. Using the simple method, we also successful produced two additional olfactory receptors. To obtain soluble olfactory receptors and to increase yield, we directly added different detergents in varying concentrations to the cell-free reaction. To identify a purification buffer system that maintained the receptor in a nonaggregated form, we developed a method that uses small-volume size-exclusion column chromatography combined with rapid and sensitive dot-blot detection. Different buffer components including salt concentration, various detergents and detergent concentration, and reducing agent and its concentrations were evaluated for their ability to maintain the cell-free produced protein stable and nonaggregated. The purified olfactory receptor displays a typical a alpha-helical CD spectrum. Surface plasmon resonance measurements were used to show binding of a known ligand undecanal to hOR17-4. Our approach to produce a high yield of purified olfactory receptor is a milestone toward obtaining a large quantity of olfactory receptors for designing bionic sensors. Furthermore, this simple approach may be broadly useful not only for other classes of GPCRs but also for other membrane proteins.

  4. Cell-free protein synthesis in micro compartments: building a minimal cell from biobricks.

    Science.gov (United States)

    Jia, Haiyang; Heymann, Michael; Bernhard, Frank; Schwille, Petra; Kai, Lei

    2017-10-25

    The construction of a minimal cell that exhibits the essential characteristics of life is a great challenge in the field of synthetic biology. Assembling a minimal cell requires multidisciplinary expertise from physics, chemistry and biology. Scientists from different backgrounds tend to define the essence of 'life' differently and have thus proposed different artificial cell models possessing one or several essential features of living cells. Using the tools and methods of molecular biology, the bottom-up engineering of a minimal cell appears in reach. However, several challenges still remain. In particular, the integration of individual sub-systems that is required to achieve a self-reproducing cell model presents a complex optimization challenge. For example, multiple self-organisation and self-assembly processes have to be carefully tuned. We review advances and developments of new methods and techniques, for cell-free protein synthesis as well as micro-fabrication, for their potential to resolve challenges and to accelerate the development of minimal cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Expression, stabilization and purification of membrane proteins via diverse protein synthesis systems and detergents involving cell-free associated with self-assembly peptide surfactants.

    Science.gov (United States)

    Zheng, Xuan; Dong, Shuangshuang; Zheng, Jie; Li, Duanhua; Li, Feng; Luo, Zhongli

    2014-01-01

    G-protein coupled receptors (GPCRs) are involved in regulating most of physiological actions and metabolism in the bodies, which have become most frequently addressed therapeutic targets for various disorders and diseases. Purified GPCR-based drug discoveries have become routine that approaches to structural study, novel biophysical and biochemical function analyses. However, several bottlenecks that GPCR-directed drugs need to conquer the problems including overexpression, solubilization, and purification as well as stabilization. The breakthroughs are to obtain efficient protein yield and stabilize their functional conformation which are both urgently requiring of effective protein synthesis system methods and optimal surfactants. Cell-free protein synthesis system is superior to the high yields and post-translation modifications, and early signs of self-assembly peptide detergents also emerged to superiority in purification of membrane proteins. We herein focus several predominant protein synthesis systems and surfactants involving the novel peptide detergents, and uncover the advantages of cell-free protein synthesis system with self-assembling peptide detergents in purification of functional GPCRs. This review is useful to further study in membrane proteins as well as the new drug exploration. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Rapid and Scalable Characterization of CRISPR Technologies Using an E. coli Cell-Free Transcription-Translation System.

    Science.gov (United States)

    Marshall, Ryan; Maxwell, Colin S; Collins, Scott P; Jacobsen, Thomas; Luo, Michelle L; Begemann, Matthew B; Gray, Benjamin N; January, Emma; Singer, Anna; He, Yonghua; Beisel, Chase L; Noireaux, Vincent

    2018-01-04

    CRISPR-Cas systems offer versatile technologies for genome engineering, yet their implementation has been outpaced by ongoing discoveries of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation (TXTL) systems to vastly improve the speed and scalability of CRISPR characterization and validation. TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression-all without protein purification or live cells. We used TXTL to measure the dynamics of DNA cleavage and gene repression for single- and multi-effector CRISPR nucleases, predict gene repression strength in E. coli, determine the specificities of 24 diverse anti-CRISPR proteins, and develop a fast and scalable screen for protospacer-adjacent motifs that was successfully applied to five uncharacterized Cpf1 nucleases. These examples underscore how TXTL can facilitate the characterization and application of CRISPR technologies across their many uses. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Cell-free protein synthesis enabled rapid prototyping for metabolic engineering and synthetic biology

    Directory of Open Access Journals (Sweden)

    Lihong Jiang

    2018-06-01

    Full Text Available Advances in metabolic engineering and synthetic biology have facilitated the manufacturing of many valuable-added compounds and commodity chemicals using microbial cell factories in the past decade. However, due to complexity of cellular metabolism, the optimization of metabolic pathways for maximal production represents a grand challenge and an unavoidable barrier for metabolic engineering. Recently, cell-free protein synthesis system (CFPS has been emerging as an enabling alternative to address challenges in biomanufacturing. This review summarizes the recent progresses of CFPS in rapid prototyping of biosynthetic pathways and genetic circuits (biosensors to speed up design-build-test (DBT cycles of metabolic engineering and synthetic biology. Keywords: Cell-free protein synthesis, Metabolic pathway optimization, Genetic circuits, Metabolic engineering, Synthetic biology

  8. Cell-free synthesis of functional antibody fragments to provide a structural basis for antibody-antigen interaction.

    Directory of Open Access Journals (Sweden)

    Takayoshi Matsuda

    Full Text Available Growing numbers of therapeutic antibodies offer excellent treatment strategies for many diseases. Elucidation of the interaction between a potential therapeutic antibody and its target protein by structural analysis reveals the mechanism of action and offers useful information for developing rational antibody designs for improved affinity. Here, we developed a rapid, high-yield cell-free system using dialysis mode to synthesize antibody fragments for the structural analysis of antibody-antigen complexes. Optimal synthesis conditions of fragments (Fv and Fab of the anti-EGFR antibody 059-152 were rapidly determined in a day by using a 30-μl-scale unit. The concentration of supplemented disulfide isomerase, DsbC, was critical to obtaining soluble antibody fragments. The optimal conditions were directly applicable to a 9-ml-scale reaction, with linear scalable yields of more than 1 mg/ml. Analyses of purified 059-152-Fv and Fab showed that the cell-free synthesized antibody fragments were disulfide-bridged, with antigen binding activity comparable to that of clinical antibodies. Examination of the crystal structure of cell-free synthesized 059-152-Fv in complex with the extracellular domain of human EGFR revealed that the epitope of 059-152-Fv broadly covers the EGF binding surface on domain III, including residues that formed critical hydrogen bonds with EGF (Asp355EGFR, Gln384EGFR, H409EGFR, and Lys465EGFR, so that the antibody inhibited EGFR activation. We further demonstrated the application of the cell-free system to site-specific integration of non-natural amino acids for antibody engineering, which would expand the availability of therapeutic antibodies based on structural information and rational design. This cell-free system could be an ideal antibody-fragment production platform for functional and structural analysis of potential therapeutic antibodies and for engineered antibody development.

  9. Parametric study on the advantages of weather-predicted control algorithm of free cooling ventilation system

    International Nuclear Information System (INIS)

    Medved, Sašo; Babnik, Miha; Vidrih, Boris; Arkar, Ciril

    2014-01-01

    Predicted climate changes and the increased intensity of urban heat islands, as well as population aging, will increase the energy demand for the cooling of buildings in the future. However, the energy demand for cooling can be efficiently reduced by low-exergy free-cooling systems, which use natural processes, like evaporative cooling or the environmental cold of ambient air during night-time ventilation for the cooling of buildings. Unlike mechanical cooling systems, the energy for the operation of free-cooling system is needed only for the transport of the cold from the environment into the building. Because the natural cold potential is time dependent, the efficiency of free-cooling systems could be improved by introducing a weather forecast into the algorithm for the controlling. In the article, a numerical algorithm for the optimization of the operation of free-cooling systems with night-time ventilation is presented and validated on a test cell with different thermal storage capacities and during different ambient conditions. As a case study, the advantage of weather-predicted controlling is presented for a summer week for typical office room. The results show the necessity of the weather-predicted controlling of free-cooling ventilation systems for achieving the highest overall energy efficiency of such systems in comparison to mechanical cooling, better indoor comfort conditions and a decrease in the primary energy needed for cooling of the buildings. - Highlights: • Energy demand for cooling will increase due to climate changes and urban heat island • Free cooling could significantly reduce energy demand for cooling of the buildings. • Free cooling is more effective if weather prediction is included in operation control. • Weather predicted free cooling operation algorithm was validated on test cell. • Advantages of free-cooling on mechanical cooling is shown with different indicators

  10. High yield cell-free production of integral membrane proteins without refolding or detergents.

    Science.gov (United States)

    Wuu, Jessica J; Swartz, James R

    2008-05-01

    Integral membrane proteins act as critical cellular components and are important drug targets. However, difficulties in producing membrane proteins have hampered investigations of structure and function. In vivo production systems are often limited by cell toxicity, and previous in vitro approaches have required unnatural folding pathways using detergents or lipid solutions. To overcome these limitations, we present an improved cell-free expression system which produces high yields of integral membrane proteins without the use of detergents or refolding steps. Our cell-free reaction activates an Escherichia coli-derived cell extract for transcription and translation. Purified E. coli inner membrane vesicles supply membrane-bound components and the lipid environment required for insertion and folding. Using this system, we demonstrated successful synthesis of two complex integral membrane transporters, the tetracycline pump (TetA) and mannitol permease (MtlA), in yields of 570+/-50 microg/mL and 130+/-30 microg/mL of vesicle-associated protein, respectively. These yields are up to 400 times typical in vivo concentrations. Insertion and folding of these proteins are verified by sucrose flotation, protease digestion, and activity assays. Whereas TetA incorporates efficiently into vesicle membranes with over two-thirds of the synthesized protein being inserted, MtlA yields appear to be limited by insufficient concentrations of a membrane-associated chaperone.

  11. Escherichia coli cell-free protein synthesis and isotope labeling of mammalian proteins.

    Science.gov (United States)

    Terada, Takaho; Yokoyama, Shigeyuki

    2015-01-01

    This chapter describes the cell-free protein synthesis method, using an Escherichia coli cell extract. This is a cost-effective method for milligram-scale protein production and is particularly useful for the production of mammalian proteins, protein complexes, and membrane proteins that are difficult to synthesize by recombinant expression methods, using E. coli and eukaryotic cells. By adjusting the conditions of the cell-free method, zinc-binding proteins, disulfide-bonded proteins, ligand-bound proteins, etc., may also be produced. Stable isotope labeling of proteins can be accomplished by the cell-free method, simply by using stable isotope-labeled amino acid(s) in the cell-free reaction. Moreover, the cell-free protein synthesis method facilitates the avoidance of stable isotope scrambling and dilution over the recombinant expression methods and is therefore advantageous for amino acid-selective stable isotope labeling. Site-specific stable isotope labeling is also possible with a tRNA molecule specific to the UAG codon. By the cell-free protein synthesis method, coupled transcription-translation is performed from a plasmid vector or a PCR-amplified DNA fragment encoding the protein. A milligram quantity of protein can be produced with a milliliter-scale reaction solution in the dialysis mode. More than a thousand solution structures have been determined by NMR spectroscopy for uniformly labeled samples of human and mouse functional domain proteins, produced by the cell-free method. Here, we describe the practical aspects of mammalian protein production by the cell-free method for NMR spectroscopy. © 2015 Elsevier Inc. All rights reserved.

  12. The scale-free dynamics of eukaryotic cells.

    Directory of Open Access Journals (Sweden)

    Miguel A Aon

    Full Text Available Temporal organization of biological processes requires massively parallel processing on a synchronized time-base. We analyzed time-series data obtained from the bioenergetic oscillatory outputs of Saccharomyces cerevisiae and isolated cardiomyocytes utilizing Relative Dispersional (RDA and Power Spectral (PSA analyses. These analyses revealed broad frequency distributions and evidence for long-term memory in the observed dynamics. Moreover RDA and PSA showed that the bioenergetic dynamics in both systems show fractal scaling over at least 3 orders of magnitude, and that this scaling obeys an inverse power law. Therefore we conclude that in S. cerevisiae and cardiomyocytes the dynamics are scale-free in vivo. Applying RDA and PSA to data generated from an in silico model of mitochondrial function indicated that in yeast and cardiomyocytes the underlying mechanisms regulating the scale-free behavior are similar. We validated this finding in vivo using single cells, and attenuating the activity of the mitochondrial inner membrane anion channel with 4-chlorodiazepam to show that the oscillation of NAD(PH and reactive oxygen species (ROS can be abated in these two evolutionarily distant species. Taken together these data strongly support our hypothesis that the generation of ROS, coupled to redox cycling, driven by cytoplasmic and mitochondrial processes, are at the core of the observed rhythmicity and scale-free dynamics. We argue that the operation of scale-free bioenergetic dynamics plays a fundamental role to integrate cellular function, while providing a framework for robust, yet flexible, responses to the environment.

  13. Synthetic surface for expansion of human mesenchymal stem cells in xeno-free, chemically defined culture conditions.

    Directory of Open Access Journals (Sweden)

    Paula J Dolley-Sonneville

    Full Text Available Human mesenchymal stem cells (HMSCS possess three properties of great interest for the development of cell therapies and tissue engineering: multilineage differentiation, immunomodulation, and production of trophic factors. Efficient ex vivo expansion of hMSCs is a challenging requirement for large scale production of clinical grade cells. Low-cost, robust, scalable culture methods using chemically defined materials need to be developed to address this need. This study describes the use of a xeno-free synthetic peptide acrylate surface, the Corning® Synthemax® Surface, for culture of hMSCs in serum-free, defined medium. Cell performance on the Corning Synthemax Surface was compared to cells cultured on biological extracellular matrix (ECM coatings in xeno-free defined medium and in traditional conditions on tissue culture treated (TCT plastic in fetal bovine serum (FBS supplemented medium. Our results show successful maintenance of hMSCs on Corning Synthemax Surface for eight passages, with cell expansion rate comparable to cells cultured on ECM and significantly higher than for cells in TCT/FBS condition. Importantly, on the Corning Synthemax Surface, cells maintained elongated, spindle-like morphology, typical hMSC marker profile and in vitro multilineage differentiation potential. We believe the Corning Synthemax Surface, in combination with defined media, provides a complete synthetic, xeno-free, cell culture system for scalable production of hMSCs.

  14. Cell-free DNA: A Neglected Source for Antibiotic Resistance Genes Spreading from WWTPs.

    Science.gov (United States)

    Zhang, Yan; Li, Aolin; Dai, Tianjiao; Li, Feifei; Xie, Hui; Chen, Lujun; Wen, Donghui

    2018-01-02

    Cell-associated ARGs in wastewater treatment plants (WWTPs) has been concerned, however, cell-free ARGs in WWTPs was rarely studied. In this study, the abundances of four representative ARGs, sulII, tetC, bla PSE-1 , and ermB, in a large municipal WWTP were investigated in both cell-associated and cell-free fractions. Cell-associated ARGs was the dominant ARGs fraction in the raw wastewater. After biological treatment, sludge settling, membrane filtration, and disinfection, cell-associated ARGs were substantially reduced, though the ratios of ARG/16S rRNA gene were increased with disinfection. Cell-free ARGs persisted in the WWTP with a removal of 0.36 log to 2.68 logs, which was much lower than the removal of cell-associated ARGs (3.21 logs to 4.14 logs). Therefore, the abundance ratio of cell-free ARGs to cell-associated ARGs increased from 0.04-1.59% to 2.00-1895.08% along the treatment processes. After 25-day-storage, cell-free ARGs in both biological effluent and disinfection effluent increased by 0.14 log to 1.99 logs and 0.12 log to 1.77 logs respectively, reflecting the persistence and low decay rate of cell-free ARGs in the discharge water. Therefore, cell-free ARGs might be a kind of important but previously neglected pollutant from WWTPs, which added potential risks to the effluent receiving environments.

  15. Vector-free and transgene-free human iPS cells differentiate into functional neurons and enhance functional recovery after ischemic stroke in mice.

    Directory of Open Access Journals (Sweden)

    Osama Mohamad

    Full Text Available Stroke is a leading cause of human death and disability in the adult population in the United States and around the world. While stroke treatment is limited, stem cell transplantation has emerged as a promising regenerative therapy to replace or repair damaged tissues and enhance functional recovery after stroke. Recently, the creation of induced pluripotent stem (iPS cells through reprogramming of somatic cells has revolutionized cell therapy by providing an unlimited source of autologous cells for transplantation. In addition, the creation of vector-free and transgene-free human iPS (hiPS cells provides a new generation of stem cells with a reduced risk of tumor formation that was associated with the random integration of viral vectors seen with previous techniques. However, the potential use of these cells in the treatment of ischemic stroke has not been explored. In the present investigation, we examined the neuronal differentiation of vector-free and transgene-free hiPS cells and the transplantation of hiPS cell-derived neural progenitor cells (hiPS-NPCs in an ischemic stroke model in mice. Vector-free hiPS cells were maintained in feeder-free and serum-free conditions and differentiated into functional neurons in vitro using a newly developed differentiation protocol. Twenty eight days after transplantation in stroke mice, hiPS-NPCs showed mature neuronal markers in vivo. No tumor formation was seen up to 12 months after transplantation. Transplantation of hiPS-NPCs restored neurovascular coupling, increased trophic support and promoted behavioral recovery after stroke. These data suggest that using vector-free and transgene-free hiPS cells in stem cell therapy are safe and efficacious in enhancing recovery after focal ischemic stroke in mice.

  16. Culture in embryonic kidney serum and xeno-free media as renal cell carcinoma and renal cell carcinoma cancer stem cells research model.

    Science.gov (United States)

    Krawczyk, Krzysztof M; Matak, Damian; Szymanski, Lukasz; Szczylik, Cezary; Porta, Camillo; Czarnecka, Anna M

    2018-04-01

    The use of fetal bovine serum hinders obtaining reproducible experimental results and should also be removed in hormone and growth factor studies. In particular hormones found in FBS act globally on cancer cell physiology and influence transcriptome and metabolome. The aim of our study was to develop a renal carcinoma serum free culture model optimized for (embryonal) renal cells in order to select the best study model for downstream auto-, para- or endocrine research. Secondary aim was to verify renal carcinoma stem cell culture for this application. In the study, we have cultured renal cell carcinoma primary tumour cell line (786-0) as well as human kidney cancer stem cells in standard 2D monolayer cultures in Roswell Park Memorial Institute Medium or Dulbecco's Modified Eagle's Medium and Complete Human Kidney Cancer Stem Cell Medium, respectively. Serum-free, animal-component free Human Embryonic Kidney 293 media were tested. Our results revealed that xeno-free embryonal renal cells optimized culture media provide a useful tool in RCC cancer biology research and at the same time enable effective growth of RCC. We propose bio-mimic RCC cell culture model with specific serum-free and xeno-free medium that promote RCC cell viability.

  17. HoloMonitor M4: holographic imaging cytometer for real-time kinetic label-free live-cell analysis of adherent cells

    Science.gov (United States)

    Sebesta, Mikael; Egelberg, Peter J.; Langberg, Anders; Lindskov, Jens-Henrik; Alm, Kersti; Janicke, Birgit

    2016-03-01

    Live-cell imaging enables studying dynamic cellular processes that cannot be visualized in fixed-cell assays. An increasing number of scientists in academia and the pharmaceutical industry are choosing live-cell analysis over or in addition to traditional fixed-cell assays. We have developed a time-lapse label-free imaging cytometer HoloMonitorM4. HoloMonitor M4 assists researchers to overcome inherent disadvantages of fluorescent analysis, specifically effects of chemical labels or genetic modifications which can alter cellular behavior. Additionally, label-free analysis is simple and eliminates the costs associated with staining procedures. The underlying technology principle is based on digital off-axis holography. While multiple alternatives exist for this type of analysis, we prioritized our developments to achieve the following: a) All-inclusive system - hardware and sophisticated cytometric analysis software; b) Ease of use enabling utilization of instrumentation by expert- and entrylevel researchers alike; c) Validated quantitative assay end-points tracked over time such as optical path length shift, optical volume and multiple derived imaging parameters; d) Reliable digital autofocus; e) Robust long-term operation in the incubator environment; f) High throughput and walk-away capability; and finally g) Data management suitable for single- and multi-user networks. We provide examples of HoloMonitor applications of label-free cell viability measurements and monitoring of cell cycle phase distribution.

  18. Free-zone electrophoresis of animal cells. 1: Experiments on cell-cell interactions

    Science.gov (United States)

    Todd, P. W.; Hjerten, S.

    1985-01-01

    The electrophoretically migrating zones wasa monitored. The absence of fluid flows in the direction of migration permits direct measurement of electrophoretic velocities of any material. Sedimentation is orthogonal to electrokinetic motion and the effects of particle-particle interaction on electrophoretic mobility is studied by free zone electrophoresis. Fixed erythrocytes at high concentrations, mixtures of fixed erythrocytes from different animal species, and mixtures of cultured human cells were studied in low ionic strength buffers. The electrophoretic velocity of fixed erythrocytes was not altered by increasing cell concentration or by the mixing of erythrocytes from different species. When zones containing cultured human glial cells and neuroblastoma cells are permitted to interact during electrophoresis, altered migration patterns occur. It is found that cell-cell interactions depends upon cell type.

  19. Cell-Free Expression, Purification, and Characterization of the Functional β2-Adrenergic Receptor for Multianalyte Detection of β-Agonists.

    Science.gov (United States)

    Wang, Jian; Liu, Yuan; Zhang, Junhua; Han, Zhengzheng; Wang, Wei; Liu, Yang; Wei, Dong; Huang, Wei

    2017-11-01

    Large-scale expression of β 2 -adrenergic receptor (β 2 -AR) in functional form is necessary for establishment of receptor assays for detecting illegally abused β-adrenergic agonists (β-agonists). Cell-based heterologous expression systems have manycritical difficulties in synthesizing this membrane protein, such as low protein yields and aberrant folding. To overcome these challenges, the main objective of the present work was to synthesize large amounts of functional β 2 -AR in a cell-free system based on Escherichia coli extracts. A codon-optimized porcine β 2 -AR gene (codon adaptation index: 0.96) suitable for high expression in E. coli was synthesized and transcribed to the cell-free system, which contributed to increase the expression up to 1.1 mg/ml. After purification using Ni-affinity chromatography, the bioactivity of the purified receptor was measured by novel enzyme-linked receptor assays. It was determined that the relative affinities of the purified β 2 -AR for β-agonists in descending order were as follows: clenbuterol > salbutamol > ractopamine. Moreover, their IC 50 values were 45.99, 60.38, and 78.02 µg/liter, respectively. Although activity of the cell-free system was slightly lower than activity of systems based on insect and mammalian cells, this system should allow production of β 2 -AR in bulk amounts sufficient for the development of multianalyte screening methods for detecting β-agonist residues.

  20. Centrifugation-free washing: A novel approach for removing immunoglobulin A from stored red blood cells.

    Science.gov (United States)

    Vörös, Eszter; Piety, Nathaniel Z; Strachan, Briony C; Lu, Madeleine; Shevkoplyas, Sergey S

    2018-08-01

    Washed red blood cells (RBCs) are indicated for immunoglobulin A (IgA) deficient recipients. Centrifugation-based cell processors commonly used by hospital blood banks cannot consistently reduce IgA below the recommended levels, hence double washing is frequently required. Here, we describe a prototype of a simple, portable, disposable system capable of washing stored RBCs without centrifugation, while reducing IgA below 0.05 mg/dL in a single run. Samples from RBC units (n = 8, leukoreduced, 4-6 weeks storage duration) were diluted with normal saline to a hematocrit of 10%, and then washed using either the prototype washing system, or via conventional centrifugation. The efficiency of the two washing methods was quantified and compared by measuring several key in vitro quality metrics. The prototype of the washing system was able to process stored RBCs at a rate of 300 mL/hour, producing a suspension of washed RBCs with 43 ± 3% hematocrit and 86 ± 7% cell recovery. Overall, the two washing methods performed similarly for most measured parameters, lowering the concentration of free hemoglobin by >4-fold and total free protein by >10-fold. Importantly, the new washing system reduced the IgA level to 0.02 ± 0.01 mg/mL, a concentration 5-fold lower than that produced by conventional centrifugation. This proof-of-concept study showed that centrifugation may be unnecessary for washing stored RBCs. A simple, disposable, centrifugation-free washing system could be particularly useful in smaller medical facilities and resource limited settings that may lack access to centrifugation-based cell processors. © 2017 Wiley Periodicals, Inc.

  1. Roll-coating fabrication of flexible organic solar cells: comparison of fullerene and fullerene-free systems

    DEFF Research Database (Denmark)

    Liu, Kuan; Larsen-Olsen, Thue Trofod; Lin, Yuze

    2016-01-01

    Flexible organic solar cells (OSCs) based on a blend of low-bandgap polymer donor PTB7-TH and nonfullerene small molecule acceptor IEIC were fabricated via a roll-coating process under ambient atmosphere. Both an indium tin oxide (ITO)-free substrate and a flexible ITO substrate were employed...... in these inverted OSCs. OSCs with flexible ITO and ITO-free substrates exhibited power conversion efficiencies (PCEs) up to 2.26% and 1.79%, respectively, which were comparable to those of the reference devices based on fullerene acceptors under the same conditions. This is the first example for all roll......-coating fabrication procedures for flexible OSCs based on non-fullerene acceptors with the PCE exceeding 2%. The fullerene-free OSCs exhibited better dark storage stability than the fullerene-based control devices....

  2. Watch Out for the “Living Dead”: Cell-Free Enzymes and Their Fate

    Directory of Open Access Journals (Sweden)

    Federico Baltar

    2018-01-01

    Full Text Available Microbes are the engines driving biogeochemical cycles. Microbial extracellular enzymatic activities (EEAs are the “gatekeepers” of the carbon cycle. The total EEA is the sum of cell-bound (i.e., cell-attached, and dissolved (i.e., cell-free enzyme activities. Cell-free enzymes make up a substantial proportion (up to 100% of the total marine EEA. Although we are learning more about how microbial diversity and function (including total EEA will be affected by environmental changes, little is known about what factors control the importance of the abundant cell-free enzymes. Since cell-attached EEAs are linked to the cell, their fate will likely be linked to the factors controlling the cell’s fate. In contrast, cell-free enzymes belong to a kind of “living dead” realm because they are not attached to a living cell but still are able to perform their function away from the cell; and as such, the factors controlling their activity and fate might differ from those affecting cell-attached enzymes. This article aims to place cell-free EEA into the wider context of hydrolysis of organic matter, deal with recent studies assessing what controls the production, activity and lifetime of cell-free EEA, and what their fate might be in response to environmental stressors. This perspective article advocates the need to go “beyond the living things,” studying the response of cells/organisms to different stressors, but also to study cell-free enzymes, in order to fully constrain the future and evolution of marine biogeochemical cycles.

  3. Watch Out for the “Living Dead”: Cell-Free Enzymes and Their Fate

    Science.gov (United States)

    Baltar, Federico

    2018-01-01

    Microbes are the engines driving biogeochemical cycles. Microbial extracellular enzymatic activities (EEAs) are the “gatekeepers” of the carbon cycle. The total EEA is the sum of cell-bound (i.e., cell-attached), and dissolved (i.e., cell-free) enzyme activities. Cell-free enzymes make up a substantial proportion (up to 100%) of the total marine EEA. Although we are learning more about how microbial diversity and function (including total EEA) will be affected by environmental changes, little is known about what factors control the importance of the abundant cell-free enzymes. Since cell-attached EEAs are linked to the cell, their fate will likely be linked to the factors controlling the cell’s fate. In contrast, cell-free enzymes belong to a kind of “living dead” realm because they are not attached to a living cell but still are able to perform their function away from the cell; and as such, the factors controlling their activity and fate might differ from those affecting cell-attached enzymes. This article aims to place cell-free EEA into the wider context of hydrolysis of organic matter, deal with recent studies assessing what controls the production, activity and lifetime of cell-free EEA, and what their fate might be in response to environmental stressors. This perspective article advocates the need to go “beyond the living things,” studying the response of cells/organisms to different stressors, but also to study cell-free enzymes, in order to fully constrain the future and evolution of marine biogeochemical cycles. PMID:29354095

  4. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis.

    Directory of Open Access Journals (Sweden)

    Nitzan Krinsky

    Full Text Available Cell-free protein synthesis (CFPS systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3 and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa. This system was able to produce 40-150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins.

  5. A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis

    Science.gov (United States)

    Kaduri, Maya; Shainsky-Roitman, Janna; Goldfeder, Mor; Ivanir, Eran; Benhar, Itai; Shoham, Yuval; Schroeder, Avi

    2016-01-01

    Cell-free protein synthesis (CFPS) systems are important laboratory tools that are used for various synthetic biology applications. Here, we present a simple and inexpensive laboratory-scale method for preparing a CFPS system from E. coli. The procedure uses basic lab equipment, a minimal set of reagents, and requires less than one hour to process the bacterial cell mass into a functional S30-T7 extract. BL21(DE3) and MRE600 E. coli strains were used to prepare the S30-T7 extract. The CFPS system was used to produce a set of fluorescent and therapeutic proteins of different molecular weights (up to 66 kDa). This system was able to produce 40–150 μg-protein/ml, with variations depending on the plasmid type, expressed protein and E. coli strain. Interestingly, the BL21-based CFPS exhibited stability and increased activity at 40 and 45°C. To the best of our knowledge, this is the most rapid and affordable lab-scale protocol for preparing a cell-free protein synthesis system, with high thermal stability and efficacy in producing therapeutic proteins. PMID:27768741

  6. Mechanistic insights of the Min oscillator via cell-free reconstitution and imaging

    Science.gov (United States)

    Mizuuchi, Kiyoshi; Vecchiarelli, Anthony G.

    2018-05-01

    The MinD and MinE proteins of Escherichia coli self-organize into a standing-wave oscillator on the membrane to help align division at mid-cell. When unleashed from cellular confines, MinD and MinE form a spectrum of patterns on artificial bilayers—static amoebas, traveling waves, traveling mushrooms, and bursts with standing-wave dynamics. We recently focused our cell-free studies on bursts because their dynamics recapitulate many features of Min oscillation observed in vivo. The data unveiled a patterning mechanism largely governed by MinE regulation of MinD interaction with membrane. We proposed that the MinD to MinE ratio on the membrane acts as a toggle switch between MinE-stimulated recruitment and release of MinD from the membrane. In this review, we summarize cell-free data on the Min system and expand upon a molecular mechanism that provides a biochemical explanation as to how these two ‘simple’ proteins can form the remarkable spectrum of patterns.

  7. Integrated dual-tomography for refractive index analysis of free-floating single living cell with isotropic superresolution.

    Science.gov (United States)

    B, Vinoth; Lai, Xin-Ji; Lin, Yu-Chih; Tu, Han-Yen; Cheng, Chau-Jern

    2018-04-13

    Digital holographic microtomography is a promising technique for three-dimensional (3D) measurement of the refractive index (RI) profiles of biological specimens. Measurement of the RI distribution of a free-floating single living cell with an isotropic superresolution had not previously been accomplished. To the best of our knowledge, this is the first study focusing on the development of an integrated dual-tomographic (IDT) imaging system for RI measurement of an unlabelled free-floating single living cell with an isotropic superresolution by combining the spatial frequencies of full-angle specimen rotation with those of beam rotation. A novel 'UFO' (unidentified flying object) like shaped coherent transfer function is obtained. The IDT imaging system does not require any complex image-processing algorithm for 3D reconstruction. The working principle was successfully demonstrated and a 3D RI profile of a single living cell, Candida rugosa, was obtained with an isotropic superresolution. This technology is expected to set a benchmark for free-floating single live sample measurements without labeling or any special sample preparations for the experiments.

  8. Scaffold Free Bio-orthogonal Assembly of 3-Dimensional Cardiac Tissue via Cell Surface Engineering

    Science.gov (United States)

    Rogozhnikov, Dmitry; O'Brien, Paul J.; Elahipanah, Sina; Yousaf, Muhammad N.

    2016-12-01

    There has been tremendous interest in constructing in vitro cardiac tissue for a range of fundamental studies of cardiac development and disease and as a commercial system to evaluate therapeutic drug discovery prioritization and toxicity. Although there has been progress towards studying 2-dimensional cardiac function in vitro, there remain challenging obstacles to generate rapid and efficient scaffold-free 3-dimensional multiple cell type co-culture cardiac tissue models. Herein, we develop a programmed rapid self-assembly strategy to induce specific and stable cell-cell contacts among multiple cell types found in heart tissue to generate 3D tissues through cell-surface engineering based on liposome delivery and fusion to display bio-orthogonal functional groups from cell membranes. We generate, for the first time, a scaffold free and stable self assembled 3 cell line co-culture 3D cardiac tissue model by assembling cardiomyocytes, endothelial cells and cardiac fibroblast cells via a rapid inter-cell click ligation process. We compare and analyze the function of the 3D cardiac tissue chips with 2D co-culture monolayers by assessing cardiac specific markers, electromechanical cell coupling, beating rates and evaluating drug toxicity.

  9. An optimized rapid bisulfite conversion method with high recovery of cell-free DNA.

    Science.gov (United States)

    Yi, Shaohua; Long, Fei; Cheng, Juanbo; Huang, Daixin

    2017-12-19

    Methylation analysis of cell-free DNA is a encouraging tool for tumor diagnosis, monitoring and prognosis. Sensitivity of methylation analysis is a very important matter due to the tiny amounts of cell-free DNA available in plasma. Most current methods of DNA methylation analysis are based on the difference of bisulfite-mediated deamination of cytosine between cytosine and 5-methylcytosine. However, the recovery of bisulfite-converted DNA based on current methods is very poor for the methylation analysis of cell-free DNA. We optimized a rapid method for the crucial steps of bisulfite conversion with high recovery of cell-free DNA. A rapid deamination step and alkaline desulfonation was combined with the purification of DNA on a silica column. The conversion efficiency and recovery of bisulfite-treated DNA was investigated by the droplet digital PCR. The optimization of the reaction results in complete cytosine conversion in 30 min at 70 °C and about 65% of recovery of bisulfite-treated cell-free DNA, which is higher than current methods. The method allows high recovery from low levels of bisulfite-treated cell-free DNA, enhancing the analysis sensitivity of methylation detection from cell-free DNA.

  10. Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy

    Science.gov (United States)

    Kemper, Björn; Bauwens, Andreas; Vollmer, Angelika; Ketelhut, Steffi; Langehanenberg, Patrik; Müthing, Johannes; Karch, Helge; von Bally, Gert

    2010-05-01

    Digital holographic microscopy (DHM) enables quantitative multifocus phase contrast imaging for nondestructive technical inspection and live cell analysis. Time-lapse investigations on human brain microvascular endothelial cells demonstrate the use of DHM for label-free dynamic quantitative monitoring of cell division of mother cells into daughter cells. Cytokinetic DHM analysis provides future applications in toxicology and cancer research.

  11. Defined xenogeneic-free and hypoxic environment provides superior conditions for long-term expansion of human adipose-derived stem cells.

    Science.gov (United States)

    Yang, Sufang; Pilgaard, Linda; Chase, Lucas G; Boucher, Shayne; Vemuri, Mohan C; Fink, Trine; Zachar, Vladimir

    2012-08-01

    Development and implementation of therapeutic protocols based on stem cells or tissue-engineered products relies on methods that enable the production of substantial numbers of cells while complying with stringent quality and safety demands. In the current study, we aimed to assess the benefits of maintaining cultures of adipose-derived stem cells (ASCs) in a defined culture system devoid of xenogeneic components (xeno-free) and hypoxia over a 49-day growth period. Our data provide evidence that conditions involving StemPro mesenchymal stem cells serum-free medium (SFM) Xeno-Free and hypoxia (5% oxygen concentration) in the culture atmosphere provide a superior proliferation rate compared to a standard growth environment comprised of alpha-modified Eagle medium (A-MEM) supplemented with fetal calf serum (FCS) and ambient air (20% oxygen concentration) or that of A-MEM supplemented with FCS and hypoxia. Furthermore, a flow cytometric analysis and in vitro differentiation assays confirmed the immunophenotype stability and maintained multipotency of ASCs when expanded under xeno-free conditions and hypoxia. In conclusion, our data demonstrate that growth conditions utilizing a xeno-free and hypoxic environment not only provide an improved environment for the expansion of ASCs, but also set the stage as a culture system with the potential broad spectrum utility for regenerative medicine and tissue engineering applications.

  12. Nucleotide-excision repair of DNA in cell-free extracts of the yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Wang, Z.; Wu, X.; Friedberg, E.C.

    1993-01-01

    A wide spectrum of DNA lesions are repaired by the nucleotide-excision repair (NER) pathway in both eukaryotic and prokaryotic cells. We have developed a cell-free system in Saccharomyces cerevisiae that supports NER. NER was monitored by measuring repair synthesis in DNA treated with cisplatin or with UV radiation. Repair synthesis in vitro was defective in extracts of rad1, rad2, and rad10 mutant cells, all of which have mutations in genes whose products are known to be required for NER in vivo. Additionally, repair synthesis was complemented by mixing different mutant extracts, or by adding purified Rad1 or Rad10 protein to rad1 or rad10 mutant extracts, respectively. The latter observation demonstrates that the Rad1 and Rad10 proteins directly participate in the biochemical pathway of NER. NER supported by nuclear extracts requires ATP and Mg 2+ and is stimulated by polyethylene glycol and by small amounts of whole cell extract containing overexpressed Rad2 protein. The nuclear extracts also contain base-excision repair activity that is present at wild-type levels in rad mutant extracts. This cell-free system is expected to facilitate studies on the biochemical pathway of NER in S. cerevisiae

  13. Cell-free mitochondrial DNA copy number variation in head and neck squamous cell carcinoma: A study of non-invasive biomarker from Northeast India.

    Science.gov (United States)

    Kumar, Manish; Srivastava, Shilpee; Singh, Seram Anil; Das, Anup Kumar; Das, Ganesh Chandra; Dhar, Bishal; Ghosh, Sankar Kumar; Mondal, Rosy

    2017-10-01

    Head and neck squamous cell carcinoma is the most commonly diagnosed cancer worldwide. The lifestyle, food habits, and customary practices manifest the Northeast Indian population toward higher susceptibility to develop head and neck squamous cell carcinoma. Here, we have investigated the association of smoke and smokeless tobacco, and alcohol with copy number variation of cell-free mitochondrial DNA and cell-free nuclear DNA in cases and controls. Cell-free DNA from plasma was isolated from 50 head and neck squamous cell carcinoma cases and 50 controls with informed written consent using QIAamp Circulating Nucleic Acid Kit. Real-time polymerase chain reaction was done for copy number variation in cell-free mitochondrial DNA and cell-free nuclear DNA. Receiver operating characteristic curve analysis was performed to evaluate the diagnostic application between the two study groups using clinicopathological parameters. The levels of cell-free nuclear DNA and cell-free mitochondrial DNA of cases in association with smoke and smokeless tobacco, alcohol with smoking (p squamous cell carcinoma cases and controls, we distinguished cell-free mitochondrial DNA (cutoff: 19.84 raw Ct; sensitivity: 84%; specificity: 100%; p < 0.001) and cell-free nuclear DNA (cutoff: 463,282 genomic equivalent/mL; sensitivity: 53%; specificity: 87%; p < 0.001). The copy number variation in cases (cell-free nuclear DNA: 5451.66 genomic equivalent/mL and cell-free mitochondrial DNA: 29,103,476.15 genomic equivalent/mL) and controls (cell-free nuclear DNA: 1650.9 genomic equivalent/mL and cell-free mitochondrial DNA: 9,189,312.54 genomic equivalent/mL), respectively. Our result indicates that the cell-free mitochondrial DNA content is highly associated with smoke and smokeless tobacco, betel quid chewing, and alcohol which shows greater promises, holding the key characteristics of diagnostic biomarkers, that is, minimal invasiveness, high specificity, and sensitivity.

  14. Mesostructured platinum-free anode and carbon-free cathode catalysts for durable proton exchange membrane fuel cells.

    Science.gov (United States)

    Cui, Xiangzhi; Shi, Jianlin; Wang, Yongxia; Chen, Yu; Zhang, Lingxia; Hua, Zile

    2014-01-01

    As one of the most important clean energy sources, proton exchange membrane fuel cells (PEMFCs) have been a topic of extensive research focus for decades. Unfortunately, several critical technique obstacles, such as the high cost of platinum electrode catalysts, performance degradation due to the CO poisoning of the platinum anode, and carbon corrosion by oxygen in the cathode, have greatly impeded its commercial development. A prototype of a single PEMFC catalyzed by a mesostructured platinum-free WO3/C anode and a mesostructured carbon-free Pt/WC cathode catalysts is reported herein. The prototype cell exhibited 93% power output of a standard PEMFC using commercial Pt/C catalysts at 50 and 70 °C, and more importantly, CO poisoning-free and carbon corrosion-resistant characters of the anode and cathode, respectively. Consequently, the prototype cell demonstrated considerably enhanced cell operation durability. The mesostructured electrode catalysts are therefore highly promising in the future development and application of PEMFCs. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Advancement on Lead-Free Organic-Inorganic Halide Perovskite Solar Cells: A Review.

    Science.gov (United States)

    Sani, Faruk; Shafie, Suhaidi; Lim, Hong Ngee; Musa, Abubakar Ohinoyi

    2018-06-14

    Remarkable attention has been committed to the recently discovered cost effective and solution processable lead-free organic-inorganic halide perovskite solar cells. Recent studies have reported that, within five years, the reported efficiency has reached 9.0%, which makes them an extremely promising and fast developing candidate to compete with conventional lead-based perovskite solar cells. The major challenge associated with the conventional perovskite solar cells is the toxic nature of lead (Pb) used in the active layer of perovskite material. If lead continues to be used in fabricating solar cells, negative health impacts will result in the environment due to the toxicity of lead. Alternatively, lead free perovskite solar cells could give a safe way by substituting low-cost, abundant and non toxic material. This review focuses on formability of lead-free organic-inorganic halide perovskite, alternative metal cations candidates to replace lead (Pb), and possible substitutions of organic cations, as well as halide anions in the lead-free organic-inorganic halide perovskite architecture. Furthermore, the review gives highlights on the impact of organic cations, metal cations and inorganic anions on stability and the overall performance of lead free perovskite solar cells.

  16. The value of cell-free DNA for molecular pathology.

    Science.gov (United States)

    Stewart, Caitlin M; Kothari, Prachi D; Mouliere, Florent; Mair, Richard; Somnay, Saira; Benayed, Ryma; Zehir, Ahmet; Weigelt, Britta; Dawson, Sarah-Jane; Arcila, Maria E; Berger, Michael F; Tsui, Dana Wy

    2018-04-01

    Over the past decade, advances in molecular biology and genomics techniques have revolutionized the diagnosis and treatment of cancer. The technological advances in tissue profiling have also been applied to the study of cell-free nucleic acids, an area of increasing interest for molecular pathology. Cell-free nucleic acids are released from tumour cells into the surrounding body fluids and can be assayed non-invasively. The repertoire of genomic alterations in circulating tumour DNA (ctDNA) is reflective of both primary tumours and distant metastatic sites, and ctDNA can be sampled multiple times, thereby overcoming the limitations of the analysis of single biopsies. Furthermore, ctDNA can be sampled regularly to monitor response to treatment, to define the evolution of the tumour genome, and to assess the acquisition of resistance and minimal residual disease. Recently, clinical ctDNA assays have been approved for guidance of therapy, which is an exciting first step in translating cell-free nucleic acid research tests into clinical use for oncology. In this review, we discuss the advantages of cell-free nucleic acids as analytes in different body fluids, including blood plasma, urine, and cerebrospinal fluid, and their clinical applications in solid tumours and haematological malignancies. We will also discuss practical considerations for clinical deployment, such as preanalytical factors and regulatory requirements. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  17. Label-free cell-cycle analysis by high-throughput quantitative phase time-stretch imaging flow cytometry

    Science.gov (United States)

    Mok, Aaron T. Y.; Lee, Kelvin C. M.; Wong, Kenneth K. Y.; Tsia, Kevin K.

    2018-02-01

    Biophysical properties of cells could complement and correlate biochemical markers to characterize a multitude of cellular states. Changes in cell size, dry mass and subcellular morphology, for instance, are relevant to cell-cycle progression which is prevalently evaluated by DNA-targeted fluorescence measurements. Quantitative-phase microscopy (QPM) is among the effective biophysical phenotyping tools that can quantify cell sizes and sub-cellular dry mass density distribution of single cells at high spatial resolution. However, limited camera frame rate and thus imaging throughput makes QPM incompatible with high-throughput flow cytometry - a gold standard in multiparametric cell-based assay. Here we present a high-throughput approach for label-free analysis of cell cycle based on quantitative-phase time-stretch imaging flow cytometry at a throughput of > 10,000 cells/s. Our time-stretch QPM system enables sub-cellular resolution even at high speed, allowing us to extract a multitude (at least 24) of single-cell biophysical phenotypes (from both amplitude and phase images). Those phenotypes can be combined to track cell-cycle progression based on a t-distributed stochastic neighbor embedding (t-SNE) algorithm. Using multivariate analysis of variance (MANOVA) discriminant analysis, cell-cycle phases can also be predicted label-free with high accuracy at >90% in G1 and G2 phase, and >80% in S phase. We anticipate that high throughput label-free cell cycle characterization could open new approaches for large-scale single-cell analysis, bringing new mechanistic insights into complex biological processes including diseases pathogenesis.

  18. Rho kinase inhibitor Y-27632 promotes the differentiation of human bone marrow mesenchymal stem cells into keratinocyte-like cells in xeno-free conditioned medium.

    Science.gov (United States)

    Li, Zhenzhen; Han, Shichao; Wang, Xingqin; Han, Fu; Zhu, Xiongxiang; Zheng, Zhao; Wang, Hongtao; Zhou, Qin; Wang, Yunchuan; Su, Linlin; Shi, Jihong; Tang, Chaowu; Hu, Dahai

    2015-03-11

    Bone marrow mesenchymal stem cells (BMSCs), which have the ability to self-renew and to differentiate into multiple cell types, have recently become a novel strategy for cell-based therapies. The differentiation of BMSCs into keratinocytes may be beneficial for patients with burns, disease, or trauma. However, the currently available cells are exposed to animal materials during their cultivation and induction. These xeno-contaminations severely limit their clinical outcomes. Previous studies have shown that the Rho kinase (ROCK) inhibitor Y-27632 can promote induction efficiency and regulate the self-renewal and differentiation of stem cells. In the present study, we attempted to establish a xeno-free system for the differentiation of BMSCs into keratinocytes and to investigate whether Y-27632 can facilitate this differentiation. BMSCs isolated from patients were cultured by using a xeno-free system and characterised by using flow cytometric analysis and adipogenic and osteogenic differentiation assays. Human primary keratinocytes were also isolated from patients. Then, the morphology, population doubling time, and β-galactosidase staining level of these cells were evaluated in the presence or absence of Y-27632 to determine the effects of Y-27632 on the state of the keratinocytes. Keratinocyte-like cells (KLCs) were detected at different time points by immunocytofluorescence analysis. Moreover, the efficiency of BMSC differentiation under different conditions was measured by quantitative real-time-polymerase chain reaction (RT-PCR) and Western blot analyses. The ROCK inhibitor Y-27632 promoted the proliferation and lifespan of human primary keratinocytes. In addition, we showed that keratinocyte-specific markers could be detected in BMSCs cultured in a xeno-free system using keratinocyte-conditioned medium (KCM) independent of the presence of Y-27632. However, the efficiency of the differentiation of BMSCs into KLCs was significantly higher in the presence of Y

  19. Evolution of taste and solitary chemoreceptor cell systems.

    Science.gov (United States)

    Finger, T E

    1997-01-01

    Vertebrates possess four distinct chemosensory systems distinguishable on the basis of structure, innervation and utilization: olfaction, taste, solitary chemoreceptor cells (SCC) and the common chemical sense (free nerve endings). Of these, taste and the SCC sense rely on secondary receptor cells situated in the epidermis and synapsing on sensory nerve fibers innervating them near their base. The SCC sense occurs in anamniote aquatic craniates, including hagfish, and may be used for feeding or predator avoidance. The sense of taste occurs only in vertebrates and is always utilized for feeding. The SCC system achieves a high degree of specialization in two teleosts: sea robins (Prionotus) and rocklings (Ciliata). In sea robins, SCCs are abundant on the three anterior fin rays of the pectoral fin which are free of fin webbing and are used in active exploration of the substrate. Behavioral and physiological studies show that this SCC system responds to feeding cues and drives feeding behavior. It is connected centrally like a somatosensory system. In contrast, the specialized SCC system of rocklings occurs on the anterior dorsal fin which actively samples the surrounding water. This system responds to mucus substances and may serve as a predator detector. The SCC system in rocklings is connected centrally like a gustatory system. Taste buds contain multiple receptor cell types, including a serotonergic Merkel-like cell. Taste receptor cells respond to nutritionally relevant substances. Due to similarities between SCCs and one type of taste receptor cell, the suggestion is made that taste buds may be compound sensory organs that include some cells related to SCCs and others related to cutaneous Merkel cells. The lack of taste buds in hagfish and their presence in all vertebrates may indicate that the phylogenetic development of taste buds coincided with the elaboration of head structures at the craniate-vertebrate transition.

  20. Preparative scale production of functional mouse aquaporin 4 using different cell-free expression modes.

    Directory of Open Access Journals (Sweden)

    Lei Kai

    Full Text Available The continuous progress in the structural and functional characterization of aquaporins increasingly attracts attention to study their roles in certain mammalian diseases. Although several structures of aquaporins have already been solved by crystallization, the challenge of producing sufficient amounts of functional proteins still remains. CF (cell free expression has emerged in recent times as a promising alternative option in order to synthesize large quantities of membrane proteins, and the focus of this report was to evaluate the potential of this technique for the production of eukaryotic aquaporins. We have selected the mouse aquaporin 4 as a representative of mammalian aquaporins. The protein was synthesized in an E. coli extract based cell-free system with two different expression modes, and the efficiencies of two modes were compared. In both, the P-CF (cell-free membrane protein expression as precipitate mode generating initial aquaporin precipitates as well as in the D-CF (cell-free membrane protein expression in presence of detergent mode, generating directly detergent solubilized samples, we were able to obtain mg amounts of protein per ml of cell-free reaction. Purified aquaporin samples solubilized in different detergents were reconstituted into liposomes, and analyzed for the water channel activity. The calculated P(f value of proteoliposome samples isolated from the D-CF mode was 133 µm/s at 10°C, which was 5 times higher as that of the control. A reversible inhibitory effect of mercury chloride was observed, which is consistent with previous observations of in vitro reconstituted aquaporin 4. In this study, a fast and convenient protocol was established for functional expression of aquaporins, which could serve as basis for further applications such as water filtration.

  1. xCELLigence system for real-time label-free monitoring of growth and viability of cell lines from hematological malignancies.

    Science.gov (United States)

    Martinez-Serra, Jordi; Gutierrez, Antonio; Muñoz-Capó, Saúl; Navarro-Palou, María; Ros, Teresa; Amat, Juan Carlos; Lopez, Bernardo; Marcus, Toni F; Fueyo, Laura; Suquia, Angela G; Gines, Jordi; Rubio, Francisco; Ramos, Rafael; Besalduch, Joan

    2014-01-01

    The xCELLigence system is a new technological approach that allows the real-time cell analysis of adherent tumor cells. To date, xCELLigence has not been able to monitor the growth or cytotoxicity of nonadherent cells derived from hematological malignancies. The basis of its technology relies on the use of culture plates with gold microelectrodes located in their base. We have adapted the methodology described by others to xCELLigence, based on the pre-coating of the cell culture surface with specific substrates, some of which are known to facilitate cell adhesion in the extracellular matrix. Pre-coating of the culture plates with fibronectin, compared to laminin, collagen, or gelatin, significantly induced the adhesion of most of the leukemia/lymphoma cells assayed (Jurkat, L1236, KMH2, and K562). With a fibronectin substrate, nonadherent cells deposited in a monolayer configuration, and consequently, the cell growth and viability were robustly monitored. We further demonstrate the feasibility of xCELLigence for the real-time monitoring of the cytotoxic properties of several antineoplastic agents. In order to validate this technology, the data obtained through real-time cell analysis was compared with that obtained from using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. This provides an excellent label-free tool for the screening of drug efficacy in nonadherent cells and discriminates optimal time points for further molecular analysis of cellular events associated with treatments, reducing both time and costs.

  2. Insights into cell-free therapeutic approach: Role of stem cell "soup-ernatant".

    Science.gov (United States)

    Raik, Shalini; Kumar, Ajay; Bhattacharyya, Shalmoli

    2018-03-01

    Current advances in medicine have revolutionized the field of regenerative medicine dramatically with newly evolved therapies for repair or replacement of degenerating or injured tissues. Stem cells (SCs) can be harvested from different sources for clinical therapeutics, which include fetal tissues, umbilical cord blood, embryos, and adult tissues. SCs can be isolated and differentiated into desired lineages for tissue regeneration and cell replacement therapy. However, several loopholes need to be addressed properly before this can be extended for large-scale therapeutic application. These include a careful approach for patient safety during SC treatments and tolerance of recipients. SC treatments are associated with a number of risk factors and require successful integration and survival of transplanted cells in the desired microenvironment with concurrent tissue regeneration. Recent studies have focused on developing alternatives that can replace the cell-based therapy using paracrine factors. The development of stem "cell free" therapies can be devoted mainly to the use of soluble factors (secretome), extracellular vesicles, and mitochondrial transfer. The present review emphasizes on the paradigms related to the use of SC-based therapeutics and the potential applications of a cell-free approach as an alternative to cell-based therapy in the area of regenerative medicine. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

  3. Design and exergetic analysis of a novel carbon free tri-generation system for hydrogen, power and heat production from natural gas, based on combined solid oxide fuel and electrolyser cells

    Energy Technology Data Exchange (ETDEWEB)

    Perdikaris, N.; Hofmann, Ph.; Spyrakis, S. [Laboratory of Steam Boilers and Thermal Plants, School of Mechanical Engineering, Thermal Engineering Section, National Technical University of Athens, 9 Heroon Polytechniou Ave., Zografou, 15780 Athens (Greece); Panopoulos, K.D. [Institute for Solid Fuels Technology and Applications, Centre for Research and Technology Hellas, 4th km N.R. Ptolemais-Kozani, P.O. Box 95, 50200 Ptolemais (Greece); Kakaras, E. [Laboratory of Steam Boilers and Thermal Plants, School of Mechanical Engineering, Thermal Engineering Section, National Technical University of Athens, 9 Heroon Polytechniou Ave., Zografou, 15780 Athens (Greece); Institute for Solid Fuels Technology and Applications, Centre for Research and Technology Hellas, 4th km N.R. Ptolemais-Kozani, P.O. Box 95, 50200 Ptolemais (Greece)

    2010-03-15

    The Solid Oxide Cells (SOCs) are able to operate in two modes: (a) the Solid Oxide Fuel Cells (SOFCs) that produce electricity and heat and (b) the Solid Oxide Electrolyser Cells (SOEC) that consume electricity and heat to electrolyse water and produce hydrogen and oxygen. The present paper presents a carbon free SOEC/SOFC combined system for the production of hydrogen, electricity and heat (tri-generation) from natural gas fuel. Hydrogen can be locally used as automobile fuel whereas the oxygen produced in the SOEC is used to combust the depleted fuel from the SOFC, which is producing electricity and heat from natural gas. In order to achieve efficient carbon capture in such a system, water steam should be used as the SOEC anode sweep gas, to allow the production of nitrogen free flue gases. The SOEC and SOFC operations were matched through modeling of all components in Aspenplus trademark. The exergetic efficiency of the proposed decentralised system is 28.25% for power generation and 18.55% for production of hydrogen. The system is (a) carbon free because it offers an almost pure pressurised CO{sub 2} stream to be driven for fixation via parallel pipelines to the natural gas feed, (b) does not require any additional water for its operation and (c) offers 26.53% of its energetic input as hot water for applications. (author)

  4. 21 CFR 862.1730 - Free tyrosine test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Free tyrosine test system. 862.1730 Section 862....1730 Free tyrosine test system. (a) Identification. A free tyrosine test system is a device intended to measure free tyrosine (an amono acid) in serum and urine. Measurements obtained by this device are used in...

  5. CRISPR/Cas9-Mediated Mutagenesis of Human Pluripotent Stem Cells in Defined Xeno-Free E8 Medium.

    Science.gov (United States)

    Soh, Chew-Li; Huangfu, Danwei

    2017-01-01

    The recent advent of engineered nucleases including the CRISPR/Cas9 system has greatly facilitated genome manipulation in human pluripotent stem cells (hPSCs). In addition to facilitating hPSC-based disease studies, the application of genome engineering in hPSCs has also opened up new avenues for cell replacement therapy. To improve consistency and reproducibility of hPSC-based studies, and to meet the safety and regulatory requirements for clinical translation, it is necessary to use a defined, xeno-free cell culture system. This chapter describes protocols for CRISPR/Cas9 genome editing in an inducible Cas9 hPSC-based system, using cells cultured in chemically defined, xeno-free E8 Medium on a recombinant human vitronectin substrate. We detail procedures for the design and transfection of CRISPR guide RNAs, colony selection, and the expansion and validation of clonal mutant lines, all within this fully defined culture condition. These methods may be applied to a wide range of genome-engineering applications in hPSCs, including those that utilize different types of site-specific nucleases such as zinc finger nucleases (ZFNs) and TALENs, and form a closer step towards clinical utility of these cells.

  6. Generation of clinical-grade human induced pluripotent stem cells in Xeno-free conditions.

    Science.gov (United States)

    Wang, Juan; Hao, Jie; Bai, Donghui; Gu, Qi; Han, Weifang; Wang, Lei; Tan, Yuanqing; Li, Xia; Xue, Ke; Han, Pencheng; Liu, Zhengxin; Jia, Yundan; Wu, Jun; Liu, Lei; Wang, Liu; Li, Wei; Liu, Zhonghua; Zhou, Qi

    2015-11-12

    Human induced pluripotent stem cells (hiPSCs) are considered as one of the most promising seed cell sources in regenerative medicine. Now hiPSC-based clinical trials are underway. To ensure clinical safety, cells used in clinical trials or therapies should be generated under GMP conditions, and with Xeno-free culture media to avoid possible side effects like immune rejection that induced by the Xeno reagents. However, up to now there are no reports for hiPSC lines developed completely under GMP conditions using Xeno-free reagents. Clinical-grade human foreskin fibroblast (HFF) cells used as feeder cells and parental cells of the clinical-grade hiPSCs were isolated from human foreskin tissues and cultured in Xeno-free media. Clinical-grade hiPSCs were derived by integration-free Sendai virus-based reprogramming kit in Xeno-free pluriton™ reprogramming medium or X medium. Neural cells and cardiomyocytes differentiation were conducted following a series of spatial and temporal specific signals induction according to the corresponding lineage development signals. Biological safety evaluation of the clinical-grade HFF cells and hiPSCs were conducted following the guidance of the "Pharmacopoeia of the People's Republic of China, Edition 2010, Volume III". We have successfully derived several integration-free clinical-grade hiPSC lines under GMP-controlled conditions and with Xeno-free reagents culture media in line with the current guidance of international and national evaluation criteria. As for the source of hiPSCs and feeder cells, biological safety evaluation of the HFF cells have been strictly reviewed by the National Institutes for Food and Drug Control (NIFDC). The hiPSC lines are pluripotent and have passed the safety evaluation. Moreover, one of the randomly selected hiPSC lines was capable of differentiating into functional neural cells and cardiomyocytes in Xeno-free culture media. The clinical-grade hiPSC lines therefore could be valuable sources for

  7. F NMR measurement of intracellular free calcium in human red blood cells

    International Nuclear Information System (INIS)

    Gupta, R.K.; Schanne, F.A.X.

    1986-01-01

    Optical techniques for the measurement of intracellular Ca are not readily applicable to the human red cell because of the intense absorption of hemoglobin. The authors have therefore examined the use of 19 F NMR of 5,5'-difluoro-1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetra acetic acid (5FBAPTA) introduced non-disruptively by intracellular hydrolysis of the membrane-permeant acetoxymethyl ester derivative. 19 F NMR spectra of 5FBAPTA-containing erythrocytes at 188 MHz displayed two well resolved resonances corresponding to the free and Ca-bound forms of the chelator, the resonance of the free form being ten-fold larger than that of the Ca-bound form. Addition of the ionophore A23187 resulted in the disappearance of the resonance of the free anion and a quantitative increase in the intensity of the resonance of the Ca-complex. From these data, and a K/sub D/ of 708 nM for the Ca-5FBAPTA complex, the authors estimate red cell free Ca to be 70 nM, which is in the range of values obtained for other cells, despite the fact that the human red cell, which lacks intracellular organelles for storing Ca, possesses only 1 μmol total Ca/1. cells in comparison to mmols of total Ca found in other cells. The authors ability to use 19 F NMR to measure free Ca in the red blood cell paves the way for future NMR studies of red cell free Ca concentrations in human essential hypertension as well as in other diseases states in which alterations in cellular Ca homeostasis may be involved

  8. Rapid Detection of Cell-Free Mycobacterium tuberculosis DNA in Tuberculous Pleural Effusion.

    Science.gov (United States)

    Che, Nanying; Yang, Xinting; Liu, Zichen; Li, Kun; Chen, Xiaoyou

    2017-05-01

    Tuberculous pleurisy is one of the most common types of extrapulmonary tuberculosis, but its diagnosis remains difficult. In this study, we report for the first time on the detection of cell-free Mycobacterium tuberculosis DNA in pleural effusion and an evaluation of a newly developed molecular assay for the detection of cell-free Mycobacterium tuberculosis DNA. A total of 78 patients with pleural effusion, 60 patients with tuberculous pleurisy, and 18 patients with alternative diseases were included in this study. Mycobacterial culture, the Xpert MTB/RIF assay, the adenosine deaminase assay, the T-SPOT.TB assay, and the cell-free Mycobacterium tuberculosis DNA assay were performed on all the pleural effusion samples. The cell-free Mycobacterium tuberculosis DNA assay and adenosine deaminase assay showed significantly higher sensitivities of 75.0% and 68.3%, respectively, than mycobacterial culture and the Xpert MTB/RIF assay, which had sensitivities of 26.7% and 20.0%, respectively ( P pleural effusion showed the highest sensitivity of 95.0% but the lowest specificity of 38.9%. The cell-free Mycobacterium tuberculosis DNA assay detected as few as 1.25 copies of IS 6110 per ml of pleural effusion and showed good accordance of the results between repeated tests ( r = 0.978, P = 2.84 × 10 -10 ). These data suggest that the cell-free Mycobacterium tuberculosis DNA assay is a rapid and accurate molecular test which provides direct evidence of Mycobacterium tuberculosis etiology. Copyright © 2017 American Society for Microbiology.

  9. Content of intrinsic disorder influences the outcome of cell-free protein synthesis.

    Science.gov (United States)

    Tokmakov, Alexander A; Kurotani, Atsushi; Ikeda, Mariko; Terazawa, Yumiko; Shirouzu, Mikako; Stefanov, Vasily; Sakurai, Tetsuya; Yokoyama, Shigeyuki

    2015-09-11

    Cell-free protein synthesis is used to produce proteins with various structural traits. Recent bioinformatics analyses indicate that more than half of eukaryotic proteins possess long intrinsically disordered regions. However, no systematic study concerning the connection between intrinsic disorder and expression success of cell-free protein synthesis has been presented until now. To address this issue, we examined correlations of the experimentally observed cell-free protein expression yields with the contents of intrinsic disorder bioinformatically predicted in the expressed sequences. This analysis revealed strong relationships between intrinsic disorder and protein amenability to heterologous cell-free expression. On the one hand, elevated disorder content was associated with the increased ratio of soluble expression. On the other hand, overall propensity for detectable protein expression decreased with disorder content. We further demonstrated that these tendencies are rooted in some distinct features of intrinsically disordered regions, such as low hydrophobicity, elevated surface accessibility and high abundance of sequence motifs for proteolytic degradation, including sites of ubiquitination and PEST sequences. Our findings suggest that identification of intrinsically disordered regions in the expressed amino acid sequences can be of practical use for predicting expression success and optimizing cell-free protein synthesis.

  10. Intercellular signaling through secreted proteins induces free-energy gradient-directed cell movement.

    Science.gov (United States)

    Kravchenko-Balasha, Nataly; Shin, Young Shik; Sutherland, Alex; Levine, R D; Heath, James R

    2016-05-17

    Controlling cell migration is important in tissue engineering and medicine. Cell motility depends on factors such as nutrient concentration gradients and soluble factor signaling. In particular, cell-cell signaling can depend on cell-cell separation distance and can influence cellular arrangements in bulk cultures. Here, we seek a physical-based approach, which identifies a potential governed by cell-cell signaling that induces a directed cell-cell motion. A single-cell barcode chip (SCBC) was used to experimentally interrogate secreted proteins in hundreds of isolated glioblastoma brain cancer cell pairs and to monitor their relative motions over time. We used these trajectories to identify a range of cell-cell separation distances where the signaling was most stable. We then used a thermodynamics-motivated analysis of secreted protein levels to characterize free-energy changes for different cell-cell distances. We show that glioblastoma cell-cell movement can be described as Brownian motion biased by cell-cell potential. To demonstrate that the free-energy potential as determined by the signaling is the driver of motion, we inhibited two proteins most involved in maintaining the free-energy gradient. Following inhibition, cell pairs showed an essentially random Brownian motion, similar to the case for untreated, isolated single cells.

  11. Raman-Activated Droplet Sorting (RADS) for Label-Free High-Throughput Screening of Microalgal Single-Cells.

    Science.gov (United States)

    Wang, Xixian; Ren, Lihui; Su, Yetian; Ji, Yuetong; Liu, Yaoping; Li, Chunyu; Li, Xunrong; Zhang, Yi; Wang, Wei; Hu, Qiang; Han, Danxiang; Xu, Jian; Ma, Bo

    2017-11-21

    Raman-activated cell sorting (RACS) has attracted increasing interest, yet throughput remains one major factor limiting its broader application. Here we present an integrated Raman-activated droplet sorting (RADS) microfluidic system for functional screening of live cells in a label-free and high-throughput manner, by employing AXT-synthetic industrial microalga Haematococcus pluvialis (H. pluvialis) as a model. Raman microspectroscopy analysis of individual cells is carried out prior to their microdroplet encapsulation, which is then directly coupled to DEP-based droplet sorting. To validate the system, H. pluvialis cells containing different levels of AXT were mixed and underwent RADS. Those AXT-hyperproducing cells were sorted with an accuracy of 98.3%, an enrichment ratio of eight folds, and a throughput of ∼260 cells/min. Of the RADS-sorted cells, 92.7% remained alive and able to proliferate, which is equivalent to the unsorted cells. Thus, the RADS achieves a much higher throughput than existing RACS systems, preserves the vitality of cells, and facilitates seamless coupling with downstream manipulations such as single-cell sequencing and cultivation.

  12. Shift of microRNA profile upon glioma cell migration using patient-derived spheroids and serum-free conditions

    DEFF Research Database (Denmark)

    Munthe, Sune; Halle, Bo; Boldt, Henning B

    2017-01-01

    Glioblastoma multiforme (GBM) is the most frequent malignant primary brain tumor. A major reason for the overall median survival being only 14.6 months is migrating tumor cells left behind after surgery. Another major reason is tumor cells having a so-called cancer stem cell phenotype being...... therefore resistant towards traditional chemo- and radiotherapy. A group of novel molecular targets are microRNAs (miRNAs). MiRNAs are small non-coding RNAs exerting post-transcriptional regulation of gene expression. The aim of this study was to identify differentially expressed miRNAs in migrating GBM...... cells using serum-free stem cell conditions. We used patient-derived GBM spheroid cultures for a novel serum-free migration assay. MiRNA expression of migrating tumor cells isolated at maximum migration speed was compared with corresponding spheroids using an OpenArray Real-Time PCR System. The mi...

  13. Positive cell-free fetal DNA testing for trisomy 13 reveals confined placental mosaicism.

    Science.gov (United States)

    Hall, April L; Drendel, Holli M; Verbrugge, Jennifer L; Reese, Angela M; Schumacher, Katherine L; Griffith, Christopher B; Weaver, David D; Abernathy, Mary P; Litton, Christian G; Vance, Gail H

    2013-09-01

    We report on a case in which cell-free fetal DNA was positive for trisomy 13 most likely due to confined placental mosaicism. Cell-free fetal DNA testing analyzes DNA derived from placental trophoblast cells and can lead to incorrect results that are not representative of the fetus. We sought to confirm commercial cell-free fetal DNA testing results by chorionic villus sampling and amniocentesis. These results were followed up by postnatal chromosome analysis of cord blood and placental tissue. First-trimester cell-free fetal DNA test results were positive for trisomy 13. Cytogenetic analysis of chorionic villus sampling yielded a mosaic karyotype of 47,XY,+13[10]/46,XY[12]. G-banded analysis of amniotic fluid was normal, 46,XY. Postnatal cytogenetic analysis of cord blood was normal. Karyotyping of tissues from four quadrants of the placenta demonstrated mosaicism for trisomy 13 in two of the quadrants and a normal karyotype in the other two. Our case illustrates several important aspects of this new testing methodology: that cell-free fetal DNA may not be representative of the fetal karyotype; that follow-up with diagnostic testing of chorionic villus sampling and/or amniotic fluid for abnormal test results should be performed; and that pretest counseling regarding the full benefits, limitations, and possible testing outcomes of cell-free fetal DNA screening is important.

  14. Circulating cell free DNA as a predictor of systemic lupus ...

    African Journals Online (AJOL)

    Olfat M. Hendy

    2015-07-29

    Jul 29, 2015 ... as a potential tool to predict disease activity and treatment follow up. Subjects and ... control group. Thorough clinical examination stressing on the central nervous system, vascular, ... sis/necrosis of blood and tissue cells) and, second, active metabolic ..... alternative biomarkers should be tested. There was ...

  15. Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System.

    Science.gov (United States)

    Nandal, Anjali; Mallon, Barbara; Telugu, Bhanu P

    2017-11-08

    Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide "NGG" protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

  16. Advanced feeder-free generation of induced pluripotent stem cells directly from blood cells.

    Science.gov (United States)

    Trokovic, Ras; Weltner, Jere; Nishimura, Ken; Ohtaka, Manami; Nakanishi, Mahito; Salomaa, Veikko; Jalanko, Anu; Otonkoski, Timo; Kyttälä, Aija

    2014-12-01

    Generation of validated human induced pluripotent stem cells (iPSCs) for biobanking is essential for exploring the full potential of iPSCs in disease modeling and drug discovery. Peripheral blood mononuclear cells (PBMCs) are attractive targets for reprogramming, because blood is collected by a routine clinical procedure and is a commonly stored material in biobanks. Generation of iPSCs from blood cells has previously been reported using integrative retroviruses, episomal Sendai viruses, and DNA plasmids. However, most of the published protocols require expansion and/or activation of a specific cell population from PBMCs. We have recently collected a PBMC cohort from the Finnish population containing more than 2,000 subjects. Here we report efficient generation of iPSCs directly from PBMCs in feeder-free conditions in approximately 2 weeks. The produced iPSC clones are pluripotent and transgene-free. Together, these properties make this novel method a powerful tool for large-scale reprogramming of PBMCs and for iPSC biobanking. ©AlphaMed Press.

  17. Cloning-free template DNA preparation for cell-free protein synthesis via two-step PCR using versatile primer designs with short 3'-UTR.

    Science.gov (United States)

    Nomoto, Mika; Tada, Yasuomi

    2018-01-01

    Cell-free protein synthesis (CFPS) systems largely retain the endogenous translation machinery of the host organism, making them highly applicable for proteomics analysis of diverse biological processes. However, laborious and time-consuming cloning procedures hinder progress with CFPS systems. Herein, we report the development of a rapid and efficient two-step polymerase chain reaction (PCR) method to prepare linear DNA templates for a wheat germ CFPS system. We developed a novel, effective short 3'-untranslated region (3'-UTR) sequence that facilitates translation. Application of the short 3'-UTR to two-step PCR enabled the generation of various transcription templates from the same plasmid, including fusion proteins with N- or C-terminal tags, and truncated proteins. Our method supports the cloning-free expression of target proteins using an mRNA pool from biological material. The established system is a highly versatile platform for in vitro protein synthesis using wheat germ CFPS. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  18. Effect of Antimalarial Drugs on Plasmodia Cell-Free Protein Synthesis

    Directory of Open Access Journals (Sweden)

    Ana Ferreras

    2002-04-01

    Full Text Available A cell-free system from Plasmodium falciparum able to translate endogenous mRNA was used to determine the effect of artemisinin, chloroquine and primaquine on the protein synthesis mechanism of the parasite. The antimalarial drugs did not inhibit the incorporation of [³H] methionine into parasite proteins even at concentrations higher than the ones found to strongly inhibit the parasite growth. Results clearly indicate that these compounds do not have a direct effect on protein synthesis activity of P. falciparum coded by endogenous mRNA.

  19. Label-free and live cell imaging by interferometric scattering microscopy.

    Science.gov (United States)

    Park, Jin-Sung; Lee, Il-Buem; Moon, Hyeon-Min; Joo, Jong-Hyeon; Kim, Kyoung-Hoon; Hong, Seok-Cheol; Cho, Minhaeng

    2018-03-14

    Despite recent remarkable advances in microscopic techniques, it still remains very challenging to directly observe the complex structure of cytoplasmic organelles in live cells without a fluorescent label. Here we report label-free and live-cell imaging of mammalian cell, Escherischia coli , and yeast, using interferometric scattering microscopy, which reveals the underlying structures of a variety of cytoplasmic organelles as well as the underside structure of the cells. The contact areas of the cells attached onto a glass substrate, e.g. , focal adhesions and filopodia, are clearly discernible. We also found a variety of fringe-like features in the cytoplasmic area, which may reflect the folded structures of cytoplasmic organelles. We thus anticipate that the label-free interferometric scattering microscopy can be used as a powerful tool to shed interferometric light on in vivo structures and dynamics of various intracellular phenomena.

  20. Serum-Free Media and the Immunoregulatory Properties of Mesenchymal Stem Cells In Vivo and In Vitro

    Directory of Open Access Journals (Sweden)

    Mei Wu

    2014-02-01

    Full Text Available Background: Mesenchymal stem cells are capable of self-renewal and multi-lineage differentiation. They are used extensively to treat several diseases. Traditionally, mesenchymal stem cells are cultured in serum-containing media, typically supplemented with fetal bovine serum (FBS. However, the variability of FBS is likely to skew experimental results. Although serum-free media used to expand mesenchymal stem cells has facilitated remarkable achievements, immunomodulation of these cells in under serum-free conditions is poorly understood. We hypothesized that mesenchymal stem cells expanded in serum-free media will retain powerful immunoregulatory functions in vitro and in vivo. Design and Methods: Immunosuppressive activity and the immunomodulatory cytokines produced by mesenchymal stem cells in serum-free media were characterized in vitro. Immunomodulation by serum-free mesenchymal stem cell expansion in monocrotaline-induced pulmonary hypertension was explored in vivo. Results: Similar to cells in serum-containing media, mesenchymal stem cells expanded in serum-free media inhibited proliferation and apoptosis of CD4+T cells. They also exhibited strong immunosuppressive activities and secreted high levels of immunomodulatory cytokines such as PGE2, IDO1, COX2, IL-6, and IL-1β, but not HGF. On the other hand, growth of mesenchymal stem cells in serum-free media attenuated pulmonary vascular remodeling and inhibited mRNA expression of proinflammatory cytokines TNF-α, IFN-γ, IL-6, IL-1β, and IL-18. Conclusions: Mesenchymal stem cells in serum-free media maintained powerful immunomodulatory function in vitro and in vivo; serum-free media may replace serum-containing media for basic research and clinical applications.

  1. Progress in Electrolyte-Free Fuel Cells

    International Nuclear Information System (INIS)

    Lu, Yuzheng; Zhu, Bin; Cai, Yixiao; Kim, Jung-Sik; Wang, Baoyuan; Wang, Jun; Zhang, Yaoming; Li, Junjiao

    2016-01-01

    Solid oxide fuel cell (SOFC) represents a clean electrochemical energy conversion technology with characteristics of high conversion efficiency and low emissions. It is one of the most important new energy technologies in the future. However, the manufacture of SOFCs based on the structure of anode/electrolyte/cathode is complicated and time-consuming. Thus, the cost for the entire fabrication and technology is too high to be affordable, and challenges still hinder commercialization. Recently, a novel type of electrolyte-free fuel cell (EFFC) with single component was invented, which could be the potential candidate for the next generation of advanced fuel cells. This paper briefly introduces the EFFC, working principle, performance, and advantages with updated research progress. A number of key R&D issues about EFFCs have been addressed, and future opportunities and challenges are discussed.

  2. Progress in Electrolyte-Free Fuel Cells

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Yuzheng [Jiangsu Provincial Key Laboratory of Solar Energy Science and Technology, School of Energy and Environment, Southeast University, Nanjing (China); Zhu, Bin, E-mail: binzhu@kth.se [Faculty of Physics and Electronic Technology, Hubei Collaborative Innovation Center for Advanced Organic Materials, Hubei University, Wuhan (China); Department of Energy Technology, Royal Institute of Technology KTH, Stockholm (Sweden); Cai, Yixiao [Ångström Laboratory, Department of Engineering Sciences, Uppsala University, Uppsala (Sweden); Kim, Jung-Sik [Department of Aeronautical and Automotive Engineering, Loughborough University, Loughborough (United Kingdom); Wang, Baoyuan [Faculty of Physics and Electronic Technology, Hubei Collaborative Innovation Center for Advanced Organic Materials, Hubei University, Wuhan (China); Department of Energy Technology, Royal Institute of Technology KTH, Stockholm (Sweden); Wang, Jun, E-mail: binzhu@kth.se; Zhang, Yaoming [Jiangsu Provincial Key Laboratory of Solar Energy Science and Technology, School of Energy and Environment, Southeast University, Nanjing (China); Li, Junjiao [Nanjing Yunna Nano Technology Co., Ltd., Nanjing (China)

    2016-05-02

    Solid oxide fuel cell (SOFC) represents a clean electrochemical energy conversion technology with characteristics of high conversion efficiency and low emissions. It is one of the most important new energy technologies in the future. However, the manufacture of SOFCs based on the structure of anode/electrolyte/cathode is complicated and time-consuming. Thus, the cost for the entire fabrication and technology is too high to be affordable, and challenges still hinder commercialization. Recently, a novel type of electrolyte-free fuel cell (EFFC) with single component was invented, which could be the potential candidate for the next generation of advanced fuel cells. This paper briefly introduces the EFFC, working principle, performance, and advantages with updated research progress. A number of key R&D issues about EFFCs have been addressed, and future opportunities and challenges are discussed.

  3. Cell-free DNA, inflammation, and the initiation of spontaneous term labor.

    Science.gov (United States)

    Herrera, Christina A; Stoerker, Jay; Carlquist, John; Stoddard, Gregory J; Jackson, Marc; Esplin, Sean; Rose, Nancy C

    2017-11-01

    Hypomethylated cell-free DNA from senescent placental trophoblasts may be involved in the activation of the inflammatory cascade to initiate labor. To determine the changes in cell-free DNA concentrations, the methylation ratio, and inflammatory markers between women in labor at term vs women without labor. In this prospective cohort study, eligible participants carried a nonanomalous singleton fetus. Women with major medical comorbidity, preterm labor, progesterone use, aneuploidy, infectious disease, vaginal bleeding, abdominal trauma, or invasive procedures during the pregnancy were excluded. Maternal blood samples were collected at 28 weeks, 36 weeks, and at admission for delivery. Total cell-free DNA concentration, methylation ratio, and interleukin-6 were analyzed. The primary outcome was the difference in methylation ratio in women with labor vs without labor. Secondary outcomes included the longitudinal changes in these biomarkers corresponding to labor status. A total of 55 women were included; 20 presented in labor on admission and 35 presented without labor. Women in labor had significantly greater methylation ratio (P = .001) and interleukin-6 (P < .001) on admission for delivery than women without labor. After we controlled for body mass index and maternal age, methylation ratio (adjusted relative risk, 1.38; 95% confidence interval, 1.13 to 1.68) and interleukin-6 (adjusted relative risk, 1.12, 95% confidence interval, 1.07 to 1.17) remained greater in women presenting in labor. Total cell-free DNA was not significantly different in women with labor compared with women without. Longitudinally, total cell-free DNA (P < .001 in labor, P = .002 without labor) and interleukin-6 (P < .001 in labor, P = .01 without labor) increased significantly across gestation in both groups. The methylation ratio increased significantly in women with labor from 36 weeks to delivery (P = .02). Spontaneous labor at term is associated with a greater cell-free DNA

  4. Transcriptional blockages in a cell-free system by sequence-selective DNA alkylating agents.

    Science.gov (United States)

    Ferguson, L R; Liu, A P; Denny, W A; Cullinane, C; Talarico, T; Phillips, D R

    2000-04-14

    There is considerable interest in DNA sequence-selective DNA-binding drugs as potential inhibitors of gene expression. Five compounds with distinctly different base pair specificities were compared in their effects on the formation and elongation of the transcription complex from the lac UV5 promoter in a cell-free system. All were tested at drug levels which killed 90% of cells in a clonogenic survival assay. Cisplatin, a selective alkylator at purine residues, inhibited transcription, decreasing the full-length transcript, and causing blockage at a number of GG or AG sequences, making it probable that intrastrand crosslinks are the blocking lesions. A cyclopropylindoline known to be an A-specific alkylator also inhibited transcription, with blocks at adenines. The aniline mustard chlorambucil, that targets primarily G but also A sequences, was also effective in blocking the formation of full-length transcripts. It produced transcription blocks either at, or one base prior to, AA or GG sequences, suggesting that intrastrand crosslinks could again be involved. The non-alkylating DNA minor groove binder Hoechst 33342 (a bisbenzimidazole) blocked formation of the full-length transcript, but without creating specific blockage sites. A bisbenzimidazole-linked aniline mustard analogue was a more effective transcription inhibitor than either chlorambucil or Hoechst 33342, with different blockage sites occurring immediately as compared with 2 h after incubation. The blockages were either immediately prior to AA or GG residues, or four to five base pairs prior to such sites, a pattern not predicted from in vitro DNA-binding studies. Minor groove DNA-binding ligands are of particular interest as inhibitors of gene expression, since they have the potential ability to bind selectively to long sequences of DNA. The results suggest that the bisbenzimidazole-linked mustard does cause alkylation and transcription blockage at novel DNA sites. in addition to sites characteristic of

  5. Investigating free radical generation in HepG2 cells using immuno-spin trapping.

    Science.gov (United States)

    Horinouchi, Yuya; Summers, Fiona A; Ehrenshaft, Marilyn; Kawazoe, Kazuyoshi; Tsuchiya, Koichiro; Tamaki, Toshiaki; Mason, Ronald P

    2014-10-01

    Oxidative stress can induce the generation of free radicals, which are believed to play an important role in both physiological and pathological processes and a number of diseases such as cancer. Therefore, it is important to identify chemicals which are capable of inducing oxidative stress. In this study, we evaluated the ability of four environmental chemicals, aniline, nitrosobenzene (NB), N,N-dimethylaniline (DMA) and N,N-dimethyl-4-nitrosoaniline (DMNA), to induce free radicals and cellular damage in the hepatoma cell line HepG2. Cytotoxicity was assessed using lactate dehydrogenase (LDH) assays and morphological changes were observed using phase contrast microscopy. Free radicals were detected by immuno-spin trapping (IST) in in-cell western experiments or in confocal microscopy experiments to determine the subcellular localization of free radical generation. DMNA induced free radical generation, LDH release and morphological changes in HepG2 cells whereas aniline, NB and DMA did not. Confocal microscopy showed that DMNA induced free radical generation mainly in the cytosol. Preincubation of HepG2 cells with N-acetylcysteine and 2,2'-dipyridyl significantly prevented free radical generation upon subsequent incubation with DMNA, whereas preincubation with apocynin and dimethyl sulfoxide did not. These results suggest that DMNA induces oxidative stress and that reactive oxygen species, metals and free radical generation play a critical role in DMNA-induced cytotoxicity. Copyright © 2014. Published by Elsevier Inc.

  6. Fluorescent in situ folding control for rapid optimization of cell-free membrane protein synthesis.

    Directory of Open Access Journals (Sweden)

    Annika Müller-Lucks

    Full Text Available Cell-free synthesis is an open and powerful tool for high-yield protein production in small reaction volumes predestined for high-throughput structural and functional analysis. Membrane proteins require addition of detergents for solubilization, liposomes, or nanodiscs. Hence, the number of parameters to be tested is significantly higher than with soluble proteins. Optimization is commonly done with respect to protein yield, yet without knowledge of the protein folding status. This approach contains a large inherent risk of ending up with non-functional protein. We show that fluorophore formation in C-terminal fusions with green fluorescent protein (GFP indicates the folding state of a membrane protein in situ, i.e. within the cell-free reaction mixture, as confirmed by circular dichroism (CD, proteoliposome reconstitution and functional assays. Quantification of protein yield and in-gel fluorescence intensity imply suitability of the method for membrane proteins of bacterial, protozoan, plant, and mammalian origin, representing vacuolar and plasma membrane localization, as well as intra- and extracellular positioning of the C-terminus. We conclude that GFP-fusions provide an extension to cell-free protein synthesis systems eliminating the need for experimental folding control and, thus, enabling rapid optimization towards membrane protein quality.

  7. A deterministic method for estimating free energy genetic network landscapes with applications to cell commitment and reprogramming paths.

    Science.gov (United States)

    Olariu, Victor; Manesso, Erica; Peterson, Carsten

    2017-06-01

    Depicting developmental processes as movements in free energy genetic landscapes is an illustrative tool. However, exploring such landscapes to obtain quantitative or even qualitative predictions is hampered by the lack of free energy functions corresponding to the biochemical Michaelis-Menten or Hill rate equations for the dynamics. Being armed with energy landscapes defined by a network and its interactions would open up the possibility of swiftly identifying cell states and computing optimal paths, including those of cell reprogramming, thereby avoiding exhaustive trial-and-error simulations with rate equations for different parameter sets. It turns out that sigmoidal rate equations do have approximate free energy associations. With this replacement of rate equations, we develop a deterministic method for estimating the free energy surfaces of systems of interacting genes at different noise levels or temperatures. Once such free energy landscape estimates have been established, we adapt a shortest path algorithm to determine optimal routes in the landscapes. We explore the method on three circuits for haematopoiesis and embryonic stem cell development for commitment and reprogramming scenarios and illustrate how the method can be used to determine sequential steps for onsets of external factors, essential for efficient reprogramming.

  8. Stem cell recovering effect of copper-free GHK in skin.

    Science.gov (United States)

    Choi, Hye-Ryung; Kang, Youn-A; Ryoo, Sun-Jong; Shin, Jung-Won; Na, Jung-Im; Huh, Chang-Hun; Park, Kyoung-Chan

    2012-11-01

    The peptide Gly-His-Lys (GHK) is a naturally occurring copper(II)-chelating motifs in human serum and cerebrospinal fluid. In industry, GHK (with or without copper) is used to make hair and skin care products. Copper-GHK plays a physiological role in the process of wound healing and tissue repair by stimulating collagen synthesis in fibroblasts. We also reported that copper-GHK promotes the survival of basal stem cells in the skin. However, the effects of copper-free GHK (GHK) have not been investigated well. In this study, the effects of GHK were studied using cultured normal human keratinocytes and skin equivalent (SE) models. In monolayer cultured keratinocytes, GHK increased the proliferation of keratinocytes. When GHK was added during the culture of SE models, the basal cells became more cuboidal than control model. In addition, there was linear and intense staining of α6 and β1 integrin along the basement membrane. The number of p63 and proliferating cell nuclear antigen positive cells was also significantly increased in GHK-treated SEs than in control SEs. Western blot and slide culture experiment showed that GHK increased the expression of integrin by keratinocytes. All these results showed that GHK increased the stemness and proliferative potential of epidermal basal cells, which is associated with increased expression of integrin. In conclusion, copper-free GHK showed similar effects with copper-GHK. Thus, it can be said that copper-free GHK can be used in industry to obtain the effects of copper-GHK in vivo. Further study is necessary to explore the relationship between copper-free GHK and copper-GHK. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

  9. Robust and highly-efficient differentiation of functional monocytic cells from human pluripotent stem cells under serum- and feeder cell-free conditions.

    Directory of Open Access Journals (Sweden)

    Masakatsu D Yanagimachi

    Full Text Available Monocytic lineage cells (monocytes, macrophages and dendritic cells play important roles in immune responses and are involved in various pathological conditions. The development of monocytic cells from human embryonic stem cells (ESCs and induced pluripotent stem cells (iPSCs is of particular interest because it provides an unlimited cell source for clinical application and basic research on disease pathology. Although the methods for monocytic cell differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established, these methods depend on the use of xenogeneic materials and, therefore, have a relatively poor-reproducibility. Here, we established a robust and highly-efficient method to differentiate functional monocytic cells from ESCs/iPSCs under serum- and feeder cell-free conditions. This method produced 1.3 × 10(6 ± 0.3 × 10(6 floating monocytes from approximately 30 clusters of ESCs/iPSCs 5-6 times per course of differentiation. Such monocytes could be differentiated into functional macrophages and dendritic cells. This method should be useful for regenerative medicine, disease-specific iPSC studies and drug discovery.

  10. Progress in Electrolyte-Free Fuel Cells

    Directory of Open Access Journals (Sweden)

    Yuzheng eLu

    2016-05-01

    Full Text Available Solid Oxide Fuel Cell (SOFC represents a clean electrochemical energy conversion technology with characteristics of high conversion efficiency and low emissions. It is one of the most important new energy technologies in the future. However, the manufacture of SOFCs based on the structure of anode/electrolyte/cathode is complicated and time-consuming. Thus, the cost for the entire fabrication and technology is too high to be affordable and challenges still hinder commercialization. Recently, a novel type of Electrolyte -free fuel cell (EFFC with single component was invented which could be the potential candidate for the next generation of advanced fuel cells. This paper briefly introduces the EFFC, working principle, performance and advantages with updated research progress. A number of key R&D issues about EFFCs have been addressed and future opportunities and challenges are discussed.

  11. Body-on-a-chip systems for animal-free toxicity testing.

    Science.gov (United States)

    Mahler, Gretchen J; Esch, Mandy B; Stokol, Tracy; Hickman, James J; Shuler, Michael L

    2016-10-01

    Body-on-a-chip systems replicate the size relationships of organs, blood distribution and blood flow, in accordance with human physiology. When operated with tissues derived from human cell sources, these systems are capable of simulating human metabolism, including the conversion of a prodrug to its effective metabolite, as well as its subsequent therapeutic actions and toxic side-effects. The system also permits the measurement of human tissue electrical and mechanical reactions, which provide a measure of functional response. Since these devices can be operated with human tissue samples or with in vitro tissues derived from induced pluripotent stem cells (iPS), they can play a significant role in determining the success of new pharmaceuticals, without resorting to the use of animals. By providing a platform for testing in the context of human metabolism, as opposed to animal models, the systems have the potential to eliminate the use of animals in preclinical trials. This article will review progress made and work achieved as a direct result of the 2015 Lush Science Prize in support of animal-free testing. 2016 FRAME.

  12. Cell-Free Synthetic Biology Chassis for Nanocatalytic Photon-to-Hydrogen Conversion.

    Science.gov (United States)

    Wang, Peng; Chang, Angela Y; Novosad, Valentyn; Chupin, Vladimir V; Schaller, Richard D; Rozhkova, Elena A

    2017-07-25

    We report on an entirely man-made nano-bio architecture fabricated through noncovalent assembly of a cell-free expressed transmembrane proton pump and TiO 2 semiconductor nanoparticles as an efficient nanophotocatalyst for H 2 evolution. The system produces hydrogen at a turnover of about 240 μmol of H 2 (μmol protein) -1 h -1 and 17.74 mmol of H 2 (μmol protein) -1 h -1 under monochromatic green and white light, respectively, at ambient conditions, in water at neutral pH and room temperature, with methanol as a sacrificial electron donor. Robustness and flexibility of this approach allow for systemic manipulation at the nanoparticle-bio interface toward directed evolution of energy transformation materials and artificial systems.

  13. Interface engineering for efficient fullerene-free organic solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Shivanna, Ravichandran; Narayan, K. S., E-mail: rajaram@jncasr.ac.in, E-mail: narayan@jncasr.ac.in [Chemistry and Physics of Materials Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064 (India); Rajaram, Sridhar, E-mail: rajaram@jncasr.ac.in, E-mail: narayan@jncasr.ac.in [International Centre for Materials Science, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064 (India)

    2015-03-23

    We demonstrate the role of zinc oxide (ZnO) morphology and addition of an acceptor interlayer to achieve high efficiency fullerene-free bulk heterojunction inverted organic solar cells. Nanopatterning of the ZnO buffer layer enhances the effective light absorption in the active layer, and the insertion of a twisted perylene acceptor layer planarizes and decreases the electron extraction barrier. Along with an increase in current homogeneity, the reduced work function difference and selective transport of electrons prevent the accumulation of charges and decrease the electron-hole recombination at the interface. These factors enable an overall increase of efficiency to 4.6%, which is significant for a fullerene-free solution-processed organic solar cell.

  14. Cytokine-free directed differentiation of human pluripotent stem cells efficiently produces hemogenic endothelium with lymphoid potential.

    Science.gov (United States)

    Galat, Yekaterina; Dambaeva, Svetlana; Elcheva, Irina; Khanolkar, Aaruni; Beaman, Kenneth; Iannaccone, Philip M; Galat, Vasiliy

    2017-03-17

    The robust generation of human hematopoietic progenitor cells from induced or embryonic pluripotent stem cells would be beneficial for multiple areas of research, including mechanistic studies of hematopoiesis, the development of cellular therapies for autoimmune diseases, induced transplant tolerance, anticancer immunotherapies, disease modeling, and drug/toxicity screening. Over the past years, significant progress has been made in identifying effective protocols for hematopoietic differentiation from pluripotent stem cells and understanding stages of mesodermal, endothelial, and hematopoietic specification. Thus, it has been shown that variations in cytokine and inhibitory molecule treatments in the first few days of hematopoietic differentiation define primitive versus definitive potential of produced hematopoietic progenitor cells. The majority of current feeder-free, defined systems for hematopoietic induction from pluripotent stem cells include prolonged incubations with various cytokines that make the differentiation process complex and time consuming. We established that the application of Wnt agonist CHIR99021 efficiently promotes differentiation of human pluripotent stem cells in the absence of any hematopoietic cytokines to the stage of hemogenic endothelium capable of definitive hematopoiesis. The hemogenic endothelium differentiation was accomplished in an adherent, serum-free culture system by applying CHIR99021. Hemogenic endothelium progenitor cells were isolated on day 5 of differentiation and evaluated for their endothelial, myeloid, and lymphoid potential. Monolayer induction based on GSK3 inhibition, described here, yielded a large number of CD31 + CD34 + hemogenic endothelium cells. When isolated and propagated in adherent conditions, these progenitors gave rise to mature endothelium. When further cocultured with OP9 mouse stromal cells, these progenitors gave rise to various cells of myeloid lineages as well as natural killer lymphoid, T

  15. Universal Approach toward Hysteresis-Free Perovskite Solar Cell via Defect Engineering.

    Science.gov (United States)

    Son, Dae-Yong; Kim, Seul-Gi; Seo, Ja-Young; Lee, Seon-Hee; Shin, Hyunjung; Lee, Donghwa; Park, Nam-Gyu

    2018-01-31

    Organic-inorganic halide perovskite is believed to be a potential candidate for high efficiency solar cells because power conversion efficiency (PCE) was certified to be more than 22%. Nevertheless, mismatch of PCE due to current density (J)-voltage (V) hysteresis in perovskite solar cells is an obstacle to overcome. There has been much lively debate on the origin of J-V hysteresis; however, effective methodology to solve the hysteric problem has not been developed. Here we report a universal approach for hysteresis-free perovskite solar cells via defect engineering. A severe hysteresis observed from the normal mesoscopic structure employing TiO 2 and spiro-MeOTAD is almost removed or does not exist upon doping the pure perovskites, CH 3 NH 3 PbI 3 and HC(NH 2 ) 2 PbI 3 , and the mixed cation/anion perovskites, FA 0.85 MA 0.15 PbI 2.55 Br 0.45 and FA 0.85 MA 0.1 Cs 0.05 PbI 2.7 Br 0.3 , with potassium iodide. Substantial reductions in low-frequency capacitance and bulk trap density are measured from the KI-doped perovskite, which is indicative of trap-hysteresis correlation. A series of experiments with alkali metal iodides of LiI, NaI, KI, RbI and CsI reveals that potassium ion is the right element for hysteresis-free perovskite. Theoretical studies suggest that the atomistic origin of the hysteresis of perovskite solar cells is not the migration of iodide vacancy but results from the formation of iodide Frenkel defect. Potassium ion is able to prevent the formation of Frenkel defect since K + energetically prefers the interstitial site. A complete removal of hysteresis is more pronounced at mixed perovskite system as compared to pure perovskites, which is explained by lower formation energy of K interstitial (-0.65 V for CH 3 NH 3 PbI 3 vs -1.17 V for mixed perovskite). The developed KI doping methodology is universally adapted for hysteresis-free perovskite regardless of perovskite composition and device structure.

  16. Flavor formation and cell physiology during the production of alcohol-free beer with immobilized Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Iersel, van M.F.M.; Dieren, van B.; Rombouts, F.M.; Abee, T.

    1999-01-01

    Production of alcohol-free beer by limited fermentation is optimally performed in a packed-bed reactor operating in downflow. This ensures a highly controllable system with optimal reactor design. In the present study, we report on changes in the physiology of immobilized yeast cells in the reactor.

  17. IRON AND FREE RADICAL OXIDATIONS IN CELL MEMBRANES

    Science.gov (United States)

    Schafer, Freya Q.; Yue Qian, Steven; Buettner, Garry R.

    2013-01-01

    Brain tissue being rich in polyunsaturated fatty acids, is very susceptible to lipid peroxidation. Iron is well known to be an important initiator of free radical oxidations. We propose that the principal route to iron-mediated lipid peroxidations is via iron-oxygen complexes rather than the reaction of iron with hydrogen peroxide, the Fenton reaction. To test this hypothesis, we enriched leukemia cells (K-562 and L1210 cells) with docosahexaenoic acid (DHA) as a model for brain tissue, increasing the amount of DHA from approximately 3 mole % to 32 mole %. These cells were then subjected to ferrous iron and dioxygen to initiate lipid peroxidation in the presence or absence of hydrogen peroxide. Lipid-derived radicals were detected using EPR spin trapping with α-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN). As expected, lipid-derived radical formation increases with increasing cellular lipid unsaturation. Experiments with Desferal demonstrate that iron is required for the formation of lipid radicals from these cells. Addition of iron to DHA-enriched L1210 cells resulted in significant amounts of radical formation; radical formation increased with increasing amount of iron. However, the exposure of cells to hydrogen peroxide before the addition of ferrous iron did not increase cellular radical formation, but actually decreased spin adduct formation. These data suggest that iron-oxygen complexes are the primary route to the initiation of biological free radical oxidations. This model proposes a mechanism to explain how catalytic iron in brain tissue can be so destructive. PMID:10872752

  18. Device-Free Indoor Activity Recognition System

    Directory of Open Access Journals (Sweden)

    Mohammed Abdulaziz Aide Al-qaness

    2016-11-01

    Full Text Available In this paper, we explore the properties of the Channel State Information (CSI of WiFi signals and present a device-free indoor activity recognition system. Our proposed system uses only one ubiquitous router access point and a laptop as a detection point, while the user is free and neither needs to wear sensors nor carry devices. The proposed system recognizes six daily activities, such as walk, crawl, fall, stand, sit, and lie. We have built the prototype with an effective feature extraction method and a fast classification algorithm. The proposed system has been evaluated in a real and complex environment in both line-of-sight (LOS and none-line-of-sight (NLOS scenarios, and the results validate the performance of the proposed system.

  19. Elevated levels of cell-free circulating DNA in patients with acute dengue virus infection.

    Directory of Open Access Journals (Sweden)

    Tran Thi Ngoc Ha

    Full Text Available BACKGROUND: Apoptosis is thought to play a role in the pathogenesis of severe dengue and the release of cell-free DNA into the circulatory system in several medical conditions. Therefore, we investigated circulating DNA as a potential biomarker for severe dengue. METHODS AND FINDINGS: A direct fluorometric degradation assay using PicoGreen was performed to quantify cell-free DNA from patient plasma. Circulating DNA levels were significantly higher in patients with dengue virus infection than with other febrile illnesses and healthy controls. Remarkably, the increase of DNA levels correlated with the severity of dengue. Additionally, multivariate logistic regression analysis showed that circulating DNA levels independently correlated with dengue shock syndrome. CONCLUSIONS: Circulating DNA levels were increased in dengue patients and correlated with dengue severity. Additional studies are required to show the benefits of this biomarker in early dengue diagnosis and for the prognosis of shock complication.

  20. Free tissue transfer in patients with sickle cell disease: Considerations for multi-disciplinary peri-operative management.

    Science.gov (United States)

    Cooper, Lilli; Seth, Rohit; Rhodes, Elizabeth; Alousi, Mohammed; Sivakumar, Bran

    2017-01-01

    Sickle cell disease (SCD) is an increasingly common condition in the UK. The safety of free tissue transfer in these patients is controversial, and no specific guidelines exist. The aim of this paper is to create recommendations for the plastic surgical multidisciplinary team for use in the assessment and management of SCD patients undergoing free tissue transfer and reconstruction. A literature review was performed in PubMed of 'sickle [TiAb] AND plast* adj3 surg*. Sickle cell disease is explained, as is the relative peri-operative risk in different genotypes of SCD. Acute and chronic manifestations of SCD are described by system, for consideration at pre-operative assessment and post-operative review. The evidence surrounding free tissue transfer and SCD is discussed and the outcomes in published cases summarised. An algorithm for peri-operative multi-disciplinary management is outlined and justified. Free tissue transfer theoretically carries a high risk of a crisis, due not only to long anaesthetic times, but the potential requirement for tourniquet use, and the relatively hypoxic state of the transferred tissue. This paper outlines a useful, practical algorithm to optimise the safety of free tissue transfer in patients with SCD. Copyright © 2016 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

  1. Cooling systems of the resting area in free stall dairy barn

    Science.gov (United States)

    Calegari, F.; Calamari, L.; Frazzi, E.

    2016-04-01

    A study during the summer season evaluated the effect of different cooling systems on behavioral and productive responses of Italian Friesian dairy cows kept in an experimental-free stall barn located in the Po Valley in Italy. The study involved 30 lactating dairy cows subdivided into two groups kept in two pens with external hard court paddock in each free stall. The same cooling system was applied in the feeding area in both pens. A different cooling system in the resting area was applied to the two pens: in the pen SW, the resting area was equipped with fans and misters; in the other, there was simple ventilation (SV). Breathing rate, rectal temperature, milk yield, and milk characteristics (fat, protein, and somatic cell count) were measured. Behavioral activities (standing and lying cows in the different areas, as well as the animals in the feed bunk) were recorded. Mild to moderate heat waves during the trial were observed. On average, the breathing rate was numerically greater in SV compared with SW cows (60.2 and 55.8 breath/min, respectively), and mean rectal temperature remained below 39 °C in both groups during the trial (on average 38.7 and 38.8 °C in SV and SW, respectively. During the hotter periods of the trial, the time spent lying indoor in the free stall was greater in SW (11.8 h/day) than SV (10.7 h/day). Conversely, the time spent standing indoor without feeding was greater in SV (4.3 h/day) than SW (3.8 h/day). Milk yield was slightly better maintained during hotter period in SW compared with SV and somatic cell count was also slightly greater in the former. In conclusion, the adoption of the cooling system by means of evaporative cooling also in the resting area reduces the alteration of time budget caused by heat stress.

  2. Detection of atomic scale changes in the free volume void size of three-dimensional colorectal cancer cell culture using positron annihilation lifetime spectroscopy.

    Science.gov (United States)

    Axpe, Eneko; Lopez-Euba, Tamara; Castellanos-Rubio, Ainara; Merida, David; Garcia, Jose Angel; Plaza-Izurieta, Leticia; Fernandez-Jimenez, Nora; Plazaola, Fernando; Bilbao, Jose Ramon

    2014-01-01

    Positron annihilation lifetime spectroscopy (PALS) provides a direct measurement of the free volume void sizes in polymers and biological systems. This free volume is critical in explaining and understanding physical and mechanical properties of polymers. Moreover, PALS has been recently proposed as a potential tool in detecting cancer at early stages, probing the differences in the subnanometer scale free volume voids between cancerous/healthy skin samples of the same patient. Despite several investigations on free volume in complex cancerous tissues, no positron annihilation studies of living cancer cell cultures have been reported. We demonstrate that PALS can be applied to the study in human living 3D cell cultures. The technique is also capable to detect atomic scale changes in the size of the free volume voids due to the biological responses to TGF-β. PALS may be developed to characterize the effect of different culture conditions in the free volume voids of cells grown in vitro.

  3. High-resolution characterization of sequence signatures due to non-random cleavage of cell-free DNA.

    Science.gov (United States)

    Chandrananda, Dineika; Thorne, Natalie P; Bahlo, Melanie

    2015-06-17

    High-throughput sequencing of cell-free DNA fragments found in human plasma has been used to non-invasively detect fetal aneuploidy, monitor organ transplants and investigate tumor DNA. However, many biological properties of this extracellular genetic material remain unknown. Research that further characterizes circulating DNA could substantially increase its diagnostic value by allowing the application of more sophisticated bioinformatics tools that lead to an improved signal to noise ratio in the sequencing data. In this study, we investigate various features of cell-free DNA in plasma using deep-sequencing data from two pregnant women (>70X, >50X) and compare them with matched cellular DNA. We utilize a descriptive approach to examine how the biological cleavage of cell-free DNA affects different sequence signatures such as fragment lengths, sequence motifs at fragment ends and the distribution of cleavage sites along the genome. We show that the size distributions of these cell-free DNA molecules are dependent on their autosomal and mitochondrial origin as well as the genomic location within chromosomes. DNA mapping to particular microsatellites and alpha repeat elements display unique size signatures. We show how cell-free fragments occur in clusters along the genome, localizing to nucleosomal arrays and are preferentially cleaved at linker regions by correlating the mapping locations of these fragments with ENCODE annotation of chromatin organization. Our work further demonstrates that cell-free autosomal DNA cleavage is sequence dependent. The region spanning up to 10 positions on either side of the DNA cleavage site show a consistent pattern of preference for specific nucleotides. This sequence motif is present in cleavage sites localized to nucleosomal cores and linker regions but is absent in nucleosome-free mitochondrial DNA. These background signals in cell-free DNA sequencing data stem from the non-random biological cleavage of these fragments. This

  4. Derivation of transgene-free human induced pluripotent stem cells from human peripheral T cells in defined culture conditions.

    Directory of Open Access Journals (Sweden)

    Yoshikazu Kishino

    Full Text Available Recently, induced pluripotent stem cells (iPSCs were established as promising cell sources for revolutionary regenerative therapies. The initial culture system used for iPSC generation needed fetal calf serum in the culture medium and mouse embryonic fibroblast as a feeder layer, both of which could possibly transfer unknown exogenous antigens and pathogens into the iPSC population. Therefore, the development of culture systems designed to minimize such potential risks has become increasingly vital for future applications of iPSCs for clinical use. On another front, although donor cell types for generating iPSCs are wide-ranging, T cells have attracted attention as unique cell sources for iPSCs generation because T cell-derived iPSCs (TiPSCs have a unique monoclonal T cell receptor genomic rearrangement that enables their differentiation into antigen-specific T cells, which can be applied to novel immunotherapies. In the present study, we generated transgene-free human TiPSCs using a combination of activated human T cells and Sendai virus under defined culture conditions. These TiPSCs expressed pluripotent markers by quantitative PCR and immunostaining, had a normal karyotype, and were capable of differentiating into cells from all three germ layers. This method of TiPSCs generation is more suitable for the therapeutic application of iPSC technology because it lowers the risks associated with the presence of undefined, animal-derived feeder cells and serum. Therefore this work will lead to establishment of safer iPSCs and extended clinical application.

  5. Influence of free fatty acids on glucose uptake in prostate cancer cells

    International Nuclear Information System (INIS)

    Andersen, Kim Francis; Divilov, Vadim; Sevak, Kuntalkumar; Koziorowski, Jacek; Lewis, Jason S.; Pillarsetty, NagaVaraKishore

    2014-01-01

    Introduction: The study focuses on the interaction between glucose and free fatty acids (FFA) in malignant human prostate cancer cell lines by an in vitro observation of uptake of fluoro-2-deoxy-D-glucose (FDG) and acetate. Methods: Human prostate cancer cell lines (PC3, CWR22Rv1, LNCaP, and DU145) were incubated for 2 h and 24 h in glucose-containing (5.5 mM) Dulbecco’s Modified Eagle’s Medium (DMEM) with varying concentrations of the free fatty acid palmitate (0–1.0 mM). Then the cells were incubated with [ 18 F]-FDG (1 μCi/mL; 0.037 MBq/mL) in DMEM either in presence or absence of glucose and in presence of varying concentrations of palmitate for 1 h. Standardized procedures regarding cell counting and measuring for 18 F radioactivity were applied. Cell uptake studies with 14 C-1-acetate under the same conditions were performed on PC3 cells. Results: In glucose containing media there was significantly increased FDG uptake after 24 h incubation in all cell lines, except DU145, when upper physiological levels of palmitate were added. A 4-fold increase of FDG uptake in PC3 cells (15.11% vs. 3.94%/10 6 cells) was observed in media with 1.0 mM palmitate compared to media with no palmitate. The same tendency was observed in PC3 and CWR22Rv1 cells after 2 h incubation. In glucose-free media no significant differences in FDG uptake after 24 h incubation were observed. The significant differences after 2 h incubation all pointed in the direction of increased FDG uptake when palmitate was added. Acetate uptake in PC3 cells was significantly lower when palmitate was added in glucose-free DMEM. No clear tendency when comparing FDG or acetate uptake in the same media at different time points of incubation was observed. Conclusions: Our results indicate a FFA dependent metabolic boost/switch of glucose uptake in PCa, with patterns reflecting the true heterogeneity of the disease

  6. xCELLigence system for real-time label-free monitoring of growth and viability of cell lines from hematological malignancies

    Directory of Open Access Journals (Sweden)

    Martinez-Serra J

    2014-06-01

    Full Text Available Jordi Martinez-Serra,1 Antonio Gutierrez,1 Saúl Muñoz-Capó,1 María Navarro-Palou,1 Teresa Ros,1 Juan Carlos Amat,1 Bernardo Lopez,1 Toni F Marcus,1 Laura Fueyo,2 Angela G Suquia,2 Jordi Gines,3 Francisco Rubio,1 Rafael Ramos,4 Joan Besalduch11Department of Hematology, 2Department of Clinical Analysis, 3Department of Pharmacy, 4Department of Pathology, University Hospital Son Espases, Palma de Mallorca, Balearic Islands, SpainAbstract: The xCELLigence system is a new technological approach that allows the real-time cell analysis of adherent tumor cells. To date, xCELLigence has not been able to monitor the growth or cytotoxicity of nonadherent cells derived from hematological malignancies. The basis of its technology relies on the use of culture plates with gold microelectrodes located in their base. We have adapted the methodology described by others to xCELLigence, based on the pre-coating of the cell culture surface with specific substrates, some of which are known to facilitate cell adhesion in the extracellular matrix. Pre-coating of the culture plates with fibronectin, compared to laminin, collagen, or gelatin, significantly induced the adhesion of most of the leukemia/lymphoma cells assayed (Jurkat, L1236, KMH2, and K562. With a fibronectin substrate, nonadherent cells deposited in a monolayer configuration, and consequently, the cell growth and viability were robustly monitored. We further demonstrate the feasibility of xCELLigence for the real-time monitoring of the cytotoxic properties of several antineoplastic agents. In order to validate this technology, the data obtained through real-time cell analysis was compared with that obtained from using the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide method. This provides an excellent label-free tool for the screening of drug efficacy in nonadherent cells and discriminates optimal time points for further molecular analysis of cellular events associated with treatments

  7. Establishment and characterization of GSA-1, a human cell line highly susceptible to apoptosis after free-fall

    International Nuclear Information System (INIS)

    Nomura, Jun; Himeda, Jyuni; Chen, Zheng; Sugaya, Shigeru; Takahashi, Shunji; Kita, Kazuko; Ichinose, Masaharu; Suzuki, Nobuo

    2002-01-01

    The induction of apoptosis by microgravity and/or gravity-changing stress is considered to be one of the important causes of cell death, although the molecular mechanisms of the apoptotic event remain unclarified. In this study, we established a cell line,GSA-1, from ethyl methanesulfonate-treated human RSa cells. GSA-1 cells were highly susceptible to apoptosis after a free-fall; 24.4% of these cells underwent apoptosis after free-fall, compared with only 6% of the RSa cells. The apoptosis of GSA-1 cells was augmented by ultraviolet (UV, principally 254-nm wavelength) irradiation before free-fall to a greater extents than those in RSa cells. The molecular mechanisms of apoptosis included p53 and Bax proteins; the expression of nuclear p53 and cytoplasmic Bax in GSA-1 cells increased at 4 h after free-fall irrespective of irradiation. In addition, the rate of removal of cyclobutane pyrimidine dimer (CPD) in UV-irradiated GSA-1 cells was higher in cells exposed to free-fall than in those under the l-G condition. Our results suggested that in GSA-1 cells, free-fall accelerates apoptosis, and that this process is associated with the accumulation of p53 and Bax, as well as CPD removal. Thus, GSA-1 cells should be useful for investigating the mechanism of cellular response, including the induction of apoptosis under gravity-changing stress. (author)

  8. Serum-free microcarrier based production of replication deficient Influenza vaccine candidate virus lacking NS1 using Vero cells

    Directory of Open Access Journals (Sweden)

    Yan Mylene L

    2011-08-01

    Full Text Available Abstract Background Influenza virus is a major health concern that has huge impacts on the human society, and vaccination remains as one of the most effective ways to mitigate this disease. Comparing the two types of commercially available Influenza vaccine, the live attenuated virus vaccine is more cross-reactive and easier to administer than the traditional inactivated vaccines. One promising live attenuated Influenza vaccine that has completed Phase I clinical trial is deltaFLU, a deletion mutant lacking the viral Nonstructural Protein 1 (NS1 gene. As a consequence of this gene deletion, this mutant virus can only propagate effectively in cells with a deficient interferon-mediated antiviral response. To demonstrate the manufacturability of this vaccine candidate, a batch bioreactor production process using adherent Vero cells on microcarriers in commercially available animal-component free, serum-free media is described. Results Five commercially available animal-component free, serum-free media (SFM were evaluated for growth of Vero cells in agitated Cytodex 1 spinner flask microcarrier cultures. EX-CELL Vero SFM achieved the highest cell concentration of 2.6 × 10^6 cells/ml, whereas other SFM achieved about 1.2 × 10^6 cells/ml. Time points for infection between the late exponential and stationary phases of cell growth had no significant effect in the final virus titres. A virus yield of 7.6 Log10 TCID50/ml was achieved using trypsin concentration of 10 μg/ml and MOI of 0.001. The Influenza vaccine production process was scaled up to a 3 liter controlled stirred tank bioreactor to achieve a cell density of 2.7 × 10^6 cells/ml and virus titre of 8.3 Log10 TCID50/ml. Finally, the bioreactor system was tested for the production of the corresponding wild type H1N1 Influenza virus, which is conventionally used in the production of inactivated vaccine. High virus titres of up to 10 Log10 TCID50/ml were achieved. Conclusions We describe for the

  9. Efficacy of a potential trivalent vaccine based on Hc fragments of botulinum toxins A, B, and E produced in a cell-free expression system.

    Science.gov (United States)

    Zichel, R; Mimran, A; Keren, A; Barnea, A; Steinberger-Levy, I; Marcus, D; Turgeman, A; Reuveny, S

    2010-05-01

    Botulinum toxins produced by the anaerobic bacterium Clostridium botulinum are the most potent biological toxins in nature. Traditionally, people at risk are immunized with a formaldehyde-inactivated toxin complex. Second generation vaccines are based on the recombinant carboxy-terminal heavy-chain (Hc) fragment of the neurotoxin. However, the materialization of this approach is challenging, mainly due to the high AT content of clostridial genes. Herein, we present an alternative strategy in which the native genes encoding Hc proteins of botulinum toxins A, B, and E were used to express the recombinant Hc fragments in a cell-free expression system. We used the unique property of this open system to introduce different combinations of chaperone systems, protein disulfide isomerase (PDI), and reducing/oxidizing environments directly to the expression reaction. Optimized expression conditions led to increased production of soluble Hc protein, which was successfully scaled up using a continuous exchange (CE) cell-free system. Hc proteins were produced at a concentration of more than 1 mg/ml and purified by one-step Ni(+) affinity chromatography. Mice immunized with three injections containing 5 microg of any of the in vitro-expressed, alum-absorbed, Hc vaccines generated a serum enzyme-linked immunosorbent assay (ELISA) titer of 10(5) against the native toxin complex, which enabled protection against a high-dose toxin challenge (10(3) to 10(6) mouse 50% lethal dose [MsLD(50)]). Finally, immunization with a trivalent HcA, HcB, and HcE vaccine protected mice against the corresponding trivalent 10(5) MsLD(50) toxin challenge. Our results together with the latest developments in scalability of the in vitro protein expression systems offer alternative routes for the preparation of botulinum vaccine.

  10. The B-domain of factor VIII reduces cell membrane attachement to host cells in serum free conditions

    DEFF Research Database (Denmark)

    Kolind, Mille Petersen; Nørby, Peder Lisby; Flintegaard, Thomas Veje

    2010-01-01

    engineered extensively throughout the years to increase the low production yields that initially were obtained from mammalian cell cultures. The scope of this work was to investigate the interaction of rFVIII with the cell membrane surface of the producing cells in serum free medium. We wondered whether...... binding of rFVIII to the cell membrane could be a factor diminishing the production yield. We studied the contribution of the rFVIII B-domain to membrane attachment by transfecting several constructs containing increasing lengths of the B-domain into cells under serum free conditions. We found that 90......% of rFVIII is attached to the cell membrane of the producing cell when the rFVIII variant contains a short B-domain (21 aa). By increasing the length of the B-domain the membrane attached fraction can be reduced to 50% of the total expressed rFVIII. Further, our studies show that the N...

  11. Current Collecting Grids for ITO-Free Solar Cells

    DEFF Research Database (Denmark)

    Galagan, Yulia; Zimmermann, Birger; Coenen, Erica W. C.

    2012-01-01

    Indium-tin-oxide (ITO) free polymer solar cells prepared by ink jet printing a composite front electrode comprising silver grid lines and a semitransparent PEDOT:PSS conductor are demonstrated. The effect of grid line density is explored for a large series of devices and a careful modeling study...

  12. Influence of free fatty acids on glucose uptake in prostate cancer cells

    DEFF Research Database (Denmark)

    Andersen, Kim Francis; Divilov, Vadim; Sevak, Kuntalkumar

    2014-01-01

    The study focuses on the interaction between glucose and free fatty acids (FFA) in malignant human prostate cancer cell lines by an in vitro observation of uptake of fluoro-2-deoxy-d-glucose (FDG) and acetate.......The study focuses on the interaction between glucose and free fatty acids (FFA) in malignant human prostate cancer cell lines by an in vitro observation of uptake of fluoro-2-deoxy-d-glucose (FDG) and acetate....

  13. Cell-free DNA in a three-dimensional spheroid cell culture model

    DEFF Research Database (Denmark)

    Aucamp, Janine; Calitz, Carlemi; Bronkhorst, Abel J.

    2017-01-01

    Background Investigating the biological functions of cell-free DNA (cfDNA) is limited by the interference of vast numbers of putative sources and causes of DNA release into circulation. Utilization of three-dimensional (3D) spheroid cell cultures, models with characteristics closer to the in vivo...... cultures can serve as effective, simplified in vivo-simulating “closed-circuit” models since putative sources of cfDNA are limited to only the targeted cells. In addition, cfDNA can also serve as an alternative or auxiliary marker for tracking spheroid growth, development and culture stability. Biological...... significance 3D cell cultures can be used to translate “closed-circuit” in vitro model research into data that is relevant for in vivo studies and clinical applications. In turn, the utilization of cfDNA during 3D culture research can optimize sample collection without affecting the stability of the growth...

  14. Hydrogen exchange during cell-free incorporation of deuterated amino acids and an approach to its inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Tonelli, Marco; Singarapu, Kiran K. [University of Wisconsin-Madison, National Magnetic Resonance Facility at Madison (NMRFAM), Department of Biochemistry (United States); Makino, Shin-ichi; Sahu, Sarata C.; Matsubara, Yuko [University of Wisconsin-Madison, Center for Eukaryotic Structural Genomics (CESG), Department of Biochemistry (United States); Endo, Yaeta [Ehime University, Cell-Free Science and Technology Research Center (Japan); Kainosho, Masatsune [Tokyo Metropolitan University, Center for Priority Areas (Japan); Markley, John L., E-mail: markley@nmrfam.wisc.edu [University of Wisconsin-Madison, National Magnetic Resonance Facility at Madison (NMRFAM), Department of Biochemistry (United States)

    2011-12-15

    Perdeuteration, selective deuteration, and stereo array isotope labeling (SAIL) are valuable strategies for NMR studies of larger proteins and membrane proteins. To minimize scrambling of the label, it is best to use cell-free methods to prepare selectively labeled proteins. However, when proteins are prepared from deuterated amino acids by cell-free translation in H{sub 2}O, exchange reactions can lead to contamination of {sup 2}H sites by {sup 1}H from the solvent. Examination of a sample of SAIL-chlorella ubiquitin prepared by Escherichia coli cell-free synthesis revealed that exchange had occurred at several residues (mainly at Gly, Ala, Asp, Asn, Glu, and Gln). We present results from a study aimed at identifying the exchanging sites and level of exchange and at testing a strategy for minimizing {sup 1}H contamination during wheat germ cell-free translation of proteins produced from deuterated amino acids by adding known inhibitors of transaminases (1 mM aminooxyacetic acid) and glutamate synthetase (0.1 mM l-methionine sulfoximine). By using a wheat germ cell-free expression system, we produced [U-{sup 2}H, {sup 15}N]-chlorella ubiquitin without and with added inhibitors, and [U-{sup 15}N]-chlorella ubiquitin as a reference to determine the extent of deuterium incorporation. We also prepared a sample of [U-{sup 13}C, {sup 15}N]-chlorella ubiquitin, for use in assigning the sites of exchange. The added inhibitors did not reduce the protein yield and were successful in blocking hydrogen exchange at C{sup {alpha}} sites, with the exception of Gly, and at C{sup {beta}} sites of Ala. We discovered, in addition, that partial exchange occurred with or without the inhibitors at certain side-chain methyl and methylene groups: Asn-H{sup {beta}}, Asp-H{sup {beta}}, Gln-H{sup {gamma}}, Glu-H{sup {gamma}}, and Lys-H{sup {epsilon}}. The side-chain labeling pattern, in particular the mixed chiral labeling resulting from partial exchange at certain sites, should be of

  15. Efficient mRNA delivery with graphene oxide-polyethylenimine for generation of footprint-free human induced pluripotent stem cells.

    Science.gov (United States)

    Choi, Hye Yeon; Lee, Tae-Jin; Yang, Gwang-Mo; Oh, Jaesur; Won, Jihye; Han, Jihae; Jeong, Gun-Jae; Kim, Jongpil; Kim, Jin-Hoi; Kim, Byung-Soo; Cho, Ssang-Goo

    2016-08-10

    Clinical applications of induced pluripotent stem cells (iPSCs) require development of technologies for the production of "footprint-free" (gene integration-free) iPSCs, which avoid the potential risk of insertional mutagenesis in humans. Previously, several studies have shown that mRNA transfer can generate "footprint-free" iPSCs, but these studies did not use a delivery vehicle and thus repetitive daily transfection was required because of mRNA degradation. Here, we report an mRNA delivery system employing graphene oxide (GO)-polyethylenimine (PEI) complexes for the efficient generation of "footprint-free" iPSCs. GO-PEI complexes were found to be very effective for loading mRNA of reprogramming transcription factors and protection from mRNA degradation by RNase. Dynamic suspension cultures of GO-PEI/RNA complexes-treated cells dramatically increased the reprogramming efficiency and successfully generated rat and human iPSCs from adult adipose tissue-derived fibroblasts without repetitive daily transfection. The iPSCs showed all the hallmarks of pluripotent stem cells including expression of pluripotency genes, epigenetic reprogramming, and differentiation into the three germ layers. These results demonstrate that mRNA delivery using GO-PEI-RNA complexes can efficiently generate "footprint-free" iPSCs, which may advance the translation of iPSC technology into the clinical settings. Copyright © 2016. Published by Elsevier B.V.

  16. Relating Structure to Efficiency in Surfactant-Free Polymer/Fullerene Nanoparticle-Based Organic Solar Cells.

    Science.gov (United States)

    Gärtner, Stefan; Clulow, Andrew J; Howard, Ian A; Gilbert, Elliot P; Burn, Paul L; Gentle, Ian R; Colsmann, Alexander

    2017-12-13

    Nanoparticle dispersions open up an ecofriendly route toward printable organic solar cells. They can be formed from a variety of organic semiconductors by using miniemulsions that employ surfactants to stabilize the nanoparticles in dispersion and to prevent aggregation. However, whenever surfactant-based nanoparticle dispersions have been used to fabricate solar cells, the reported performances remain moderate. In contrast, solar cells from nanoparticle dispersions formed by precipitation (without surfactants) can exhibit power conversion efficiencies close to those of state-of-the-art solar cells processed from blend solutions using chlorinated solvents. In this work, we use small-angle neutron scattering measurements and transient absorption spectroscopy to investigate why surfactant-free nanoparticles give rise to efficient organic solar cells. We show that surfactant-free nanoparticles comprise a uniform distribution of small semiconductor domains, similar to that of bulk-heterojunction films formed using traditional solvent processing. This observation differs from surfactant-based miniemulsion nanoparticles that typically exhibit core-shell structures. Hence, the surfactant-free nanoparticles already possess the optimum morphology for efficient energy conversion before they are assembled into the photoactive layer of a solar cell. This structural property underpins the superior performance of the solar cells containing surfactant-free nanoparticles and is an important design criterion for future nanoparticle inks.

  17. Isolation of cell-free bacterial inclusion bodies.

    Science.gov (United States)

    Rodríguez-Carmona, Escarlata; Cano-Garrido, Olivia; Seras-Franzoso, Joaquin; Villaverde, Antonio; García-Fruitós, Elena

    2010-09-17

    Bacterial inclusion bodies are submicron protein clusters usually found in recombinant bacteria that have been traditionally considered as undesirable products from protein production processes. However, being fully biocompatible, they have been recently characterized as nanoparticulate inert materials useful as scaffolds for tissue engineering, with potentially wider applicability in biomedicine and material sciences. Current protocols for inclusion body isolation from Escherichia coli usually offer between 95 to 99% of protein recovery, what in practical terms, might imply extensive bacterial cell contamination, not compatible with the use of inclusion bodies in biological interfaces. Using an appropriate combination of chemical and mechanical cell disruption methods we have established a convenient procedure for the recovery of bacterial inclusion bodies with undetectable levels of viable cell contamination, below 10⁻¹ cfu/ml, keeping the particulate organization of these aggregates regarding size and protein folding features. The application of the developed protocol allows obtaining bacterial free inclusion bodies suitable for use in mammalian cell cultures and other biological interfaces.

  18. A novel serum-free monolayer culture for orderly hematopoietic differentiation of human pluripotent cells via mesodermal progenitors.

    Directory of Open Access Journals (Sweden)

    Akira Niwa

    Full Text Available Elucidating the in vitro differentiation of human embryonic stem (ES and induced pluripotent stem (iPS cells is important for understanding both normal and pathological hematopoietic development in vivo. For this purpose, a robust and simple hematopoietic differentiation system that can faithfully trace in vivo hematopoiesis is necessary. In this study, we established a novel serum-free monolayer culture that can trace the in vivo hematopoietic pathway from ES/iPS cells to functional definitive blood cells via mesodermal progenitors. Stepwise tuning of exogenous cytokine cocktails induced the hematopoietic mesodermal progenitors via primitive streak cells. These progenitors were then differentiated into various cell lineages depending on the hematopoietic cytokines present. Moreover, single cell deposition assay revealed that common bipotential hemoangiogenic progenitors were induced in our culture. Our system provides a new, robust, and simple method for investigating the mechanisms of mesodermal and hematopoietic differentiation.

  19. Cytotoxicity of cadmium-free quantum dots and their use in cell bioimaging.

    Science.gov (United States)

    Soenen, Stefaan J; Manshian, Bella B; Aubert, Tangi; Himmelreich, Uwe; Demeester, Jo; De Smedt, Stefaan C; Hens, Zeger; Braeckmans, Kevin

    2014-06-16

    The use of quantum dots (QDots) as bright and photostable probes for long-term fluorescence imaging is gaining more interest. Thus far, (pre)clinical use of QDots remains limited, which is primarily caused by the potential toxicity of QDots. Most QDots consist of Cd2+ ions, which are known to cause high levels of toxicity. In order to overcome this problem, several strategies have been tested, such as the generation of cadmium-free QDots. In the present study, two types of cadmium-free QDots, composed of ZnSe/ZnS (QDotZnSe) and InP/ZnS (QDotInP), were studied with respect to their cytotoxicity and cellular uptake in a variety of cell types. A multiparametric cytotoxicity approach is used, where the QDots are studied with respect to cell viability, oxidative stress, cell morphology, stem cell differentiation, and neurite outgrowth. The data reveal slight differences in uptake levels for both types of QDots (maximal for QDotZnSe), but clear differences in cytotoxicity and cell functionality effects exist, with highest toxicity for QDotZnSe. Differences between cell types and between both types of QDots can be explained by the intrinsic sensitivity of certain cell types and chemical composition of the QDots. At concentrations at which no toxic effects can be observed, the functionality of the QDots for fluorescence cell visualization is evaluated, revealing that the higher brightness of QDotZnSe overcomes most of the toxicity issues compared to that of QDotInP. Comparing the results obtained with common Cd2+-containing QDots tested under identical conditions, the importance of particle functionality is demonstrated, revealing that cadmium-free QDots tested in this study are not significantly better than Cd2+-containing QDots for long-term cell imaging and that more work needs to be performed in optimizing the brightness and surface chemistry of cadmium-free QDots for them to replace currently used Cd2+-containing QDots.

  20. Bacterial production and growth rate estimation from [3H]thymidine incorporation for attached and free-living bacteria in aquatic systems

    International Nuclear Information System (INIS)

    Iriberri, J.; Unanue, M.; Ayo, B.; Barcina, I.; Egea, L.

    1990-01-01

    Production and specific growth rates of attached and free-living bacteria were estimated in an oligotrophic marine system, La Salvaje Beach, Vizcaya, Spain, and in a freshwater system having a higher nutrient concentration, Butron River, Vizcaya, Spain. Production was calculated from [methyl- 3 H]thymidine incorporation by estimating specific conversion factors (cells or micrograms of C produced per mole of thymidine incorporated) for attached and free-living bacteria, respectively, in each system. Conversion factors were not statistically different between attached and free-living bacteria: 6.812 x 10 11 and 8.678 x 10 11 μg of C mol -1 for free-living and attached bacteria in the freshwater system, and 1.276 x 10 11 and 1.354 x 10 11 μg of C mol -1 for free-living and attached bacteria in the marine system. Therefore, use of a unique conversion factor for the mixed bacterial population is well founded. However, conversion factors were higher in the freshwater system than in the marine system. This could be due to the different tropic conditions of the two systems. Free-living bacteria contributed the most to production in the two systems (85% in the marine system and 67% in the freshwater system) because of their greater contribution to total biomass. Specific growth rates calculated from production data and biomass data were similar for attached and free-living bacteria

  1. Elimination of remaining undifferentiated induced pluripotent stem cells in the process of human cardiac cell sheet fabrication using a methionine-free culture condition.

    Science.gov (United States)

    Matsuura, Katsuhisa; Kodama, Fumiko; Sugiyama, Kasumi; Shimizu, Tatsuya; Hagiwara, Nobuhisa; Okano, Teruo

    2015-03-01

    Cardiac tissue engineering is a promising method for regenerative medicine. Although we have developed human cardiac cell sheets by integration of cell sheet-based tissue engineering and scalable bioreactor culture, the risk of contamination by induced pluripotent stem (iPS) cells in cardiac cell sheets remains unresolved. In the present study, we established a novel culture method to fabricate human cardiac cell sheets with a decreased risk of iPS cell contamination while maintaining viabilities of iPS cell-derived cells, including cardiomyocytes and fibroblasts, using a methionine-free culture condition. When cultured in the methionine-free condition, human iPS cells did not survive without feeder cells and could not proliferate or form colonies on feeder cells or in coculture with cells for cardiac cell sheet fabrication. When iPS cell-derived cells after the cardiac differentiation were transiently cultured in the methionine-free condition, gene expression of OCT3/4 and NANOG was downregulated significantly compared with that in the standard culture condition. Furthermore, in fabricated cardiac cell sheets, spontaneous and synchronous beating was observed in the whole area while maintaining or upregulating the expression of various cardiac and extracellular matrix genes. These findings suggest that human iPS cells are methionine dependent and a methionine-free culture condition for cardiac cell sheet fabrication might reduce the risk of iPS cell contamination.

  2. Enzymatic cyanide degradation by cell-free extract of Rhodococcus UKMP-5M.

    Science.gov (United States)

    Nallapan Maniyam, Maegala; Sjahrir, Fridelina; Latif Ibrahim, Abdul; Cass, Anthony E G

    2015-01-01

    The cell-free extract of locally isolated Rhodococcus UKMP-5M strain was used as an alternative to develop greener and cost effective cyanide removal technology. The present study aims to assess the viability of the cell-free extract to detoxify high concentrations of cyanide which is measured through the monitoring of protein concentration and specific cyanide-degrading activity. When cyanide-grown cells were subjected to grinding in liquid nitrogen which is relatively an inexpressive and fast cell disruption method, highest cyanide-degrading activity of 0.63 mM min(-1) mg(-1) protein was obtained in comparison to enzymatic lysis and agitation with fine glass beads. The cell-free extracts managed to degrade 80% of 20 mM KCN within 80 min and the rate of cyanide consumption increased linearly as the concentration of protein was raised. In both cases, the addition of co-factor was not required which proved to be advantageous economically. The successful formation of ammonia and formate as endproducts indicated that the degradation of cyanide by Rhodococcus UKMP-5M proceeded via the activity of cyanidase and the resulting non-toxic products are safe for disposal into the environment. Further verification with SDS-PAGE revealed that the molecular weight of the active enzyme was estimated to be 38 kDa, which is consistent with previously reported cyanidases. Thus, the utilization of cell-free extracts as an alternative to live microbial in cyanide degradation offers numerous advantageous such as the potential to tolerate and degrade higher concentration of cyanide and total reduction in the overall cost of operation since the requirement for nutrient support is irrelevant.

  3. Estimation of free-hydrocarbon recovery from dual-pump systems

    International Nuclear Information System (INIS)

    Charbeneau, R.J.

    1995-01-01

    Free-product hydrocarbon which floats on the water table may be recovered using single-pump and dual-pump systems. The factors that affect the long-term free-product recovery using dual-pump systems include the free-product thickness as measured in monitoring wells, the ground-water pumping rate, hydrocarbon density and viscosity, and the soil permeability. This paper presents a simple model for prediction of free-product recovery using dual-pump systems. The model predicts the long-term rather than short-term recovery rates, and lends itself to spreadsheet calculations on microcomputers. A particularly simple form arises for cases where the drawdown is small. An application for estimating recovery from a dual-pump system is presented, and limitations of the model are summarized

  4. Systems Modelling and the Development of Coherent Understanding of Cell Biology

    Science.gov (United States)

    Verhoeff, Roald P.; Waarlo, Arend Jan; Boersma, Kerst Th.

    2008-01-01

    This article reports on educational design research concerning a learning and teaching strategy for cell biology in upper-secondary education introducing "systems modelling" as a key competence. The strategy consists of four modelling phases in which students subsequently develop models of free-living cells, a general two-dimensional model of…

  5. Low cost label-free live cell imaging for biological samples

    Science.gov (United States)

    Seniya, C.; Towers, C. E.; Towers, D. P.

    2017-02-01

    This paper reports the progress to develop a practical phase measuring microscope offering new capabilities in terms of phase measurement accuracy and quantification of cell:cell interactions over the longer term. A novel, low cost phase interference microscope for imaging live cells (label-free) is described. The method combines the Zernike phase contrast approach with a dual mirror design to enable phase modulation between the scattered and un-scattered optical fields. Two designs are proposed and demonstrated, one of which retains the common path nature of Zernike's original microscopy concept. In both setups the phase shift is simple to control via a piezoelectric driven mirror in the back focal plane of the imaging system. The approach is significantly cheaper to implement than those based on spatial light modulators (SLM) at approximately 20% of the cost. A quantitative assessment of the performance of a set of phase shifting algorithms is also presented, specifically with regard to broad bandwidth illumination in phase contrast microscopy. The simulation results show that the phase measurement accuracy is strongly dependent on the algorithm selected and the optical path difference in the sample.

  6. Failure analysis in ITO-free all-solution processed organic solar cells

    NARCIS (Netherlands)

    Galagan, Y.; Eggenhuisen, T.M.; Coenen, M.J.J.; Biezemans, A.F.K.V.; Verhees, W.J.H.; Veenstra, S.C.; Groen, W.A.; Andriessen, R.; Janssen, R.A.J.

    2015-01-01

    In this paper we discuss a problem-solving methodology and present guidance for troubleshooting defects in ITO-free all-solution processed organic solar cells with an inverted cell architecture. A systematic approach for identifying the main causes of failures in devices is presented. Comprehensive

  7. Biogrid--a microfluidic device for large-scale enzyme-free dissociation of stem cell aggregates.

    Science.gov (United States)

    Wallman, Lars; Åkesson, Elisabet; Ceric, Dario; Andersson, Per Henrik; Day, Kelly; Hovatta, Outi; Falci, Scott; Laurell, Thomas; Sundström, Erik

    2011-10-07

    Culturing stem cells as free-floating aggregates in suspension facilitates large-scale production of cells in closed systems, for clinical use. To comply with GMP standards, the use of substances such as proteolytic enzymes should be avoided. Instead of enzymatic dissociation, the growing cell aggregates may be mechanically cut at passage, but available methods are not compatible with large-scale cell production and hence translation into the clinic becomes a severe bottle-neck. We have developed the Biogrid device, which consists of an array of micrometerscale knife edges, micro-fabricated in silicon, and a manifold in which the microgrid is placed across the central fluid channel. By connecting one side of the Biogrid to a syringe or a pump and the other side to the cell culture, the culture medium with suspended cell aggregates can be aspirated, forcing the aggregates through the microgrid, and ejected back to the cell culture container. Large aggregates are thereby dissociated into smaller fragments while small aggregates pass through the microgrid unaffected. As proof-of-concept, we demonstrate that the Biogrid device can be successfully used for repeated passage of human neural stem/progenitor cells cultured as so-called neurospheres, as well as for passage of suspension cultures of human embryonic stem cells. We also show that human neural stem/progenitor cells tolerate transient pressure changes far exceeding those that will occur in a fluidic system incorporating the Biogrid microgrids. Thus, by using the Biogrid device it is possible to mechanically passage large quantities of cells in suspension cultures in closed fluidic systems, without the use of proteolytic enzymes.

  8. A phosphorus-free anolyte to enhance coulombic efficiency of microbial fuel cells

    Science.gov (United States)

    Tang, Xinhua; Li, Haoran; Du, Zhuwei; Ng, How Yong

    2014-12-01

    In this study, a phosphorus-free anolyte is prepared by using bicarbonate to replace phosphate buffer for application in two chamber microbial fuel cells (MFCs). Optical density test and Bradford protein assay shows that this phosphorus-free anolyte effectively inhibits the growth and reproduction of microorganisms suspended in the solution and greatly reduces the suspended cell mass. As a result, it considerably enhances the coulombic efficiency (CE) of MFCs. When the acetate concentration is 11 mM, the CE of the MFC using the pH 7 phosphate-containing anolyte is 9.7% and the CE with the pH 8.3 phosphate-containing anolyte is 9.1%, while the CE of the MFC using the phosphorus-free anolyte (pH 8.3) achieves 26.6%. This study demonstrates that this phosphorus-free anolyte holds the potential to enhance the feasibility for practical applications of MFCs.

  9. Value of urinary topoisomerase-IIA cell-free DNA for diagnosis of bladder cancer.

    Science.gov (United States)

    Kim, Ye-Hwan; Yan, Chunri; Lee, Il-Seok; Piao, Xuan-Mei; Byun, Young Joon; Jeong, Pildu; Kim, Won Tae; Yun, Seok-Joong; Kim, Wun-Jae

    2016-03-01

    Topoisomerase-II alpha (TopoIIA ), a DNA gyrase isoform that plays an important role in the cell cycle, is present in normal tissues and various human cancers, and can show altered expression in both. The aim of the current study was to examine the value of urinary TopoIIA cell-free DNA as a noninvasive diagnosis of bladder cancer (BC). Two patient cohorts were examined. Cohort 1 (73 BC patients and seven controls) provided bladder tissue samples, whereas cohort 2 (83 BC patients, 54 nonmalignant hematuric patients, and 61 normal controls) provided urine samples. Real-time quantitative polymerase chain reaction was used to measure expression of TopoIIA mRNA in tissues and TopoIIA cell-free DNA in urine samples. The results showed that expression of TopoIIA mRNA in BC tissues was significantly higher than that in noncancer control tissues (pbladder cancer (MIBC) when compared with nonmuscle invasive bladder cancer (NMIBC) (p=0.002). Receiver operating characteristics (ROC) curve analysis was performed to examine the sensitivity/specificity of urinary TopoIIA cell-free DNA for diagnosing BC, NMIBC, and MIBC. The areas under the ROC curve for BC, NMIBC, and MIBC were 0.741, 0.701, and 0.838, respectively. In summary, the results of this study provide evidence that cell-free TopoIIA DNA may be a potential biomarker for BC.

  10. Adult T-cell leukemia-associated antigen (ATLA): detection of a glycoprotein in cell- and virus-free supernatant.

    Science.gov (United States)

    Yamamoto, N; Schneider, J; Hinuma, Y; Hunsmann, G

    1982-01-01

    A glycoprotein of an apparent molecular mass of 46,000, gp 46, was enriched by affinity chromatography from the virus- and cell-free culture medium of adult T-cell leukemia virus (ATLV) infected cells. gp 46 was specifically precipitated with sera from patients with adult T-cell leukemia associated antigen (ATLA). Thus, gp 46 is a novel component of the ATLA antigen complex.

  11. Direct Reprogramming of Human Bone Marrow Stromal Cells into Functional Renal Cells Using Cell-free Extracts

    Directory of Open Access Journals (Sweden)

    Evangelia Papadimou

    2015-04-01

    Full Text Available The application of cell-based therapies in regenerative medicine is gaining recognition. Here, we show that human bone marrow stromal cells (BMSCs, also known as bone-marrow-derived mesenchymal cells, can be reprogrammed into renal proximal tubular-like epithelial cells using cell-free extracts. Streptolysin-O-permeabilized BMSCs exposed to HK2-cell extracts underwent morphological changes—formation of “domes” and tubule-like structures—and acquired epithelial functional properties such as transepithelial-resistance, albumin-binding, and uptake and specific markers E-cadherin and aquaporin-1. Transmission electron microscopy revealed the presence of brush border microvilli and tight intercellular contacts. RNA sequencing showed tubular epithelial transcript abundance and revealed the upregulation of components of the EGFR pathway. Reprogrammed BMSCs integrated into self-forming kidney tissue and formed tubular structures. Reprogrammed BMSCs infused in immunodeficient mice with cisplatin-induced acute kidney injury engrafted into proximal tubuli, reduced renal injury and improved function. Thus, reprogrammed BMSCs are a promising cell resource for future cell therapy.

  12. Dynamic quantitative analysis of adherent cell cultures by means of lens-free video microscopy

    Science.gov (United States)

    Allier, C.; Vincent, R.; Navarro, F.; Menneteau, M.; Ghenim, L.; Gidrol, X.; Bordy, T.; Hervé, L.; Cioni, O.; Bardin, S.; Bornens, M.; Usson, Y.; Morales, S.

    2018-02-01

    We present our implementation of lens-free video microscopy setup for the monitoring of adherent cell cultures. We use a multi-wavelength LED illumination together with a dedicated holographic reconstruction algorithm that allows for an efficient removal of twin images from the reconstructed phase image for densities up to those of confluent cell cultures (>500 cells/mm2). We thereby demonstrate that lens-free video microscopy, with a large field of view ( 30 mm2) can enable us to capture the images of thousands of cells simultaneously and directly inside the incubator. It is then possible to trace and quantify single cells along several cell cycles. We thus prove that lens-free microscopy is a quantitative phase imaging technique enabling estimation of several metrics at the single cell level as a function of time, for example the area, dry mass, maximum thickness, major axis length and aspect ratio of each cell. Combined with cell tracking, it is then possible to extract important parameters such as the initial cell dry mass (just after cell division), the final cell dry mass (just before cell division), the average cell growth rate, and the cell cycle duration. As an example, we discuss the monitoring of a HeLa cell cultures which provided us with a data-set featuring more than 10 000 cell cycle tracks and more than 2x106 cell morphological measurements in a single time-lapse.

  13. Systems and methods for free space optical communication

    Science.gov (United States)

    Harper, Warren W [Benton City, WA; Aker, Pamela M [Richland, WA; Pratt, Richard M [Richland, WA

    2011-05-10

    Free space optical communication methods and systems, according to various aspects are described. The methods and systems are characterized by transmission of data through free space with a digitized optical signal acquired using wavelength modulation, and by discrimination between bit states in the digitized optical signal using a spectroscopic absorption feature of a chemical substance.

  14. Gastric washing by distilled water can reduce free gastric cancer cells exfoliated into the stomach lumen.

    Science.gov (United States)

    Ohki, Atsuko; Abe, Nobutsugu; Yoshimoto, Eri; Hashimoto, Yoshikazu; Takeuchi, Hirohisa; Nagao, Gen; Masaki, Tadahiko; Mori, Toshiyuki; Ohkura, Yasuo; Sugiyama, Masanori

    2018-04-25

    Intragastric free cancer cells in patients with gastric cancer have rarely been studied. The purpose of this study was to investigate the detection rate of intragastric free cancer cells in gastric washes using two types of solutions during endoscopic examination. We further clarified risk factors affecting the presence of exfoliated free cancer cells. A total of 175 patients with gastric cancer were enrolled. Lactated Ringer's solution (N = 89) or distilled water (DW; N = 86) via endoscopic working channel was sprayed onto the tumor surface, and the resultant fluid was collected for cytological examination. We compared the cancer-cell positivity rate between the two (Ringer and DW) groups. We also tested the correlation between cancer-cell positivity and clinicopathological factors in the Ringer group to identify risk factors for the presence of exfoliated cancer cells. The cancer-cell positivity rate was significantly higher in the Ringer group than that in the DW group (58 vs 6%). Cytomorphology in the Ringer group was well maintained, but not in the DW group. The larger tumor size (≥ 20 mm) and positive lymphatic involvement were significant risk factors of exfoliated free cancer cells. Cancer cells can be highly exfoliated from the tumor surface into the gastric lumen by endoscopic irrigation in large gastric cancer with lymphatic involvement. Gastric washing by DW can lead to cytoclasis of free cancer cells; therefore, it may minimize the possibility of cancer-cell seeding in procedures carrying potential risks of tumor-cell seeding upon transluminal communication, such as endoscopic full-thickness resection and laparoscopy-endoscopy cooperative surgery.

  15. Cell-free soluble-phase radioimmunoassay for Thy-1 antigen

    Energy Technology Data Exchange (ETDEWEB)

    Shalev, A.; Zuckerman, F. (Ben-Gurion Univ. of the Negev, Beersheba (Israel))

    1983-12-01

    A cell-free, soluble-phase, radioimmunoassay has been developed for Thy-1 antigen. The method is based on immunoprecipitation of radiolabelled Thy-1 molecules with specific antibodies, antiimmunoglobulin serum and polyethyleneglycol (PEG). The method can be used with convenience to screen for the presence of Thy-1 in various fluids as well as on cell surfaces for qualitative or quantitative purposes. Presence of antibodies or autoantibodies against Thy-1 can also be detected specifically. Evidence that the dog, carp, hamster and goldfish carry Thy-1-like molecules on neuronal (brain) cells is demonstrated by this method.

  16. Surface free energy for systems with integrable boundary conditions

    International Nuclear Information System (INIS)

    Goehmann, Frank; Bortz, Michael; Frahm, Holger

    2005-01-01

    The surface free energy is the difference between the free energies for a system with open boundary conditions and the same system with periodic boundary conditions. We use the quantum transfer matrix formalism to express the surface free energy in the thermodynamic limit of systems with integrable boundary conditions as a matrix element of certain projection operators. Specializing to the XXZ spin-1/2 chain we introduce a novel 'finite temperature boundary operator' which characterizes the thermodynamical properties of surfaces related to integrable boundary conditions

  17. Generation of megakaryocytic progenitors from human embryonic stem cells in a feeder- and serum-free medium.

    Directory of Open Access Journals (Sweden)

    Marjorie Pick

    Full Text Available BACKGROUND: The production of human platelets from embryonic stem cells in a defined culture system is a prerequisite for the generation of platelets for therapeutic use. As an important step towards this goal, we report the differentiation of human embryonic stem cells (hESCs towards the megakaryocyte (Mk lineage using a 'spin embryoid body' method in serum-free differentiation medium. METHODOLOGY AND PRINCIPAL FINDINGS: Immunophenotypic analyses of differentiating hESC identified a subpopulation of cells expressing high levels of CD41a that expressed other markers associated with the Mk lineage, including CD110, CD42b and CD61. Differentiated cells were sorted on the basis of their expression of CD41a, CD34 and CD45 and assessed for Mk colony formation, expression of myeloid and Mk genes and ability to endoreplicate DNA. In a collagen-based colony assay, the CD41a⁺ cells sorted from these differentiation cultures produced 100-800 Mk progenitors at day 13 and 25-160 Mk progenitors at day 20 of differentiation per 100,000 cells assayed. Differentiated Mk cells produced platelet-like particles which expressed CD42b and were activated by ADP, similar to platelets generated from precursors in cord blood. These studies were complemented by real time PCR analyses showing that subsets of cells enriched for CD41a⁺ Mk precursors expressed high levels of Mk associated genes such as PF4 and MPL. Conversely, high levels of myeloid and erythroid related transcripts, such as GATA1, TAL1/SCL and PU.1, were detected in sorted fractions containing CD34⁺ and CD45⁺ cells. CONCLUSIONS: We describe a serum- and feeder-free culture system that enabled the generation of Mk progenitors from human embryonic stem cells. These cells formed colonies that included differentiated Mks that fragmented to form platelet-like particles. This protocol represents an important step towards the generation of human platelets for therapeutic use.

  18. Poking cells for efficient vector-free intracellular delivery

    Science.gov (United States)

    Wang, Ying; Yang, Yang; Yan, Li; Kwok, So Ying; Li, Wei; Wang, Zhigang; Zhu, Xiaoyue; Zhu, Guangyu; Zhang, Wenjun; Chen, Xianfeng; Shi, Peng

    2014-07-01

    Techniques for introducing foreign molecules and materials into living cells are of great value in cell biology research. A major barrier for intracellular delivery is to cross the cell membrane. Here we demonstrate a novel platform utilizing diamond nanoneedle arrays to facilitate efficient vector-free cytosolic delivery. Using our technique, cellular membrane is deformed by an array of nanoneedles with a force on the order of a few nanonewtons. We show that this technique is applicable to deliver a broad range of molecules and materials into different types of cells, including primary neurons in adherent culture. Especially, for delivering plasmid DNAs into neurons, our technique produces at least eightfold improvement (~45% versus ~1-5%) in transfection efficiency with a dramatically shorter experimental protocol, when compared with the commonly used lipofection approach. It is anticipated that our technique will greatly benefit basic research in cell biology and also a wide variety of clinical applications.

  19. Activity targets for nanostructured platinum-group-metal-free catalysts in hydroxide exchange membrane fuel cells

    Science.gov (United States)

    Setzler, Brian P.; Zhuang, Zhongbin; Wittkopf, Jarrid A.; Yan, Yushan

    2016-12-01

    Fuel cells are the zero-emission automotive power source that best preserves the advantages of gasoline automobiles: low upfront cost, long driving range and fast refuelling. To make fuel-cell cars a reality, the US Department of Energy has set a fuel cell system cost target of US$30 kW-1 in the long-term, which equates to US$2,400 per vehicle, excluding several major powertrain components (in comparison, a basic, but complete, internal combustion engine system costs approximately US$3,000). To date, most research for automotive applications has focused on proton exchange membrane fuel cells (PEMFCs), because these systems have demonstrated the highest power density. Recently, however, an alternative technology, hydroxide exchange membrane fuel cells (HEMFCs), has gained significant attention, because of the possibility to use stable platinum-group-metal-free catalysts, with inherent, long-term cost advantages. In this Perspective, we discuss the cost profile of PEMFCs and the advantages offered by HEMFCs. In particular, we discuss catalyst development needs for HEMFCs and set catalyst activity targets to achieve performance parity with state-of-the-art automotive PEMFCs. Meeting these targets requires careful optimization of nanostructures to pack high surface areas into a small volume, while maintaining high area-specific activity and favourable pore-transport properties.

  20. Efficient production of isotopically labeled proteins by cell-free synthesis: A practical protocol

    Energy Technology Data Exchange (ETDEWEB)

    Torizawa, Takuya; Shimizu, Masato [Crest, Jst (Japan); Taoka, Masato [Tokyo Metropolitan University, Graduate School of Science (Japan); Miyano, Hiroshi [Ajinomoto Co., Inc. Institute of Life Sciences (Japan); Kainosho, Masatsune [Crest, Jst (Japan)], E-mail: kainosho@nmr.chem.metro-u.ac.jp

    2004-11-15

    We provide detailed descriptions of our refined protocols for the cell-free production of labeled protein samples for NMR spectroscopy. These methods are efficient and overcome two critical problems associated with the use of conventional Escherichia coli extract systems. Endogenous amino acids normally present in E. coli S30 extracts dilute the added labeled amino acids and degrade the quality of NMR spectra of the target protein. This problem was solved by altering the protocol used in preparing the S30 extract so as to minimize the content of endogenous amino acids. The second problem encountered in conventional E. coli cell-free protein production is non-uniformity in the N-terminus of the target protein, which can complicate the NMR spectra. This problem was solved by adding a DNA sequence to the construct that codes for a cleavable N-terminal peptide tag. Addition of the tag serves to increase the yield of the protein as well as to ensure a homogeneous protein product following tag cleavage. We illustrate the method by describing its stepwise application to the production of calmodulin samples with different stable isotope labeling patterns for NMR analysis.

  1. Efficient production of isotopically labeled proteins by cell-free synthesis: A practical protocol

    International Nuclear Information System (INIS)

    Torizawa, Takuya; Shimizu, Masato; Taoka, Masato; Miyano, Hiroshi; Kainosho, Masatsune

    2004-01-01

    We provide detailed descriptions of our refined protocols for the cell-free production of labeled protein samples for NMR spectroscopy. These methods are efficient and overcome two critical problems associated with the use of conventional Escherichia coli extract systems. Endogenous amino acids normally present in E. coli S30 extracts dilute the added labeled amino acids and degrade the quality of NMR spectra of the target protein. This problem was solved by altering the protocol used in preparing the S30 extract so as to minimize the content of endogenous amino acids. The second problem encountered in conventional E. coli cell-free protein production is non-uniformity in the N-terminus of the target protein, which can complicate the NMR spectra. This problem was solved by adding a DNA sequence to the construct that codes for a cleavable N-terminal peptide tag. Addition of the tag serves to increase the yield of the protein as well as to ensure a homogeneous protein product following tag cleavage. We illustrate the method by describing its stepwise application to the production of calmodulin samples with different stable isotope labeling patterns for NMR analysis

  2. Antioxidant Activity of Ixora parviflora in a Cell/Cell-Free System and in UV-Exposed Human Fibroblasts

    Directory of Open Access Journals (Sweden)

    Hsiu-Mei Chiang

    2011-07-01

    Full Text Available Polyphenols and flavonoids possess a variety of biological activities including antioxidant and anti-tumor activities. Ixora parviflora is a member of the flavonoid-rich Rubiaceae family of flowering plants and used as folk medicine in India. The aim of this study was to investigate the antioxidant activity of Ixora parviflora extract (IPE in a cell-free system and erythrocytes, and the ability of IPE to inhibit reactive oxygen species (ROS generation in human fibroblasts (Hs68 after ultraviolet (UV exposure. Various in vitro antioxidant assays were employed in this study. The extraction yield of IPE was 17.4 ± 3.9%, the total phenolic content of IPE was 26.2 μg gallic acid equivalent (GAE/mg leaves dry weight and the total flavonoids content was 54.2 ± 4.4 μg quercetin equvalent (QE/mg extract. The content of chlorogenic acid was 9.7 ± 1.2 mg/g extract. IPE at 1000 μg/mL exhibited a reducing capacity of 90.5 ± 0.6%, a 1,1-diphenyl-2-picrylhydrazy (DPPH radical scavenging activity of 96.0 ± 0.4%, a ferrous chelating activity of 72.2 ± 3.5%, a hydroxyl radical scavenging activity of 96.8 ± 1.4%, and a hydrogen peroxide scavenging activity of 99.5 ± 3.3%. IPE at 500 μg/mL also possessed inhibitory activity against 2,2'-azobis (2-methylpropionamidine dihydrochloride (AAPH-induced hemolysis of erythrocytes (89.4 ± 1.8% and resulted in a 52.9% reduction in ROS generation in UV-exposed fibroblasts. According to our findings, IPE is a potent antioxidant and a potential anti-photoaging agent.

  3. Release of endothelial cell lipoprotein lipase by plasma lipoproteins and free fatty acids

    International Nuclear Information System (INIS)

    Saxena, U.; Witte, L.D.; Goldberg, I.J.

    1989-01-01

    Lipoprotein lipase (LPL) bound to the lumenal surface of vascular endothelial cells is responsible for the hydrolysis of triglycerides in plasma lipoproteins. Studies were performed to investigate whether human plasma lipoproteins and/or free fatty acids would release LPL which was bound to endothelial cells. Purified bovine milk LPL was incubated with cultured porcine aortic endothelial cells resulting in the association of enzyme activity with the cells. When the cells were then incubated with media containing chylomicrons or very low density lipoproteins (VLDL), a concentration-dependent decrease in the cell-associated LPL enzymatic activity was observed. In contrast, incubation with media containing low density lipoproteins or high density lipoproteins produced a much smaller decrease in the cell-associated enzymatic activity. The addition of increasing molar ratios of oleic acid:bovine serum albumin to the media also reduced enzyme activity associated with the endothelial cells. To determine whether the decrease in LPL activity was due to release of the enzyme from the cells or inactivation of the enzyme, studies were performed utilizing radioiodinated bovine LPL. Radiolabeled LPL protein was released from endothelial cells by chylomicrons, VLDL, and by free fatty acids (i.e. oleic acid bound to bovine serum albumin). The release of radiolabeled LPL by VLDL correlated with the generation of free fatty acids from the hydrolysis of VLDL triglyceride by LPL bound to the cells. Inhibition of LPL enzymatic activity by use of a specific monoclonal antibody, reduced the extent of release of 125 I-LPL from the endothelial cells by the added VLDL. These results demonstrated that LPL enzymatic activity and protein were removed from endothelial cells by triglyceride-rich lipoproteins (chylomicrons and VLDL) and oleic acid

  4. Label-free imaging of arterial cells and extracellular matrix using a multimodal CARS microscope

    Science.gov (United States)

    Wang, Han-Wei; Le, Thuc T.; Cheng, Ji-Xin

    2008-04-01

    A multimodal nonlinear optical imaging system that integrates coherent anti-Stokes Raman scattering (CARS), sum-frequency generation (SFG), and two-photon excitation fluorescence (TPEF) on the same platform was developed and applied to visualize single cells and extracellular matrix in fresh carotid arteries. CARS signals arising from CH 2-rich membranes allowed visualization of endothelial cells and smooth muscle cells of the arterial wall. Additionally, CARS microscopy allowed vibrational imaging of elastin and collagen fibrils which are also rich in CH 2 bonds. The extracellular matrix organization was further confirmed by TPEF signals arising from elastin's autofluorescence and SFG signals arising from collagen fibrils' non-centrosymmetric structure. Label-free imaging of significant components of arterial tissues suggests the potential application of multimodal nonlinear optical microscopy to monitor onset and progression of arterial diseases.

  5. Patient-specific cardiovascular progenitor cells derived from integration-free induced pluripotent stem cells for vascular tissue regeneration.

    Science.gov (United States)

    Hu, Jiang; Wang, Yongyu; Jiao, Jiao; Liu, Zhongning; Zhao, Chao; Zhou, Zhou; Zhang, Zhanpeng; Forde, Kaitlynn; Wang, Lunchang; Wang, Jiangang; Baylink, David J; Zhang, Xiao-Bing; Gao, Shaorong; Yang, Bo; Chen, Y Eugene; Ma, Peter X

    2015-12-01

    Tissue-engineered blood vessels (TEBVs) are promising in regenerating a live vascular replacement. However, the vascular cell source is limited, and it is crucial to develop a scaffold that accommodates new type of vascular progenitor cells and facilitates in vivo lineage specification of the cells into functional vascular smooth muscle cells (VSMCs) to regenerate vascular tissue. In the present study, integration-free human induced pluripotent stem cells (hiPSCs) were established from patient peripheral blood mononuclear cells through episomal vector nucleofection of reprogramming factors. The established hiPSCs were then induced into mesoderm-originated cardiovascular progenitor cells (CVPCs) with a highly efficient directed lineage specification method. The derived CVPCs were demonstrated to be able to differentiate into functional VSMCs. Subcutaneous implantation of CVPCs seeded on macroporous nanofibrous poly(l-lactide) scaffolds led to in vivo VSMC lineage specification and matrix deposition inside the scaffolds. In summary, we established integration-free patient-specific hiPSCs from peripheral blood mononuclear cells, derived CVPCs through directed lineage specification, and developed an advanced scaffold for these progenitor cells to further differentiate in vivo into VSMCs and regenerate vascular tissue in a subcutaneous implantation model. This study has established an efficient patient-specific approach towards in vivo regeneration of vascular tissue. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. 3D+time acquisitions of 3D cell culture by means of lens-free tomographic microscopy

    Science.gov (United States)

    Berdeu, Anthony; Laperrousaz, Bastien; Bordy, Thomas; Morales, S.; Gidrol, Xavier; Picollet-D'hahan, Nathalie; Allier, Cédric

    2018-02-01

    We propose a three-dimensional (3D) imaging platform based on lens-free microscopy to perform multi-angle acquisitions on 3D cell cultures embedded in extracellular matrix (ECM). We developed algorithms based on the Fourier diffraction theorem to perform fully 3D reconstructions of biological samples and we adapted the lens-free microscope to incubator conditions. Here we demonstrate for the first time, 3D+time lens-free acquisitions of 3D cell culture over 8 days directly into the incubator. The 3D reconstructed volume is as large as 5 mm3 and provides a unique way to observe in the same 3D cell culture experiment multiple cell migration strategies. Namely, in a 3D cell culture of prostate epithelial cells embedded within a Matrigel® matrix, we are able to distinguish single cell 'leaders', migration of cell clusters, migration of large aggregates of cells, and also close-gap and large-scale branching. In addition, we observe long-scale 3D deformations of the ECM that modify the geometry of the 3D cell culture. Interestingly, we also observed the opposite, i.e. we found that large aggregates of cells may deform the ECM by generating traction forces over very long distances. In sum we put forward a novel 3D lens-free microscopy tomographic technique to study the single and collective cell migrations, the cell-to-cell interactions and the cell-to-matrix interactions.

  7. Preparation of nano-biomaterials with Leptolyngbia foveolarum and heavy metal biosorption by free and immobilized algal cells

    International Nuclear Information System (INIS)

    Toncheva-Panova, T.; Pouneva, I.; Sholeva, M.; Chernev, G.

    2010-01-01

    Using the sol-gel procedure nano-biomaterials with incorporation of Leptolyngbia foveolarum in the silica matrix were manufactured. The immobilization of algal cells was confirmed with Scanning Electron Microscopy (SEM) investigations and photos. Observation of nano-biomaterials with Atomic Force Microscopy (AFM) shows nanostructure with well-defined nanounits and their aggregates. The potential of the Antarctic isolate L. foveolarum for sorption of Cu 2+ and Cd 2+ was studied by incubation of free algal cells and those immobilized in nano-biomaterials in the salts solutions of the two heavy metals. The rest of the heavy metal was determined with inductively coupled plasma atomic emission spectrometer (ICP-AES). It was established that the heavy metal biosorption capacity demonstrated by the free Leptolyngbia cells was retained after their incorporation in the nano-matrices. Free cells as well as embedded in silica nano-matrix sequestered the two heavy metals with greater affinity for copper. The highest binding capacity, 76% of the initial Cu 2+ concentration possessed nano-biomaterials with incorporated vegetative L. foveolarum cells, compared to 68% of free cells. For cadmium the degree of biosorption was lower - 35% by free cells and 30.2% by those incorporated in the biocer. (authors)

  8. Optimization of cell line development in the GS-CHO expression system using a high-throughput, single cell-based clone selection system.

    Science.gov (United States)

    Nakamura, Tsuyoshi; Omasa, Takeshi

    2015-09-01

    Therapeutic antibodies are commonly produced by high-expressing, clonal and recombinant Chinese hamster ovary (CHO) cell lines. Currently, CHO cells dominate as a commercial production host because of their ease of use, established regulatory track record, and safety profile. CHO-K1SV is a suspension, protein-free-adapted CHO-K1-derived cell line employing the glutamine synthetase (GS) gene expression system (GS-CHO expression system). The selection of high-producing mammalian cell lines is a crucial step in process development for the production of therapeutic antibodies. In general, cloning by the limiting dilution method is used to isolate high-producing monoclonal CHO cells. However, the limiting dilution method is time consuming and has a low probability of monoclonality. To minimize the duration and increase the probability of obtaining high-producing clones with high monoclonality, an automated single cell-based clone selector, the ClonePix FL system, is available. In this study, we applied the high-throughput ClonePix FL system for cell line development using CHO-K1SV cells and investigated efficient conditions for single cell-based clone selection. CHO-K1SV cell growth at the pre-picking stage was improved by optimizing the formulation of semi-solid medium. The efficiency of picking and cell growth at the post-picking stage was improved by optimization of the plating time without decreasing the diversity of clones. The conditions for selection, including the medium formulation, were the most important factors for the single cell-based clone selection system to construct a high-producing CHO cell line. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  9. Free radical generation from an aniline derivative in HepG2 cells: a possible captodative effect.

    Science.gov (United States)

    Horinouchi, Yuya; Summers, Fiona A; Ehrenshaft, Marilyn; Mason, Ronald P

    2015-01-01

    Xenobiotic metabolism can induce the generation of protein radicals, which are believed to play an important role in the toxicity of chemicals and drugs. It is therefore important to identify chemical structures capable of inducing macromolecular free radical formation in living cells. In this study, we evaluated the ability of four structurally related environmental chemicals, aniline, nitrosobenzene, N,N-dimethylaniline, and N,N-dimethyl-4-nitrosoaniline (DMNA), to induce free radicals and cellular damage in the hepatoma cell line HepG2. Cytotoxicity was assessed using lactate dehydrogenase assays, and morphological changes were observed using phase contrast microscopy. Protein free radicals were detected by immuno-spin trapping using in-cell western experiments and confocal microscopy to determine the subcellular locale of free radical generation. DMNA induced free radical generation, lactate dehydrogenase release, and morphological changes in HepG2 cells, whereas aniline, nitrosobenzene, N,N-dimethylaniline did not. Confocal microscopy showed that DMNA induced free radical generation mainly in the cytosol. Preincubation of HepG2 cells with N-acetylcysteine and 2,2'-dipyridyl significantly prevented free radical generation on subsequent incubation with DMNA, whereas preincubation with apocynin and dimethyl sulfoxide had no effect. These results suggest that DMNA is metabolized to reactive free radicals capable of generating protein radicals which may play a critical role in DMNA toxicity. We propose that the captodative effect, the combined action of the electron-releasing dimethylamine substituent, and the electron-withdrawing nitroso substituent, leads to a thermodynamically stabilized radical, facilitating enhanced protein radical formation by DMNA. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Production and characterization of high-titer serum-free cell culture grown hepatitis C virus particles of genotype 1-6

    DEFF Research Database (Denmark)

    Mathiesen, Christian K; Jensen, Tanja B; Prentoe, Jannick

    2014-01-01

    Recently, cell culture systems producing hepatitis C virus particles (HCVcc) were developed. Establishment of serum-free culture conditions is expected to facilitate development of a whole-virus inactivated HCV vaccine. We describe generation of genotype 1-6 serum-free HCVcc (sf-HCVcc) from Huh7.......5 hepatoma cells cultured in adenovirus expression medium. Compared to HCVcc, sf-HCVcc showed 0.6-2.1 log10 higher infectivity titers (4.7-6.2 log10 Focus Forming Units/mL), possibly due to increased release and specific infectivity of sf-HCVcc. In contrast to HCVcc, sf-HCVcc had a homogeneous single......-peak density profile. Entry of sf-HCVcc depended on HCV co-receptors CD81, LDLr, and SR-BI, and clathrin-mediated endocytosis. HCVcc and sf-HCVcc were neutralized similarly by chronic-phase patient sera and by human monoclonal antibodies targeting conformational epitopes. Thus, we developed serum-free culture...

  11. Dopant-Free and Carrier-Selective Heterocontacts for Silicon Solar Cells: Recent Advances and Perspectives.

    Science.gov (United States)

    Gao, Pingqi; Yang, Zhenhai; He, Jian; Yu, Jing; Liu, Peipei; Zhu, Juye; Ge, Ziyi; Ye, Jichun

    2018-03-01

    By combining the most successful heterojunctions (HJ) with interdigitated back contacts, crystalline silicon (c-Si) solar cells (SCs) have recently demonstrated a record efficiency of 26.6%. However, such SCs still introduce optical/electrical losses and technological issues due to parasitic absorption/Auger recombination inherent to the doped films and the complex process of integrating discrete p + - and n + -HJ contacts. These issues have motivated the search for alternative new functional materials and simplified deposition technologies, whereby carrier-selective contacts (CSCs) can be formed directly with c-Si substrates, and thereafter form IBC cells, via a dopant-free method. Screening and modifying CSC materials in a wider context is beneficial for building dopant-free HJ contacts with better performance, shedding new light on the relatively mature Si photovoltaic field. In this review, a significant number of achievements in two representative dopant-free hole-selective CSCs, i.e . , poly(3,4-ethylene dioxythiophene):poly(styrenesulfonate)/Si and transition metal oxides/Si, have been systemically presented and surveyed. The focus herein is on the latest advances in hole-selective materials modification, interfacial passivation, contact resistivity, light-trapping structure and device architecture design, etc. By analyzing the structure-property relationships of hole-selective materials and assessing their electrical transport properties, promising functional materials as well as important design concepts for such CSCs toward high-performance SCs have been highlighted.

  12. Secretory vesicles in live cells are not free-floating but tethered to filamentous structures: A study using photonic force microscopy

    International Nuclear Information System (INIS)

    Abu-Hamdah, Rania; Cho, Won Jin; Hoerber, J.K.H.; Jena, Bhanu P.

    2006-01-01

    It is well established that actin and microtubule cytoskeletal systems are involved in organelle transport and membrane trafficking in cells. This is also true for the transport of secretory vesicles in neuroendocrine cells and neurons. It was however unclear whether secretory vesicles remain free-floating, only to associate with such cytoskeletal systems when needing transport. This hypothesis was tested using live pancreatic acinar cells in physiological buffer solutions, using the photonic force microscope (PFM). When membrane-bound secretory vesicles (0.2-1.2 μm in diameter) in live pancreatic acinar cells were trapped at the laser focus of the PFM and pulled, they were all found tethered to filamentous structures. Mild exposure of cells to nocodazole and cytochalasin B, disrupts the tether. Immunoblot analysis of isolated secretory vesicles, further demonstrated the association of actin, myosin V, and kinesin. These studies demonstrate for the first time that secretory vesicles in live pancreatic acinar cells are tethered and not free-floating, suggesting that following vesicle biogenesis, they are placed on their own railroad track, ready to be transported to their final destination within the cell when required. This makes sense, since precision and regulation are the hallmarks of all cellular process, and therefore would hold true for the transport and localization of subcellular organelles such as secretory vesicles

  13. Secretory vesicles in live cells are not free-floating but tethered to filamentous structures: A study using photonic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Abu-Hamdah, Rania [Department of Physiology, Wayne State University School of Medicine, 5245 Scott Hall, 540 E. Canfield, Detroit, MI 48201 (United States); Cho, Won Jin [Department of Physiology, Wayne State University School of Medicine, 5245 Scott Hall, 540 E. Canfield, Detroit, MI 48201 (United States); Hoerber, J.K.H. [Department of Physics, University of Bristol, Bristol BS8 1TD (United Kingdom); Jena, Bhanu P. [Department of Physiology, Wayne State University School of Medicine, 5245 Scott Hall, 540 E. Canfield, Detroit, MI 48201 (United States)]. E-mail: bjena@med.wayne.edu

    2006-06-15

    It is well established that actin and microtubule cytoskeletal systems are involved in organelle transport and membrane trafficking in cells. This is also true for the transport of secretory vesicles in neuroendocrine cells and neurons. It was however unclear whether secretory vesicles remain free-floating, only to associate with such cytoskeletal systems when needing transport. This hypothesis was tested using live pancreatic acinar cells in physiological buffer solutions, using the photonic force microscope (PFM). When membrane-bound secretory vesicles (0.2-1.2 {mu}m in diameter) in live pancreatic acinar cells were trapped at the laser focus of the PFM and pulled, they were all found tethered to filamentous structures. Mild exposure of cells to nocodazole and cytochalasin B, disrupts the tether. Immunoblot analysis of isolated secretory vesicles, further demonstrated the association of actin, myosin V, and kinesin. These studies demonstrate for the first time that secretory vesicles in live pancreatic acinar cells are tethered and not free-floating, suggesting that following vesicle biogenesis, they are placed on their own railroad track, ready to be transported to their final destination within the cell when required. This makes sense, since precision and regulation are the hallmarks of all cellular process, and therefore would hold true for the transport and localization of subcellular organelles such as secretory vesicles.

  14. Solution-Grown Monocrystalline Hybrid Perovskite Films for Hole-Transporter-Free Solar Cells

    KAUST Repository

    Peng, Wei; Wang, Lingfei; Banavoth, Murali; Ho, Kang-Ting; Bera, Ashok; Cho, Nam Chul; Kang, Chen-Fang; Burlakov, Victor M.; Pan, Jun; Sinatra, Lutfan; Ma, Chun; Xu, Wei; Shi, Dong; Alarousu, Erkki; Goriely, Alain; He, Jr-Hau; Mohammed, Omar F.; Wu, Tao; Bakr, Osman

    2016-01-01

    conversion efficiencies (PCEs) >5%, and superior stability for prototype cells. Furthermore, the monocrystalline devices using a hole-transporter-free structure yield PCEs ≈6.5%, the highest among other similar-structured CH3NH3PbBr3 solar cells to date.

  15. Purification-Free, Target-Selective Immobilization of a Protein from Cell Lysates.

    Science.gov (United States)

    Cha, Jaehyun; Kwon, Inchan

    2018-02-27

    Protein immobilization has been widely used for laboratory experiments and industrial processes. Preparation of a recombinant protein for immobilization usually requires laborious and expensive purification steps. Here, a novel purification-free, target-selective immobilization technique of a protein from cell lysates is reported. Purification steps are skipped by immobilizing a target protein containing a clickable non-natural amino acid (p-azidophenylalanine) in cell lysates onto alkyne-functionalized solid supports via bioorthogonal azide-alkyne cycloaddition. In order to achieve a target protein-selective immobilization, p-azidophenylalanine was introduced into an exogenous target protein, but not into endogenous non-target proteins using host cells with amber codon-free genomic DNAs. Immobilization of superfolder fluorescent protein (sfGFP) from cell lysates is as efficient as that of the purified sfGFP. Using two fluorescent proteins (sfGFP and mCherry), the authors also demonstrated that the target proteins are immobilized with a minimal immobilization of non-target proteins (target-selective immobilization). © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Microemulsion-based synergistic dual-drug codelivery system for enhanced apoptosis of tumor cells.

    Science.gov (United States)

    Qu, Ding; Ma, Yihua; Sun, Wenjie; Chen, Yan; Zhou, Jing; Liu, Congyan; Huang, Mengmeng

    2015-01-01

    A microemulsion-based synergistic dual-drug codelivery system was developed for enhanced cell apoptosis by transporting coix seed oil and etoposide into A549 (human lung carcinoma) cells simultaneously. Results obtained by dynamic light scattering showed that an etoposide (VP16)-loaded coix seed oil microemulsion (EC-ME) delivery system had a small size around 35 nm, a narrow polydispersity index, and a slightly negative surface charge. The encapsulating efficiency and total drug loading rate were 97.01% and 45.48%, respectively, by high-performance liquid chromatography. The release profiles at various pH values showed an obvious pH-responsive difference, with the accumulated amount of VP16 released at pH 4.5 (and pH 5.5) being 2.7-fold higher relative to that at pH 7.4. Morphologic alteration (particle swelling) associated with a mildly acidic pH environment was found on transmission electron microscopy. In the cell study, the EC-ME system showed a significantly greater antiproliferative effect toward A549 cells in comparison with free VP16 and the mixture of VP16 and coix seed oil. The half-maximal inhibitory concentration of the EC-ME system was 3.9-fold and 10.4-fold lower relative to that of free VP16 and a mixture of VP16 and coix seed oil, respectively. Moreover, fluorescein isothiocyanate and VP16 (the green fluorescent probe and entrapped drug, respectively) were efficiently internalized into the cells by means of coix seed oil microemulsion through intuitive observation and quantitative measurement. Importantly, an EC-ME system containing 20 μg/mL of VP16 showed a 3.3-fold and 3.5-fold improvement in induction of cell apoptosis compared with the VP-16-loaded microemulsion and free VP16, respectively. The EC-ME combination strategy holds promise as an efficient drug delivery system for induction of apoptosis and treatment of lung cancer.

  17. Label-free detection of HIV-1 infected cells via integration of optical tweezers and photoluminescence spectroscopy

    Science.gov (United States)

    Lugongolo, Masixole Yvonne; Ombinda-Lemboumba, Saturnin; Noto, Luyanda Lunga; Maaza, Malik; Mthunzi-Kufa, Patience

    2018-02-01

    The human immunodeficiency virus-1 (HIV-1) is currently detected using conventional qualitative and quantitative tests to determine the presence or absence of HIV in blood samples. However, the approach of these tests detects the presence of either viral antibodies or viral RNA that require labelling which may be costly, sophisticated and time consuming. A label-free approach of detecting the presence of HIV is therefore desirable. Of note optical tweezers can be coupled with other technologies including spectroscopy, which also investigates light-matter interactions. For example, coupling of optical tweezers with luminescence spectroscopy techniques has emerged as a powerful tool in biology for micro-manipulation, detection and analysis of individual cells. Integration of optical techniques has enabled studying biological particles in a label-free manner, whilst detecting functional groups and other essential molecules within mixed populations of cells. In the current study, an optical trapping system coupled to luminescence spectroscopy was utilised to detect the presence of HIV infection in TZM-bl cells in vitro. This was performed by infecting TZM-bl cells with the ZM53 HIV-1 pseudovirus, and incubating them for 48 hours prior analysis. The differences between infected and uninfected cells were thereafter displayed as shown by the spectrographs obtained. Combination of these two techniques has a potential in the field of infectious disease diagnostics.

  18. Defined culture medium for stem cell differentiation: applicability of serum-free conditions in the mouse embryonic stem cell test.

    Science.gov (United States)

    Riebeling, Christian; Schlechter, Katharina; Buesen, Roland; Spielmann, Horst; Luch, Andreas; Seiler, Andrea

    2011-06-01

    The embryonic stem cell test (EST) is a validated method to assess the developmental toxicity potency of chemicals. It was developed to reduce animal use and allow faster testing for hazard assessment. The cells used in this method are maintained and differentiated in media containing foetal calf serum. This animal product is of considerable variation in quality, and individual batches require extensive testing for their applicability in the EST. Moreover, its production involves a large number of foetuses and possible animal suffering. We demonstrate the serum-free medium and feeder cell-free maintenance of the mouse embryonic stem cell line D3 and investigate the use of specific growth factors for induction of cardiac differentiation. Using a combination of bone morphogenetic protein-2, bone morphogenetic protein-4, activin A and ascorbic acid, embryoid bodies efficiently differentiated into contracting myocardium. Additionally, examining levels of intracellular marker proteins by flow cytometry not only confirmed differentiation into cardiomyocytes, but demonstrated significant differentiation into neuronal cells in the same time frame. Thus, this approach might allow for simultaneous detection of developmental effects on both early mesodermal and neuroectodermal differentiation. The serum-free conditions for maintenance and differentiation of D3 cells described here enhance the transferability and standardisation and hence the performance of the EST. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. Investigation of Halohydrins Degradation by Whole Cells and Cell-free Extract of Pseudomonas putida DSM 437: A Kinetic Approach

    Directory of Open Access Journals (Sweden)

    A. Konti

    2017-10-01

    Full Text Available The biodegradation of two halohydrins (1,3-dichloro-2-propanol and 3-chloro-1,2-propanediol by P. putida DSM 437 was investigated. Intact cells of previously acclimatized P. putida DSM 437 as well as cell-free extracts were used in order to study the degradation kinetics. When whole cells were used, a maximum biodegradation rate of 3-CPD (vmax = 1.28.10–5 mmol mg–1 DCW h–1 was determined, which was more than 4 times higher than that of 1,3-DCP. However, the affinity towards both halohydrins (Km was practically the same. When using cell-free extract, the apparent vmax and Km values for 1,3-DCP were estimated at 9.61.10–6 mmol mg–1 protein h–1 and 8.00 mM, respectively, while for 3-CPD the corresponding values were 2.42.10–5 mmol mg–1 protein h–1 and 9.07 mM. GC-MS analysis of cell-free extracts samples spiked with 1,3-DCP revealed the presence of 3-CPD and glycerol, intermediates of 1,3-DCP degradation pathway. 3-CPD degradation was strongly inhibited by the presence of epichlorohydrin and to a lesser extent by glycidol, intermediates of dehalogenation pathway.

  20. Control of protein synthesis in cell-free extracts of sea urchin embryos

    International Nuclear Information System (INIS)

    Hansen, L.J.; Huang, W.I.; Jagus, R.

    1986-01-01

    Although the increase in protein synthesis that occurs after fertilization of sea urchin eggs results from increased utilization of stored maternal mRNA, the underlying mechanism is unknown. The authors have prepared cell-free extracts from S.purpuratus and A.puctulata unfertilized eggs and 2-cell embryos that retain the protein synthetic differences observed in vivo. The method is based on that of Dr. Alina Lopo. 35 S methionine incorporation is linear during a 30 min incubation and is 10-20 fold higher in extracts from 2-cell embryos than unfertilized eggs. Addition of purified mRNA does not stimulate these systems, suggesting a regulatory mechanism other than mRNA masking. Addition of rabbit reticulocyte ribosomal salt wash stimulated protein synthesis in extracts from eggs but not embryos, suggesting deficiencies in translational components in unfertilized eggs. Mixing of egg and embryo lysates indicated the presence of a weak protein synthesis inhibitor in eggs. Translational control in developing sea urchin embryos thus appears to be complex, involving both stimulatory and inhibitory factors

  1. Application of dopant-free hole transport materials for perovskite solar cells

    International Nuclear Information System (INIS)

    Franckevincius, M.; Gulbinas, V.; Gratzel, M.; Zakeeruddin, S.; Pauerle, P.; Mishra, A.; Steck, C.

    2015-01-01

    In this work we present the synthesis, characterization and application of a series of additive and dopant free hole transport materials (HTM) for solid-state perovskite-based solar cells. Newly synthesized HTMs showed strong absorption in the visible spectral range and suitable HOMO-LUMO energy levels for the application for methylammonium lead(II) iodide (CH_3NH_3PbI_3) perovskite. Dopant-free perovskite solar cells have been fabricated using CH_3NH_3PbI_3 perovskite and the newly synthesized HTMs following sequential deposition method, which allows us to reach power conversion efficiencies as high as 11.4 %. The easy of synthesis, low cost and relatively high performance of newly synthesized HTMs has great prospects for commercial applications in the near-future. (authors)

  2. Clinical evaluation of the FreeStyle Precision Pro system.

    Science.gov (United States)

    Brazg, Ronald; Hughes, Kristen; Martin, Pamela; Coard, Julie; Toffaletti, John; McDonnell, Elizabeth; Taylor, Elizabeth; Farrell, Lausanne; Patel, Mona; Ward, Jeanne; Chen, Ting; Alva, Shridhara; Ng, Ronald

    2013-06-05

    A new version of international standard (ISO 15197) and CLSI Guideline (POCT12) with more stringent accuracy criteria are near publication. We evaluated the glucose test performance of the FreeStyle Precision Pro system, a new blood glucose monitoring system (BGMS) designed to enhance accuracy for point-of-care testing (POCT). Precision, interference and system accuracy with 503 blood samples from capillary, venous and arterial sources were evaluated in a multicenter study. Study results were analyzed and presented in accordance with the specifications and recommendations of the final draft ISO 15197 and the new POCT12. The FreeStyle Precision Pro system demonstrated acceptable precision (CV FreeStyle Precision Pro system met the tighter accuracy requirements, providing a means for enhancing accuracy for point-of-care blood glucose monitoring. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Label free imaging of cell-substrate contacts by holographic total internal reflection microscopy.

    Science.gov (United States)

    Mandracchia, Biagio; Gennari, Oriella; Marchesano, Valentina; Paturzo, Melania; Ferraro, Pietro

    2017-09-01

    The study of cell adhesion contacts is pivotal to understand cell mechanics and interaction at substrates or chemical and physical stimuli. We designed and built a HoloTIR microscope for label-free quantitative phase imaging of total internal reflection. Here we show for the first time that HoloTIR is a good choice for label-free study of focal contacts and of cell/substrate interaction as its sensitivity is enhanced in comparison with standard TIR microscopy. Finally, the simplicity of implementation and relative low cost, due to the requirement of less optical components, make HoloTIR a reasonable alternative, or even an addition, to TIRF microscopy for mapping cell/substratum topography. As a proof of concept, we studied the formation of focal contacts of fibroblasts on three substrates with different levels of affinity for cell adhesion. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Dynamic Modeling of Cell-Free Biochemical Networks Using Effective Kinetic Models

    Science.gov (United States)

    2015-03-03

    based whole-cell models of E. coli [6]. Conversely , highly abstracted kinetic frameworks, such as the cybernetic framework, represented a paradigm shift...metabolic objective function has been the optimization of biomass formation [18], although other metabolic objectives have also been estimated [19...experimental data. Toward these questions, we explored five hypothetical cell-free networks. Each network shared the same enzymatic connectivity, but

  5. Application of fuel cells with heat recovery for integrated utility systems

    Science.gov (United States)

    Shields, V.; King, J. M., Jr.

    1975-01-01

    This paper presents the results of a study of fuel cell powerplants with heat recovery for use in an integrated utility system. Such a design provides for a low pollution, noise-free, highly efficient integrated utility. Use of the waste heat from the fuel cell powerplant in an integrated utility system for the village center complex of a new community results in a reduction in resource consumption of 42 percent compared to conventional methods. In addition, the system has the potential of operating on fuels produced from waste materials (pyrolysis and digester gases); this would provide further reduction in energy consumption.

  6. Cell-Free DNA, High-Mobility Group Box-1, and Procalcitonin Concentrations in Dogs With Gastric Dilatation–Volvulus Syndrome

    OpenAIRE

    Roberta Troia; Massimo Giunti; Stefano Calipa; Robert Goggs

    2018-01-01

    Canine gastric dilatation–volvulus (GDV) is a life-threatening disease characterized by extensive tissue ischemia, tissue hypoperfusion, and systemic inflammation. Biomarkers that better reflect the severity of gastric necrosis and systemic inflammation would aid clinicians in the management of these patients. This study aimed to investigate the prognostic significance of cell-free DNA (cfDNA), high-mobility group box-1 (HMGB1), and procalcitonin (PCT) in dogs with GDV. Concentrations of cfDN...

  7. Dopant‐Free and Carrier‐Selective Heterocontacts for Silicon Solar Cells: Recent Advances and Perspectives

    Science.gov (United States)

    Yang, Zhenhai; He, Jian; Yu, Jing; Liu, Peipei; Zhu, Juye; Ge, Ziyi; Ye, Jichun

    2017-01-01

    Abstract By combining the most successful heterojunctions (HJ) with interdigitated back contacts, crystalline silicon (c‐Si) solar cells (SCs) have recently demonstrated a record efficiency of 26.6%. However, such SCs still introduce optical/electrical losses and technological issues due to parasitic absorption/Auger recombination inherent to the doped films and the complex process of integrating discrete p+‐ and n+‐HJ contacts. These issues have motivated the search for alternative new functional materials and simplified deposition technologies, whereby carrier‐selective contacts (CSCs) can be formed directly with c‐Si substrates, and thereafter form IBC cells, via a dopant‐free method. Screening and modifying CSC materials in a wider context is beneficial for building dopant‐free HJ contacts with better performance, shedding new light on the relatively mature Si photovoltaic field. In this review, a significant number of achievements in two representative dopant‐free hole‐selective CSCs, i.e., poly(3,4‐ethylene dioxythiophene):poly(styrenesulfonate)/Si and transition metal oxides/Si, have been systemically presented and surveyed. The focus herein is on the latest advances in hole‐selective materials modification, interfacial passivation, contact resistivity, light‐trapping structure and device architecture design, etc. By analyzing the structure–property relationships of hole‐selective materials and assessing their electrical transport properties, promising functional materials as well as important design concepts for such CSCs toward high‐performance SCs have been highlighted. PMID:29593956

  8. Efficiency and fidelity of cell-free protein synthesis by transfer RNA from aged mice

    Energy Technology Data Exchange (ETDEWEB)

    Foote, R.S.; Stulberg, M.P.

    1980-01-01

    Transfer RNAs (tRNAs) from heart, kidney, liver, and spleen of mature (10 to 12 months old) and aged (29 months old) C57BL/6 mice were tested for their ability to translate encephalomyocarditis viral RNA in a tRNA-dependent cell-free system derived from mouse ascites tumor cells. The rates of in vitro protein synthesis were compared as a function of tRNA concentration, and the fidelity of translation was examined by sodium dodecyl sulfate gel electrophoresis and isoelectric focusing of the viral polypeptides synthesized in vitro. No significant age-related differences in either the efficiency or fidelity of synthesis were discovered, indicating that alternations in tRNAs are probably not involved in the cellular aging of these tissues.

  9. Fully Automated On-Chip Imaging Flow Cytometry System with Disposable Contamination-Free Plastic Re-Cultivation Chip

    Directory of Open Access Journals (Sweden)

    Tomoyuki Kaneko

    2011-06-01

    Full Text Available We have developed a novel imaging cytometry system using a poly(methyl methacrylate (PMMA based microfluidic chip. The system was contamination-free, because sample suspensions contacted only with a flammable PMMA chip and no other component of the system. The transparency and low-fluorescence of PMMA was suitable for microscopic imaging of cells flowing through microchannels on the chip. Sample particles flowing through microchannels on the chip were discriminated by an image-recognition unit with a high-speed camera in real time at the rate of 200 event/s, e.g., microparticles 2.5 μm and 3.0 μm in diameter were differentiated with an error rate of less than 2%. Desired cells were separated automatically from other cells by electrophoretic or dielectrophoretic force one by one with a separation efficiency of 90%. Cells in suspension with fluorescent dye were separated using the same kind of microfluidic chip. Sample of 5 μL with 1 × 106 particle/mL was processed within 40 min. Separated cells could be cultured on the microfluidic chip without contamination. The whole operation of sample handling was automated using 3D micropipetting system. These results showed that the novel imaging flow cytometry system is practically applicable for biological research and clinical diagnostics.

  10. Interactions between Skeletal Muscle Myoblasts and their Extracellular Matrix Revealed by a Serum Free Culture System.

    Science.gov (United States)

    Chaturvedi, Vishal; Dye, Danielle E; Kinnear, Beverley F; van Kuppevelt, Toin H; Grounds, Miranda D; Coombe, Deirdre R

    2015-01-01

    Decellularisation of skeletal muscle provides a system to study the interactions of myoblasts with muscle extracellular matrix (ECM). This study describes the efficient decellularisation of quadriceps muscle with the retention of matrix components and the use of this matrix for myoblast proliferation and differentiation under serum free culture conditions. Three decellularisation approaches were examined; the most effective was phospholipase A2 treatment, which removed cellular material while maximizing the retention of ECM components. Decellularised muscle matrices were then solubilized and used as substrates for C2C12 mouse myoblast serum free cultures. The muscle matrix supported myoblast proliferation and differentiation equally as well as collagen and fibronectin. Immunofluorescence analyses revealed that myoblasts seeded on muscle matrix and fibronectin differentiated to form long, well-aligned myotubes, while myoblasts seeded on collagen were less organized. qPCR analyses showed a time dependent increase in genes involved in skeletal muscle differentiation and suggested that muscle-derived matrix may stimulate an increased rate of differentiation compared to collagen and fibronectin. Decellularized whole muscle three-dimensional scaffolds also supported cell adhesion and spreading, with myoblasts aligning along specific tracts of matrix proteins within the scaffolds. Thus, under serum free conditions, intact acellular muscle matrices provided cues to direct myoblast adhesion and migration. In addition, myoblasts were shown to rapidly secrete and organise their own matrix glycoproteins to create a localized ECM microenvironment. This serum free culture system has revealed that the correct muscle ECM facilitates more rapid cell organisation and differentiation than single matrix glycoprotein substrates.

  11. Stepwise Differentiation of Pluripotent Stem Cells into Osteoblasts Using Four Small Molecules under Serum-free and Feeder-free Conditions

    Directory of Open Access Journals (Sweden)

    Kosuke Kanke

    2014-06-01

    Full Text Available Pluripotent stem cells are a promising tool for mechanistic studies of tissue development, drug screening, and cell-based therapies. Here, we report an effective and mass-producing strategy for the stepwise differentiation of mouse embryonic stem cells (mESCs and mouse and human induced pluripotent stem cells (miPSCs and hiPSCs, respectively into osteoblasts using four small molecules (CHIR99021 [CHIR], cyclopamine [Cyc], smoothened agonist [SAG], and a helioxanthin-derivative 4-(4-methoxyphenylpyrido[4′,3′:4,5]thieno[2,3-b]pyridine-2-carboxamide [TH] under serum-free and feeder-free conditions. The strategy, which consists of mesoderm induction, osteoblast induction, and osteoblast maturation phases, significantly induced expressions of osteoblast-related genes and proteins in mESCs, miPSCs, and hiPSCs. In addition, when mESCs defective in runt-related transcription factor 2 (Runx2, a master regulator of osteogenesis, were cultured by the strategy, they molecularly recapitulated osteoblast phenotypes of Runx2 null mice. The present strategy will be a platform for biological and pathological studies of osteoblast development, screening of bone-augmentation drugs, and skeletal regeneration.

  12. Free boundary problems in PDEs and particle systems

    CERN Document Server

    Carinci, Gioia; Giardinà, Cristian; Presutti, Errico

    2016-01-01

    In this volume a theory for models of transport in the presence of a free boundary is developed. Macroscopic laws of transport are described by PDE's. When the system is open, there are several mechanisms to couple the system with the external forces. Here a class of systems where the interaction with the exterior takes place in correspondence of a free boundary is considered. Both continuous and discrete models sharing the same structure are analysed. In Part I a free boundary problem related to the Stefan Problem is worked out in all details. For this model a new notion of relaxed solution is proposed for which global existence and uniqueness is proven. It is also shown that this is the hydrodynamic limit of the empirical mass density of the associated particle system. In Part II several other models are discussed. The expectation is that the results proved for the basic model extend to these other cases. All the models discussed in this volume have an interest in problems arising in several research fields...

  13. Differentiation of Dental Pulp Stem Cells into Neuron-Like Cells in Serum-Free Medium

    Directory of Open Access Journals (Sweden)

    Shahrul Hisham Zainal Ariffin

    2013-01-01

    Full Text Available Dental pulp tissue contains dental pulp stem cells (DPSCs. Dental pulp cells (also known as dental pulp-derived mesenchymal stem cells are capable of differentiating into multilineage cells including neuron-like cells. The aim of this study was to examine the capability of DPSCs to differentiate into neuron-like cells without using any reagents or growth factors. DPSCs were isolated from teeth extracted from 6- to 8-week-old mice and maintained in complete medium. The cells from the fourth passage were induced to differentiate by culturing in medium without serum or growth factors. RT-PCR molecular analysis showed characteristics of Cd146+, Cd166+, and Cd31− in DPSCs, indicating that these cells are mesenchymal stem cells rather than hematopoietic stem cells. After 5 days of neuronal differentiation, the cells showed neuron-like morphological changes and expressed MAP2 protein. The activation of Nestin was observed at low level prior to differentiation and increased after 5 days of culture in differentiation medium, whereas Tub3 was activated only after 5 days of neuronal differentiation. The proliferation of the differentiated cells decreased in comparison to that of the control cells. Dental pulp stem cells are induced to differentiate into neuron-like cells when cultured in serum- and growth factor-free medium.

  14. The design and implementation of the FreeBSD operating system

    CERN Document Server

    McKusick, Marshall Kirk; Watson, Robert N M

    2015-01-01

    The most complete, authoritative technical guide to the FreeBSD kernel's internal structure has now been extensively updated to cover all major improvements between Versions 5 and 11. Approximately one-third of this edition's content is completely new, and another one-third has been extensively rewritten. Three long-time FreeBSD project leaders begin with a concise overview of the FreeBSD kernel's current design and implementation. Next, they cover the FreeBSD kernel from the system-call level down-from the interface to the kernel to the hardware. Explaining key design decisions, they detail the concepts, data structures, and algorithms used in implementing each significant system facility, including process management, security, virtual memory, the I/O system, filesystems, socket IPC, and networking. This Second Edition * Explains highly scalable and lightweight virtualization using FreeBSD jails, and virtual-machine acceleration with Xen and Virtio device paravirtualization * Describes new security features...

  15. Industrially feasible, dopant-free, carrier-selective contacts for high-efficiency silicon solar cells

    KAUST Repository

    Yang, Xinbo

    2017-05-31

    Dopant-free, carrier-selective contacts (CSCs) on high efficiency silicon solar cells combine ease of deposition with potential optical benefits. Electron-selective titanium dioxide (TiO) contacts, one of the most promising dopant-free CSC technologies, have been successfully implemented into silicon solar cells with an efficiency over 21%. Here, we report further progress of TiO contacts for silicon solar cells and present an assessment of their industrial feasibility. With improved TiO contact quality and cell processing, a remarkable efficiency of 22.1% has been achieved using an n-type silicon solar cell featuring a full-area TiO contact. Next, we demonstrate the compatibility of TiO contacts with an industrial contact-firing process, its low performance sensitivity to the wafer resistivity, its applicability to ultrathin substrates as well as its long-term stability. Our findings underscore the great appeal of TiO contacts for industrial implementation with their combination of high efficiency with robust fabrication at low cost.

  16. Photo-degradation of high efficiency fullerene-free polymer solar cells.

    Science.gov (United States)

    Upama, Mushfika Baishakhi; Wright, Matthew; Mahmud, Md Arafat; Elumalai, Naveen Kumar; Mahboubi Soufiani, Arman; Wang, Dian; Xu, Cheng; Uddin, Ashraf

    2017-12-07

    Polymer solar cells are a promising technology for the commercialization of low cost, large scale organic solar cells. With the evolution of high efficiency (>13%) non-fullerene polymer solar cells, the stability of the cells has become a crucial parameter to be considered. Among the several degradation mechanisms of polymer solar cells, burn-in photo-degradation is relatively less studied. Herein, we present the first systematic study of photo-degradation of novel PBDB-T:ITIC fullerene-free polymer solar cells. The thermally treated and as-prepared PBDB-T:ITIC solar cells were exposed to continuous 1 sun illumination for 5 hours. The aged devices exhibited rapid losses in the short-circuit current density and fill factor. The severe short-circuit current and fill factor burn in losses were attributed to trap mediated charge recombination, as evidenced by an increase in Urbach energy for aged devices.

  17. Generation of Human Induced Pluripotent Stem Cells Using RNA-Based Sendai Virus System and Pluripotency Validation of the Resulting Cell Population.

    Science.gov (United States)

    Chichagova, Valeria; Sanchez-Vera, Irene; Armstrong, Lyle; Steel, David; Lako, Majlinda

    2016-01-01

    Human induced pluripotent stem cells (hiPSCs) provide a platform for studying human disease in vitro, increase our understanding of human embryonic development, and provide clinically relevant cell types for transplantation, drug testing, and toxicology studies. Since their discovery, numerous advances have been made in order to eliminate issues such as vector integration into the host genome, low reprogramming efficiency, incomplete reprogramming and acquisition of genomic instabilities. One of the ways to achieve integration-free reprogramming is by using RNA-based Sendai virus. Here we describe a method to generate hiPSCs with Sendai virus in both feeder-free and feeder-dependent culture systems. Additionally, we illustrate methods by which to validate pluripotency of the resulting stem cell population.

  18. Innovative Free-range Resonant Electrical Energy Delivery system (FREE-D System) for a ventricular assist device using wireless power.

    Science.gov (United States)

    Waters, Benjamin H; Smith, Joshua R; Bonde, Pramod

    2014-01-01

    Technological innovation of a smaller, single moving part has an advantage over earlier large pulsatile ventricular assist devices (VADs) prone to mechanical failure. Drivelines limit the potential for extended patient survival durations with newer pumps and act as source for infection, increased morbidity, rehospitalizations, and reduced quality of life. The Free-range Resonant Electrical Energy Delivery (FREE-D) wireless power system uses magnetically coupled resonators to efficiently transfer power. We demonstrate the efficiency over distance of this system. The experimental setup consists of an radiofrequency amplifier and control board which drives the transmit resonator coil, and a receiver unit consisting of a resonant coil attached to a radiofrequency rectifier and power management module. The power management module supplies power to the axial pump, which was set at 9,600 rpm. To achieve a seamless wireless delivery in any room size, we introduced a third relay coil. This relay coil can be installed throughout a room, whereas a single relay coil could be built into a jacket worn by the patient, which would always be within range of the receive coil implanted in the patient's body. The power was delivered over a meter distance without interruptions or fluctuations with coil, rectifier, and regulator efficiency more than 80% and overall system efficiency of 61%. The axial pump worked well throughout the 8 hours of continuous operation. Having same setup on the opposite side can double the distance. A tether-free operation of a VAD can be achieved by FREE-D system in room-size distances. It has the potential to make the VAD therapy more acceptable from the patient perspective.

  19. Integrated Front–Rear-Grid Optimization of Free-Form Solar Cells

    NARCIS (Netherlands)

    Gupta, D.K.; Barink, M.; Galagan, Y.; Langelaar, M.

    2016-01-01

    Free-form solar cells expand solar power beyond traditional rectangular geometries. With the flexibility of being installed on objects of daily use, they allow making better use of available space and are expected to bring in new possibilities of generating solar power in the coming future. In

  20. Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21)

    Science.gov (United States)

    Nollevaux, Géraldine; Devillé, Christelle; El Moualij, Benaïssa; Zorzi, Willy; Deloyer, Patricia; Schneider, Yves-Jacques; Peulen, Olivier; Dandrifosse, Guy

    2006-01-01

    Background The absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. The cell lineage Caco-2 is a model commonly used to reproduce the features of the bowel epithelium. However, there is a strong debate regarding the value of Caco-2 cell culture to mimick in vivo situation. Indeed, some authors report in Caco-2 a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. The HT29-5M21 intestinal cell lineage is a mucin-secreting cellular population. A co-culture system carried out in a serum-free medium and comprising both Caco-2 and HT29-5M21 cells was developed. The systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure. Results In this study, we investigated the activity and localization of the alkaline phosphatase and the expression of IAP and MUC5AC genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of Caco-2 and HT29-5M21 cells. We observed that the culture conditions used (serum-free medium) did not change the phenotype of each cell type, and produced a reproducible model. The alkaline phosphatase expression characterizing Caco-2 cells was influenced by the presence of HT29-5M21 cells. Conclusion The culture formed by 75% Caco-2 and 25% HT29-5M21 produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules. PMID:16670004

  1. Aberrant methylation of cell-free circulating DNA in plasma predicts poor outcome in diffuse large B cell lymphoma

    DEFF Research Database (Denmark)

    Sommer Kristensen, Lasse; Hansen, Jakob Werner; Kristensen, Søren Sommer

    2016-01-01

    BACKGROUND: The prognostic value of aberrant DNA methylation of cell-free circulating DNA in plasma has not previously been evaluated in diffuse large B cell lymphoma (DLBCL). The aim of this study was to investigate if aberrant promoter DNA methylation can be detected in plasma from DLBCL patients...

  2. Responding to chromosomal breakage during M-phase: insights from a cell-free system

    Directory of Open Access Journals (Sweden)

    Costanzo Vincenzo

    2009-07-01

    Full Text Available Abstract DNA double strand breaks (DSBs activate ATM and ATR dependent checkpoints that prevent the onset of mitosis. However, how cells react to DSBs occurring when they are already in mitosis is poorly understood. The Xenopus egg extract has been utilized to study cell cycle progression and DNA damage checkpoints. Recently this system has been successfully used to uncover an ATM and ATR dependent checkpoint affecting centrosome driven spindle assembly. These studies have led to the identification of XCEP63 as major target of this pathway. XCEP63 is a coiled-coil rich protein localized at centrosome essential for proper spindle assembly. ATM and ATR directly phosphorylate XCEP63 on serine 560 inducing its delocalization from centrosome, which in turn delays spindle assembly. This pathway might contribute to regulate DNA repair or mitotic cell survival in the presence of chromosome breakage.

  3. Generation and periodontal differentiation of human gingival fibroblasts-derived integration-free induced pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Xiaohui [Department of Periodontology, School and Hospital of Stomatology, Peking University, 22 South Avenue Zhong-Guan-Cun, Beijing 100081 (China); Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Li, Yang [Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Li, Jingwen [Department of Periodontology, School and Hospital of Stomatology, Peking University, 22 South Avenue Zhong-Guan-Cun, Beijing 100081 (China); Li, Peng [Faculty of Dentistry, The University of Hong Kong, 34 Hospital Road, Hong Kong SAR (China); Liu, Yinan [Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Wen, Jinhua, E-mail: jhwen@bjmu.edu.cn [Peking University Stem Cell Research Center and Department of Cell Biology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Road, Beijing 100191 (China); Luan, Qingxian, E-mail: kqluanqx@126.com [Department of Periodontology, School and Hospital of Stomatology, Peking University, 22 South Avenue Zhong-Guan-Cun, Beijing 100081 (China)

    2016-05-06

    Induced pluripotent stem cells (iPSCs) have been recognized as a promising cell source for periodontal tissue regeneration. However, the conventional virus-based reprogramming approach is associated with a high risk of genetic mutation and limits their therapeutic utility. Here, we successfully generated iPSCs from readily accessible human gingival fibroblasts (hGFs) through an integration-free and feeder-free approach via delivery of reprogramming factors of Oct4, Sox2, Klf4, L-myc, Lin28 and TP53 shRNA with episomal plasmid vectors. The iPSCs presented similar morphology and proliferation characteristics as embryonic stem cells (ESCs), and expressed pluripotent markers including Oct4, Tra181, Nanog and SSEA-4. Additionally, these cells maintained a normal karyotype and showed decreased CpG methylation ratio in the promoter regions of Oct4 and Nanog. In vivo teratoma formation assay revealed the development of tissues representative of three germ layers, confirming the acquisition of pluripotency. Furthermore, treatment of the iPSCs in vitro with enamel matrix derivative (EMD) or growth/differentiation factor-5 (GDF-5) significantly up-regulated the expression of periodontal tissue markers associated with bone, periodontal ligament and cementum respectively. Taken together, our data demonstrate that hGFs are a valuable cell source for generating integration-free iPSCs, which could be sequentially induced toward periodontal cells under the treatment of EMD and GDF-5. - Highlights: • Integration-free iPSCs are successfully generated from hGFs via an episomal approach. • EMD promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • GDF-5 promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • hGFs-derived iPSCs could be a promising cell source for periodontal regeneration.

  4. Generation and periodontal differentiation of human gingival fibroblasts-derived integration-free induced pluripotent stem cells

    International Nuclear Information System (INIS)

    Yin, Xiaohui; Li, Yang; Li, Jingwen; Li, Peng; Liu, Yinan; Wen, Jinhua; Luan, Qingxian

    2016-01-01

    Induced pluripotent stem cells (iPSCs) have been recognized as a promising cell source for periodontal tissue regeneration. However, the conventional virus-based reprogramming approach is associated with a high risk of genetic mutation and limits their therapeutic utility. Here, we successfully generated iPSCs from readily accessible human gingival fibroblasts (hGFs) through an integration-free and feeder-free approach via delivery of reprogramming factors of Oct4, Sox2, Klf4, L-myc, Lin28 and TP53 shRNA with episomal plasmid vectors. The iPSCs presented similar morphology and proliferation characteristics as embryonic stem cells (ESCs), and expressed pluripotent markers including Oct4, Tra181, Nanog and SSEA-4. Additionally, these cells maintained a normal karyotype and showed decreased CpG methylation ratio in the promoter regions of Oct4 and Nanog. In vivo teratoma formation assay revealed the development of tissues representative of three germ layers, confirming the acquisition of pluripotency. Furthermore, treatment of the iPSCs in vitro with enamel matrix derivative (EMD) or growth/differentiation factor-5 (GDF-5) significantly up-regulated the expression of periodontal tissue markers associated with bone, periodontal ligament and cementum respectively. Taken together, our data demonstrate that hGFs are a valuable cell source for generating integration-free iPSCs, which could be sequentially induced toward periodontal cells under the treatment of EMD and GDF-5. - Highlights: • Integration-free iPSCs are successfully generated from hGFs via an episomal approach. • EMD promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • GDF-5 promotes differentiation of the hGFs-derived iPSCs toward periodontal cells. • hGFs-derived iPSCs could be a promising cell source for periodontal regeneration.

  5. Solvent-free ZnO dye-sensitised solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Guillen, E.; Anta, J.A. [Departamento de Sistemas Fisicos, Quimicos y Naturales, Universidad Pablo de Olavide, 41013 Sevilla (Spain); Fernandez-Lorenzo, C.; Alcantara, R.; Martin-Calleja, J. [Departamento de Quimica Fisica, Universidad de Cadiz, Cadiz (Spain)

    2009-10-15

    Dye-sensitised solar cells (DSSC) based on commercial nanostructured zinc oxide combined with imidazolium-based room temperature ionic-liquid electrolytes are characterized. The electrolytes are based on a binary mixture of two ionic liquids, one of them used as source of iodide ions. The composition of this solvent-free electrolyte is optimized with respect to the concentration of iodine and iodide and the effect of additives such as lithium and tert-butylpyridine (TBP) on the photovoltaic performance and the recombination rate is analyzed and discussed. A maximum photoconversion efficiency of 3.4% at 1 sun illumination has been obtained for cells of 0.64 cm{sup 2} active area with the best performing compositions. Diffusion limitations due to slow transport processes are analyzed and discussed. (author)

  6. Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope

    Science.gov (United States)

    Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

    2015-01-01

    Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications. PMID:26525841

  7. Dynamic nano-imaging of label-free living cells using electron beam excitation-assisted optical microscope.

    Science.gov (United States)

    Fukuta, Masahiro; Kanamori, Satoshi; Furukawa, Taichi; Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

    2015-11-03

    Optical microscopes are effective tools for cellular function analysis because biological cells can be observed non-destructively and non-invasively in the living state in either water or atmosphere condition. Label-free optical imaging technique such as phase-contrast microscopy has been analysed many cellular functions, and it is essential technology for bioscience field. However, the diffraction limit of light makes it is difficult to image nano-structures in a label-free living cell, for example the endoplasmic reticulum, the Golgi body and the localization of proteins. Here we demonstrate the dynamic imaging of a label-free cell with high spatial resolution by using an electron beam excitation-assisted optical (EXA) microscope. We observed the dynamic movement of the nucleus and nano-scale granules in living cells with better than 100 nm spatial resolution and a signal-to-noise ratio (SNR) around 10. Our results contribute to the development of cellular function analysis and open up new bioscience applications.

  8. The research progress of several kinds of free radical scavengers

    International Nuclear Information System (INIS)

    Qian Liren; Huang Yuecheng; Cai Jianming

    2009-01-01

    Ionization radiation can generate free radicals in biological system, which could induce lipid peroxi-dation, biomacromolecule and biomembrane damage, lost of cell function, cell cycle disturbance, genetic mutation and so on. The scavenging free radicals can protect organism from radiation damage. Many radio-protective agents, such as amylase, hydroxyl-benzene derivatives, hormone, vitamin, have great abilities to protect organism from radiation via scavenging free radicals. In this paper, we mainly review the free radical scavenging effects of several kinds of radio-protective agents. (authors)

  9. Label-Free Imaging of Umbilical Cord Tissue Morphology and Explant-Derived Cells

    Directory of Open Access Journals (Sweden)

    Raf Donders

    2016-01-01

    Full Text Available In situ detection of MSCs remains difficult and warrants additional methods to aid with their characterization in vivo. Two-photon confocal laser scanning microscopy (TPM and second harmonic generation (SHG could fill this gap. Both techniques enable the detection of cells and extracellular structures, based on intrinsic properties of the specific tissue and intracellular molecules under optical irradiation. TPM imaging and SHG imaging have been used for label-free monitoring of stem cells differentiation, assessment of their behavior in biocompatible scaffolds, and even cell tracking in vivo. In this study, we show that TPM and SHG can accurately depict the umbilical cord architecture and visualize individual cells both in situ and during culture initiation, without the use of exogenously applied labels. In combination with nuclear DNA staining, we observed a variance in fluorescent intensity in the vessel walls. In addition, antibody staining showed differences in Oct4, αSMA, vimentin, and ALDH1A1 expression in situ, indicating functional differences among the umbilical cord cell populations. In future research, marker-free imaging can be of great added value to the current antigen-based staining methods for describing tissue structures and for the identification of progenitor cells in their tissue of origin.

  10. Free radical scavenger edaravone suppresses X-ray-induced apoptosis through p53 inhibition in MOLT-4 cells

    International Nuclear Information System (INIS)

    Sasano, Nakashi; Shiraishi, Kenshiro; Igaki, Hiroshi; Nakagawa, Keiichi; Enomoto, Atsushi; Hosoi, Yoshio; Matsumoto, Yoshihisa; Miyagawa, Kiyoshi; Katsumura, Yosuke

    2007-01-01

    Edaravone, a clinical drug used widely for the treatment of acute cerebral infarction, is reported to scavenge free radicals. In the present study, we investigated the radioprotective effect of edaravone on X-ray-induced apoptosis in MOLT-4 cells. Apoptosis was determined by the dye exclusion test, Annexin V binding assay, cleavage of caspase, and DNA fragmentation. We found that edaravone significantly suppressed the X-ray-induced apoptosis. The amount of intracellular reactive oxygen species (ROS) production was determined by the chloromethyl-2', 7'-dichlorodihydro-fluorescein diacetate system. We found that the intracellular ROS production by X-irradiation was completely suppressed by the addition of edaravone. The accumulation and phosphorylation of p53 and the expression of p21 WAF1 , a target protein of p53, which were induced by X-irradiation, were also suppressed by adding edaravone. We conclude that the free radical scavenger edaravone suppresses X-ray-induced apoptosis in MOLT-4 cells by inhibiting p53. (author)

  11. Free radical scavenger edaravone suppresses X-ray-induced apoptosis through p53 inhibition in MOLT-4 cells

    Energy Technology Data Exchange (ETDEWEB)

    Sasano, Nakashi; Shiraishi, Kenshiro; Igaki, Hiroshi; Nakagawa, Keiichi [Tokyo Univ., Graduate School of Medicine, Tokyo (Japan); Enomoto, Atsushi; Hosoi, Yoshio; Matsumoto, Yoshihisa; Miyagawa, Kiyoshi [Tokyo Univ., Faculty of Medicine, Tokyo (Japan); Katsumura, Yosuke [Tokyo Univ., Graduate School of Engineering, Tokyo (Japan)

    2007-11-15

    Edaravone, a clinical drug used widely for the treatment of acute cerebral infarction, is reported to scavenge free radicals. In the present study, we investigated the radioprotective effect of edaravone on X-ray-induced apoptosis in MOLT-4 cells. Apoptosis was determined by the dye exclusion test, Annexin V binding assay, cleavage of caspase, and DNA fragmentation. We found that edaravone significantly suppressed the X-ray-induced apoptosis. The amount of intracellular reactive oxygen species (ROS) production was determined by the chloromethyl-2', 7'-dichlorodihydro-fluorescein diacetate system. We found that the intracellular ROS production by X-irradiation was completely suppressed by the addition of edaravone. The accumulation and phosphorylation of p53 and the expression of p21{sup WAF1}, a target protein of p53, which were induced by X-irradiation, were also suppressed by adding edaravone. We conclude that the free radical scavenger edaravone suppresses X-ray-induced apoptosis in MOLT-4 cells by inhibiting p53. (author)

  12. Using Potentiometric Free Drug Sensors to Determine the Free Concentration of Ionizable Drugs in Colloidal Systems

    DEFF Research Database (Denmark)

    Tran, Thuy; Chakraborty, Anjan; Xi, Xi

    2018-01-01

    The present study investigates the use of free drug sensors (FDS) to measure free ionized drug concentrations in colloidal systems, including micellar solutions, emulsions, and lipid formulations during in vitro lipolysis. Diphenhydramine hydrochloride (DPH) and loperamide hydrochloride (LOP) wer...

  13. Autofluorescence-Free Live-Cell Imaging Using Terbium Nanoparticles.

    Science.gov (United States)

    Cardoso Dos Santos, M; Goetz, J; Bartenlian, H; Wong, K-L; Charbonnière, L J; Hildebrandt, N

    2018-04-18

    Fluorescent nanoparticles (NPs) have become irreplaceable tools for advanced cellular and subcellular imaging. While very bright NPs require excitation with UV or visible light, which can create strong autofluorescence of biological components, NIR-excitable NPs without autofluorescence issues exhibit much lower brightness. Here, we show the application of a new type of surface-photosensitized terbium NPs (Tb-NPs) for autofluorescence-free intracellular imaging in live HeLa cells. The combination of exceptionally high brightness, high photostability, and long photoluminecence (PL) lifetimes for highly efficient suppression of the short-lived autofluorescence allowed for time-gated PL imaging of intracellular vesicles over 72 h without toxicity and at extremely low Tb-NP concentrations down to 12 pM. Detection of highly resolved long-lifetime (ms) PL decay curves from small (∼10 μm 2 ) areas within single cells within a few seconds emphasized the unprecedented photophysical properties of Tb-NPs for live-cell imaging that extend well beyond currently available nanometric imaging agents.

  14. Early Prognostic Value of Monitoring Serum Free Light Chain in Patients with Multiple Myeloma Undergoing Autologous Stem Cell Transplantation.

    Science.gov (United States)

    Özkurt, Zübeyde Nur; Sucak, Gülsan Türköz; Akı, Şahika Zeynep; Yağcı, Münci; Haznedar, Rauf

    2017-03-16

    We hypothesized the levels of free light chains obtained before and after autologous stem cell transplantation can be useful in predicting transplantation outcome. We analyzed 70 multiple myeloma patients. Abnormal free light chain ratios before stem cell transplantation were found to be associated early progression, although without any impact on overall survival. At day +30, the normalization of levels of involved free light chain related with early progression. According to these results almost one-third reduction of free light chain levels can predict favorable prognosis after autologous stem cell transplantation.

  15. Positive association of free triiodothyronine with pancreatic β-cell function in people with prediabetes.

    Science.gov (United States)

    Oda, T; Taneichi, H; Takahashi, K; Togashi, H; Hangai, M; Nakagawa, R; Ono, M; Matsui, M; Sasai, T; Nagasawa, K; Honma, H; Kajiwara, T; Takahashi, Y; Takebe, N; Ishigaki, Y; Satoh, J

    2015-02-01

    To analyse the effects of thyroid hormones on β-cell function and glucose metabolism in people with prediabetes who are euthyroid. A total of 111 people who were euthyroid underwent 75-g oral glucose tolerance tests, of whom 52 were assigned to the normal glucose tolerance and 59 to the prediabetes groups. Homeostatic model assessment of β-cell function, insulinogenic index and areas under the curve for insulin and glucose were evaluated as indices of pancreatic β-cell function. In both groups, BMI, fasting insulin, homeostasis model assessment ratio and HDL cholesterol correlated significantly with all indices of pancreatic β-cell function. Free triiodothyronine correlated positively with all insulin secretion indices in the prediabetes group. Multiple linear regression analysis showed that free triiodothyronine was an independent variable that had a positive correlation with all indices of β-cell function in the prediabetes group. By contrast, no such correlation was found in the normal glucose tolerance group. Free triiodothyronine is associated with both basal and glucose-stimulated insulin secretion in people with prediabetes who are euthyroid; therefore, the regulation of insulin secretion by thyroid hormones is a potentially novel therapeutic target for the treatment of diabetes. © 2014 The Authors. Diabetic Medicine © 2014 Diabetes UK.

  16. Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model

    Directory of Open Access Journals (Sweden)

    Alvaro Plaza Reyes

    2016-01-01

    Full Text Available Human embryonic stem cell (hESC-derived retinal pigment epithelial (RPE cells could replace lost tissue in geographic atrophy (GA but efficacy has yet to be demonstrated in a large-eyed model. Also, production of hESC-RPE has not yet been achieved in a xeno-free and defined manner, which is critical for clinical compliance and reduced immunogenicity. Here we describe an effective differentiation methodology using human laminin-521 matrix with xeno-free and defined medium. Differentiated cells exhibited characteristics of native RPE including morphology, pigmentation, marker expression, monolayer integrity, and polarization together with phagocytic activity. Furthermore, we established a large-eyed GA model that allowed in vivo imaging of hESC-RPE and host retina. Cells transplanted in suspension showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity. We have developed a xeno-free and defined hESC-RPE differentiation method and present evidence of functional integration of clinically compliant hESC-RPE in a large-eyed disease model.

  17. Label-free all-electronic biosensing in microfluidic systems

    Science.gov (United States)

    Stanton, Michael A.

    Label-free, all-electronic detection techniques offer great promise for advancements in medical and biological analysis. Electrical sensing can be used to measure both interfacial and bulk impedance changes in conducting solutions. Electronic sensors produced using standard microfabrication processes are easily integrated into microfluidic systems. Combined with the sensitivity of radiofrequency electrical measurements, this approach offers significant advantages over competing biological sensing methods. Scalable fabrication methods also provide a means of bypassing the prohibitive costs and infrastructure associated with current technologies. We describe the design, development and use of a radiofrequency reflectometer integrated into a microfluidic system towards the specific detection of biologically relevant materials. We developed a detection protocol based on impedimetric changes caused by the binding of antibody/antigen pairs to the sensing region. Here we report the surface chemistry that forms the necessary capture mechanism. Gold-thiol binding was utilized to create an ordered alkane monolayer on the sensor surface. Exposed functional groups target the N-terminus, affixing a protein to the monolayer. The general applicability of this method lends itself to a wide variety of proteins. To demonstrate specificity, commercially available mouse anti- Streptococcus Pneumoniae monoclonal antibody was used to target the full-length recombinant pneumococcal surface protein A, type 2 strain D39 expressed by Streptococcus Pneumoniae. We demonstrate the RF response of the sensor to both the presence of the surface decoration and bound SPn cells in a 1x phosphate buffered saline solution. The combined microfluidic sensor represents a powerful platform for the analysis and detection of cells and biomolecules.

  18. Heavy quark free energies for three quark systems at finite temperature

    International Nuclear Information System (INIS)

    Huebner, Kay; Karsch, Frithjof; Kaczmarek, Olaf; Vogt, Oliver

    2008-01-01

    We study the free energy of static three quark systems in singlet, octet, decuplet, and average color channels in the quenched approximation and in 2-flavor QCD at finite temperature. We show that in the high temperature phase singlet and decuplet free energies of three quark systems are well described by the sum of the free energies of three diquark systems plus self-energy contributions of the three quarks. In the confining low temperature phase we find evidence for a Y-shaped flux tube in SU(3) pure gauge theory, which is less evident in 2-flavor QCD due to the onset of string breaking. We also compare the short distance behavior of octet and decuplet free energies to the free energies of single static quarks in the corresponding color representations.

  19. LISA Pathfinder drag-free control and system implications

    International Nuclear Information System (INIS)

    Fichter, Walter; Gath, Peter; Vitale, Stefano; Bortoluzzi, Daniele

    2005-01-01

    The top-level requirement of the LISA Pathfinder mission is the verification of pure relative free fall between two test masses with an accuracy of about 3 x 10 -14 m s -2 Hz -1/2 in a measurement bandwidth between 1 mHz and 30 mHz. The drag-free control system is one of the key technology elements that shall be verified. Its design is strongly connected to the overall system and experimental design, in particular, via the following issues: the differential test mass motion and thus the science measurements depend on the control system; design constraints, such as negative stiffness of test masses and electrostatic actuation cross-talk, have an impact on science and control system performance; derived requirements for control system components, in particular, the micro-propulsion system, must be within reasonable and feasible limits. In this paper, the control design approach is outlined and the system-related issues are addressed

  20. Control system for JAERI Free Electron Laser

    International Nuclear Information System (INIS)

    Sugimoto, Masayoshi

    1992-01-01

    A control system comprising of the personal computers network and the CAMAC stations for the JAERI Free Electron Laser is designed and is in the development stage. It controls the equipment and analyzes the electron and optical beam experiments. The concept and the prototype of the control system are described. (author)

  1. Hematopoietic stem cell cytokines and fibroblast growth factor-2 stimulate human endothelial cell-pericyte tube co-assembly in 3D fibrin matrices under serum-free defined conditions.

    Directory of Open Access Journals (Sweden)

    Annie O Smith

    Full Text Available We describe a novel 3D fibrin matrix model using recombinant hematopoietic stem cell cytokines under serum-free defined conditions which promotes the assembly of human endothelial cell (EC tubes with co-associated pericytes. Individual ECs and pericytes are randomly mixed together and EC tubes form that is accompanied by pericyte recruitment to the EC tube abluminal surface over a 3-5 day period. These morphogenic processes are stimulated by a combination of the hematopoietic stem cell cytokines, stem cell factor, interleukin-3, stromal derived factor-1α, and Flt-3 ligand which are added in conjunction with fibroblast growth factor (FGF-2 into the fibrin matrix. In contrast, this tube morphogenic response does not occur under serum-free defined conditions when VEGF and FGF-2 are added together in the fibrin matrices. We recently demonstrated that VEGF and FGF-2 are able to prime EC tube morphogenic responses (i.e. added overnight prior to the morphogenic assay to hematopoietic stem cell cytokines in collagen matrices and, interestingly, they also prime EC tube morphogenesis in 3D fibrin matrices. EC-pericyte interactions in 3D fibrin matrices leads to marked vascular basement membrane assembly as demonstrated using immunofluorescence and transmission electron microscopy. Furthermore, we show that hematopoietic stem cell cytokines and pericytes stimulate EC sprouting in fibrin matrices in a manner dependent on the α5β1 integrin. This novel co-culture system, under serum-free defined conditions, allows for a molecular analysis of EC tube assembly, pericyte recruitment and maturation events in a critical ECM environment (i.e. fibrin matrices that regulates angiogenic events in postnatal life.

  2. ITO-Free Semitransparent Organic Solar Cells Based on Silver Thin Film Electrodes

    Directory of Open Access Journals (Sweden)

    Zhizhe Wang

    2014-01-01

    Full Text Available ITO-free semitransparent organic solar cells (OSCs based on MoO3/Ag anodes with poly(3-hexylthiophene and [6,6]-phenyl-C61-butyric acid methyl ester films as the active layer are investigated in this work. To obtain the optimal transparent (MoO3/Ag anode, ITO-free reference OSCs are firstly fabricated. The power conversion efficiency (PCE of 2.71% is obtained for OSCs based on the optimal MoO3 (2 nm/Ag (9 nm anode, comparable to that of ITO-based reference OSCs (PCE of 2.85%. Then based on MoO3 (2 nm/Ag (9 nm anode, ITO-free semitransparent OSCs with different thickness combination of Ca and Ag as the cathodes are investigated. It is observed from our results that OSCs with Ca (15 nm/Ag (15 nm cathode have the optimal transparency. Meanwhile, the PCE of 1.79% and 0.67% is obtained for illumination from the anode and cathode side, respectively, comparable to that of similar ITO-based semitransparent OSCs (PCE of 1.59% and 0.75% for illumination from the anode and cathode side, resp. (Sol. Energy Mater. Sol. Cells, 95, pp. 877–880, 2011. The transparency and PCE of ITO-free semitransparent OSCs can be further improved by introducing a light couple layer. The developed method is compatible with various substrates, which is instructive for further research of ITO-free semitransparent OSCs.

  3. Antitumor potential induction and free radicals production in melanoma cells by Boron Neutron Capture Therapy

    Energy Technology Data Exchange (ETDEWEB)

    Faiao-Flores, F. [Biochemical and Biophysical Laboratory, Butantan Institute, 1500 Vital Brasil Avenue, Sao Paulo (Brazil)] [Faculty of Medicine, University of Sao Paulo, 455 Doutor Arnaldo Avenue, Sao Paulo (Brazil); Coelho, P.R.P.; Muniz, R.O.R.; Souza, G.S. [Institute for Nuclear and Energy Research, 2242 Lineu Prestes Avenue, Sao Paulo (Brazil); Arruda-Neto, J. [Physics Institute, University of Sao Paulo, 187 Matao Street, Sao Paulo (Brazil)] [FESP, Sao Paulo Engineering School, 5520 Nove de Julho Avenue, Sao Paulo (Brazil); Maria, Durvanei A., E-mail: durvaneiaugusto@yahoo.com.br [Biochemical and Biophysical Laboratory, Butantan Institute, 1500 Vital Brasil Avenue, Sao Paulo (Brazil)

    2011-12-15

    Antiproliferative and oxidative damage effects occurring in Boron Neutron Capture Therapy (BNCT) in normal fibroblasts and melanoma cell lines were analyzed. Melanoma cells and normal fibroblasts were treated with different concentrations of Boronophenylalanine and irradiated with thermal neutron flux. The cellular viability and the oxidative stress were determined. BNCT induced free radicals production and proliferative potential inhibition in melanoma cells. Therefore, this therapeutic technique could be considered efficient to inhibit growth of melanoma with minimal effects on normal tissues. - Highlights: Black-Right-Pointing-Pointer Boron Neutron Capture Therapy (BNCT) induces melanoma cell death. Black-Right-Pointing-Pointer BNCT stimulates free radicals production and proliferative inhibition in melanoma cells. Black-Right-Pointing-Pointer It produces tumor membrane degeneration and destruction with apoptotic bodies formation. Black-Right-Pointing-Pointer This therapy damages tumor cells selectively, with minimum effects on normal adjacent tissue.

  4. Solution-Grown Monocrystalline Hybrid Perovskite Films for Hole-Transporter-Free Solar Cells

    KAUST Repository

    Peng, Wei

    2016-03-02

    High-quality perovskite monocrystalline films are successfully grown through cavitation-triggered asymmetric crystallization. These films enable a simple cell structure, ITO/CH3NH3PbBr3/Au, with near 100% internal quantum efficiency, promising power conversion efficiencies (PCEs) >5%, and superior stability for prototype cells. Furthermore, the monocrystalline devices using a hole-transporter-free structure yield PCEs ≈6.5%, the highest among other similar-structured CH3NH3PbBr3 solar cells to date.

  5. Serum-Free Cryopreservation of Five Mammalian Cell Lines in Either a Pelleted or Suspended State

    Directory of Open Access Journals (Sweden)

    Corsini Joe

    2004-01-01

    Full Text Available Herein we have explored two practical aspects of cryopreserving cultured mammalian cells during routine laboratory maintenance. First, we have examined the possibility of using a serum-free, hence more affordable, cryopreservative. Using five mammalian lines (Crandell Feline Kidney, MCF7, A72, WI 38 and NB324K, we found that the serum-free alternative preserves nearly as efficiently as the serum-containing preservatives. Second, we compared cryostorage of those cells in suspended versus a pellet form using both aforementioned cryopreservatives. Under our conditions, cells were in general recovered equally well in a suspended versus a pellet form.

  6. Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21

    Directory of Open Access Journals (Sweden)

    Schneider Yves-Jacques

    2006-05-01

    Full Text Available Abstract Background The absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. The cell lineage Caco-2 is a model commonly used to reproduce the features of the bowel epithelium. However, there is a strong debate regarding the value of Caco-2 cell culture to mimick in vivo situation. Indeed, some authors report in Caco-2 a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. The HT29-5M21 intestinal cell lineage is a mucin-secreting cellular population. A co-culture system carried out in a serum-free medium and comprising both Caco-2 and HT29-5M21 cells was developed. The systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure. Results In this study, we investigated the activity and localization of the alkaline phosphatase and the expression of IAP and MUC5AC genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of Caco-2 and HT29-5M21 cells. We observed that the culture conditions used (serum-free medium did not change the phenotype of each cell type, and produced a reproducible model. The alkaline phosphatase expression characterizing Caco-2 cells was influenced by the presence of HT29-5M21 cells. Conclusion The culture formed by 75% Caco-2 and 25% HT29-5M21 produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules.

  7. Burn-in Free Nonfullerene-Based Organic Solar Cells

    KAUST Repository

    Gasparini, Nicola; Salvador, Michael; Strohm, Sebastian; Heumueller, Thomas; Levchuk, Ievgen; Wadsworth, Andrew; Bannock, James H.; de Mello, John C.; Egelhaaf, Hans-Joachim; Baran, Derya; McCulloch, Iain; Brabec, Christoph J.

    2017-01-01

    Organic solar cells that are free of burn-in, the commonly observed rapid performance loss under light, are presented. The solar cells are based on poly(3-hexylthiophene) (P3HT) with varying molecular weights and a nonfullerene acceptor (rhodanine-benzothiadiazole-coupled indacenodithiophene, IDTBR) and are fabricated in air. P3HT:IDTBR solar cells light-soaked over the course of 2000 h lose about 5% of power conversion efficiency (PCE), in stark contrast to [6,6]-Phenyl C61 butyric acid methyl ester (PCBM)-based solar cells whose PCE shows a burn-in that extends over several hundreds of hours and levels off at a loss of ≈34%. Replacing PCBM with IDTBR prevents short-circuit current losses due to fullerene dimerization and inhibits disorder-induced open-circuit voltage losses, indicating a very robust device operation that is insensitive to defect states. Small losses in fill factor over time are proposed to originate from polymer or interface defects. Finally, the combination of enhanced efficiency and stability in P3HT:IDTBR increases the lifetime energy yield by more than a factor of 10 when compared with the same type of devices using a fullerene-based acceptor instead.

  8. Burn-in Free Nonfullerene-Based Organic Solar Cells

    KAUST Repository

    Gasparini, Nicola

    2017-07-03

    Organic solar cells that are free of burn-in, the commonly observed rapid performance loss under light, are presented. The solar cells are based on poly(3-hexylthiophene) (P3HT) with varying molecular weights and a nonfullerene acceptor (rhodanine-benzothiadiazole-coupled indacenodithiophene, IDTBR) and are fabricated in air. P3HT:IDTBR solar cells light-soaked over the course of 2000 h lose about 5% of power conversion efficiency (PCE), in stark contrast to [6,6]-Phenyl C61 butyric acid methyl ester (PCBM)-based solar cells whose PCE shows a burn-in that extends over several hundreds of hours and levels off at a loss of ≈34%. Replacing PCBM with IDTBR prevents short-circuit current losses due to fullerene dimerization and inhibits disorder-induced open-circuit voltage losses, indicating a very robust device operation that is insensitive to defect states. Small losses in fill factor over time are proposed to originate from polymer or interface defects. Finally, the combination of enhanced efficiency and stability in P3HT:IDTBR increases the lifetime energy yield by more than a factor of 10 when compared with the same type of devices using a fullerene-based acceptor instead.

  9. Low-Cost Upscaling Compatibility of Five Different ITO-Free Architectures for Polymer Solar Cells

    DEFF Research Database (Denmark)

    Angmo, Dechan; Gonzalez-Valls, Irene; Veenstra, Sjoerd

    2013-01-01

    Five different indium-tin-oxide free (ITO-free) polymer solar cell architectures provided by four participating research institutions that all presented a laboratory cell performance sufficient for use in mobile and information and communication technology (ICT) were evaluated based on photovoltaic...... performance and lifetime tests according to the ISOS protocols. The comparison of the different device architectures was performed using the same active material (P3HT: PCBM) and tested against an ITO-based reference device. The active area was 1 cm2 and rigid glass or flexible polyester substrates were...

  10. Is the climate system an anticipatory system that minimizes free energy?

    Science.gov (United States)

    Rubin, Sergio; Crucifix, Michel

    2017-04-01

    All systems, whether they are alive or not are structured determined systems, i.e. their present states [x (t)] depends of past states [x (t - α)]. However it has been suggested [Rosen, 1985; Friston, 2013] that systems that contain life are capable of anticipation and active inference. The underlying principle is that state changes in living systems are best modelled as a function of past and future states [ x(t) = f (x (t - α), x(t), x (t + β)) ]. The reason for this is that living systems contain a predictive model of their ambiance on which they are active: they appear to model their ambiance to preserve their integrity and homeorhesis. We therefore formulate the following hypothesis: can the climate system be interpreted as an anticipatory system that minimizes free energy? Can its variability (catastrophe, bifurcation and/or tipping points) be interpreted in terms of active inference and anticipation failure? Here we present a mathematical formulation of the climate system as an anticipatory system that minimizes free energy and its possible implication in the future climate predictability. References Rosen, R. (1985). Anticipatory systems. In Anticipatory systems (pp. 313-370). Springer New York. Friston, K. (2013). Life as we know it. Journal of the Royal Society Interface, 10(86), 20130475.

  11. Review of free software tools for image analysis of fluorescence cell micrographs.

    Science.gov (United States)

    Wiesmann, V; Franz, D; Held, C; Münzenmayer, C; Palmisano, R; Wittenberg, T

    2015-01-01

    An increasing number of free software tools have been made available for the evaluation of fluorescence cell micrographs. The main users are biologists and related life scientists with no or little knowledge of image processing. In this review, we give an overview of available tools and guidelines about which tools the users should use to segment fluorescence micrographs. We selected 15 free tools and divided them into stand-alone, Matlab-based, ImageJ-based, free demo versions of commercial tools and data sharing tools. The review consists of two parts: First, we developed a criteria catalogue and rated the tools regarding structural requirements, functionality (flexibility, segmentation and image processing filters) and usability (documentation, data management, usability and visualization). Second, we performed an image processing case study with four representative fluorescence micrograph segmentation tasks with figure-ground and cell separation. The tools display a wide range of functionality and usability. In the image processing case study, we were able to perform figure-ground separation in all micrographs using mainly thresholding. Cell separation was not possible with most of the tools, because cell separation methods are provided only by a subset of the tools and are difficult to parametrize and to use. Most important is that the usability matches the functionality of a tool. To be usable, specialized tools with less functionality need to fulfill less usability criteria, whereas multipurpose tools need a well-structured menu and intuitive graphical user interface. © 2014 Fraunhofer-Institute for Integrated Circuits IIS Journal of Microscopy © 2014 Royal Microscopical Society.

  12. Thermometry in dielectrophoresis chips for contact-free cell handling

    International Nuclear Information System (INIS)

    Jaeger, M S; Mueller, T; Schnelle, T

    2007-01-01

    Cell biology applications, protocols in immunology and stem cell research, require that individual cells are handled under strict control of their contacts to other cells or synthetic surfaces. Dielectrophoresis (DEP) in microfluidic chips is an established technique to investigate, group, wash, cultivate and sort cells contact-free under physiological conditions: microelectrode octode cages, versatile dielectrophoretic elements energized with radio frequency electric fields, stably trap single cells or cellular aggregates. For medical applications and cell cultivation, possible side effects of the dielectrophoretic manipulation, such as membrane polarization and Joule heating, have to be quantified. Therefore, we characterized the electric field-induced warming in dielectrophoretic cages using ohmic resistance measurements, fluorometry, liquid crystal beads, infra-red thermography and bubble size thermometry. We compare the results of these techniques with respect to the influences of voltage, electric conductivity of buffer, frequency, cage size and electrode surface. We conclude that in the culture medium thermal effects may be neglected if low voltages and an electric field-reducing phase pattern are used. Our experimental results provide explicit values for estimating the thermal effect on dielectrophoretically caged cells and show that Joule heating is best minimized by optimizing the cage geometry and reducing the buffer conductivity. The results may additionally serve to evaluate and improve theoretical predictions on field-induced effects. Based on present-day chip processing possibilities, DEP is well suited for the manipulation of cells

  13. Cell-free placental mRNA in maternal plasma to predict placental invasion in patients with placenta accreta.

    Science.gov (United States)

    El Behery, Manal M; Rasha L, Etewa; El Alfy, Yehya

    2010-04-01

    To evaluate whether measuring cell-free placental mRNA in maternal plasma improves the diagnostic accuracy of ultrasound and color Doppler in detecting placental invasion in patients at risk for placenta accreta. Thirty-five singleton pregnant women of more than 28 weeks of gestation and at risk for placenta accreta underwent ultrasound and color Doppler assessment. Cell-free placental mRNA in maternal plasma was measured using real-time reverse-transcription polymerase chain reaction. Patients were classified into 2 groups based on the findings at cesarean delivery and histological examination: women with placenta accreta (n=7) and women without placenta accreta (n=28). The median MoM (multiples of the median) value of cell-free placental mRNA was significantly higher in patients with placenta accreta than in those without placenta accreta (6.50 vs 2.60; Pplacental mRNA was significantly elevated in patients with placenta increta and percreta than in those with simple accreta. Six false-positive results were found on ultrasound, all from patients without placenta accreta and an insignificant rise in cell-free placental mRNA levels. Measuring cell-free placental mRNA in maternal plasma may increase the accuracy of ultrasound and color Doppler in prenatal prediction of placental invasion in patients with suspected placenta accreta. Copyright 2009 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.

  14. Gut microbiota and lipopolysaccharide content of the diet influence development of regulatory T cells: studies in germ-free mice.

    Science.gov (United States)

    Hrncir, Tomas; Stepankova, Renata; Kozakova, Hana; Hudcovic, Tomas; Tlaskalova-Hogenova, Helena

    2008-11-06

    Mammals are essentially born germ-free but the epithelial surfaces are promptly colonized by astounding numbers of bacteria soon after birth. The most extensive microbial community is harbored by the distal intestine. The gut microbiota outnumber ~10 times the total number of our somatic and germ cells. The host-microbiota relationship has evolved to become mutually beneficial. Studies in germ-free mice have shown that gut microbiota play a crucial role in the development of the immune system. The principal aim of the present study was to elucidate whether the presence of gut microbiota and the quality of a sterile diet containing various amounts of bacterial contaminants, measured by lipopolysaccharide (LPS) content, can influence maturation of the immune system in gnotobiotic mice. We have found that the presence of gut microbiota and to a lesser extent also the LPS-rich sterile diet drive the expansion of B and T cells in Peyer's patches and mesenteric lymph nodes. The most prominent was the expansion of CD4+ T cells including Foxp3-expressing T cells in mesenteric lymph nodes. Further, we have observed that both the presence of gut microbiota and the LPS-rich sterile diet influence in vitro cytokine profile of spleen cells. Both gut microbiota and LPS-rich diet increase the production of interleukin-12 and decrease the production of interleukin-4. In addition, the presence of gut microbiota increases the production of interleukin-10 and interferon-gamma. Our data clearly show that not only live gut microbiota but also microbial components (LPS) contained in sterile diet stimulate the development, expansion and function of the immune system. Finally, we would like to emphasize that the composition of diet should be regularly tested especially in all gnotobiotic models as the LPS content and other microbial components present in the diet may significantly alter the outcome of experiments.

  15. Label-Free Detection of Bacillus anthracis Spore Uptake in Macrophage Cells Using Analytical Optical Force Measurements.

    Science.gov (United States)

    Hebert, Colin G; Hart, Sean; Leski, Tomasz A; Terray, Alex; Lu, Qin

    2017-10-03

    Understanding the interaction between macrophage cells and Bacillus anthracis spores is of significant importance with respect to both anthrax disease progression, spore detection for biodefense, as well as understanding cell clearance in general. While most detection systems rely on specific molecules, such as nucleic acids or proteins and fluorescent labels to identify the target(s) of interest, label-free methods probe changes in intrinsic properties, such as size, refractive index, and morphology, for correlation with a particular biological event. Optical chromatography is a label free technique that uses the balance between optical and fluidic drag forces within a microfluidic channel to determine the optical force on cells or particles. Here we show an increase in the optical force experienced by RAW264.7 macrophage cells upon the uptake of both microparticles and B. anthracis Sterne 34F2 spores. In the case of spores, the exposure was detected in as little as 1 h without the use of antibodies or fluorescent labels of any kind. An increase in the optical force was also seen in macrophage cells treated with cytochalasin D, both with and without a subsequent exposure to spores, indicating that a portion of the increase in the optical force arises independent of phagocytosis. These results demonstrate the capability of optical chromatography to detect subtle biological differences in a rapid and sensitive manner and suggest future potential in a range of applications, including the detection of biological threat agents for biodefense and pathogens for the prevention of sepsis and other diseases.

  16. A defined, feeder-free, serum-free system to generate in vitro hematopoietic progenitors and differentiated blood cells from hESCs and hiPSCs.

    Directory of Open Access Journals (Sweden)

    Giorgia Salvagiotto

    2011-03-01

    Full Text Available Human ESC and iPSC are an attractive source of cells of high quantity and purity to be used to elucidate early human development processes, for drug discovery, and in clinical cell therapy applications. To efficiently differentiate pluripotent cells into a pure population of hematopoietic progenitors we have developed a new 2-dimensional, defined and highly efficient protocol that avoids the use of feeder cells, serum or embryoid body formation. Here we showed that a single matrix protein in combination with growth factors and a hypoxic environment is sufficient to generate from pluripotent cells hematopoietic progenitors capable of differentiating further in mature cell types of different lineages of the blood system. We tested the differentiation method using hESCs and 9 iPSC lines generated from different tissues. These data indicate the robustness of the protocol providing a valuable tool for the generation of clinical-grade hematopoietic cells from pluripotent cells.

  17. Nuclear delivery of recombinant OCT4 by chitosan nanoparticles for transgene-free generation of protein-induced pluripotent stem cells.

    Science.gov (United States)

    Tammam, Salma; Malak, Peter; Correa, Daphne; Rothfuss, Oliver; Azzazy, Hassan M E; Lamprecht, Alf; Schulze-Osthoff, Klaus

    2016-06-21

    Protein-based reprogramming of somatic cells is a non-genetic approach for the generation of induced pluripotent stem cells (iPSCs), whereby reprogramming factors, such as OCT4, SOX2, KLF4 and c-MYC, are delivered as functional proteins. The technique is considered safer than transgenic methods, but, unfortunately, most protein-based protocols provide very low reprogramming efficiencies. In this study, we developed exemplarily a nanoparticle (NP)-based delivery system for the reprogramming factor OCT4. To this end, we expressed human OCT4 in Sf9 insect cells using a baculoviral expression system. Recombinant OCT4 showed nuclear localization in Sf9 cells indicating proper protein folding. In comparison to soluble OCT4 protein, encapsulation of OCT4 in nuclear-targeted chitosan NPs strongly stabilized its DNA-binding activity even under cell culture conditions. OCT4-loaded NPs enabled cell treatment with high micromolar concentrations of OCT4 and successfully delivered active OCT4 into human fibroblasts. Chitosan NPs therefore provide a promising tool for the generation of transgene-free iPSCs.

  18. Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry.

    Directory of Open Access Journals (Sweden)

    Christian Niehage

    Full Text Available Multipotent mesenchymal stromal cells (MSCs are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2% or high (10% serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention.

  19. Understanding the free energy barrier and multiple timescale dynamics of charge separation in organic photovoltaic cells.

    Science.gov (United States)

    Yan, Yaming; Song, Linze; Shi, Qiang

    2018-02-28

    By employing several lattice model systems, we investigate the free energy barrier and real-time dynamics of charge separation in organic photovoltaic (OPV) cells. It is found that the combined effects of the external electric field, entropy, and charge delocalization reduce the free energy barrier significantly. The dynamic disorder reduces charge carrier delocalization and results in the increased charge separation barrier, while the effect of static disorder is more complicated. Simulation of the real-time dynamics indicates that the free charge generation process involves multiple time scales, including an ultrafast component within hundreds of femtoseconds, an intermediate component related to the relaxation of the hot charge transfer (CT) state, and a slow component on the time scale of tens of picoseconds from the thermally equilibrated CT state. Effects of hot exciton dissociation as well as its dependence on the energy offset between the Frenkel exciton and the CT state are also analyzed. The current results indicate that only a small energy offset between the band gap and the lowest energy CT state is needed to achieve efficient free charge generation in OPV devices, which agrees with recent experimental findings.

  20. Understanding the free energy barrier and multiple timescale dynamics of charge separation in organic photovoltaic cells

    Science.gov (United States)

    Yan, Yaming; Song, Linze; Shi, Qiang

    2018-02-01

    By employing several lattice model systems, we investigate the free energy barrier and real-time dynamics of charge separation in organic photovoltaic (OPV) cells. It is found that the combined effects of the external electric field, entropy, and charge delocalization reduce the free energy barrier significantly. The dynamic disorder reduces charge carrier delocalization and results in the increased charge separation barrier, while the effect of static disorder is more complicated. Simulation of the real-time dynamics indicates that the free charge generation process involves multiple time scales, including an ultrafast component within hundreds of femtoseconds, an intermediate component related to the relaxation of the hot charge transfer (CT) state, and a slow component on the time scale of tens of picoseconds from the thermally equilibrated CT state. Effects of hot exciton dissociation as well as its dependence on the energy offset between the Frenkel exciton and the CT state are also analyzed. The current results indicate that only a small energy offset between the band gap and the lowest energy CT state is needed to achieve efficient free charge generation in OPV devices, which agrees with recent experimental findings.

  1. Computer-Assisted English Learning System Based on Free Conversation by Topic

    Science.gov (United States)

    Choi, Sung-Kwon; Kwon, Oh-Woog; Kim, Young-Kil

    2017-01-01

    This paper aims to describe a computer-assisted English learning system using chatbots and dialogue systems, which allow free conversation outside the topic without limiting the learner's flow of conversation. The evaluation was conducted by 20 experimenters. The performance of the system based on a free conversation by topic was measured by the…

  2. Therapeutic potential of CAR-T cell-derived exosomes: a cell-free modality for targeted cancer therapy.

    Science.gov (United States)

    Tang, Xiang-Jun; Sun, Xu-Yong; Huang, Kuan-Ming; Zhang, Li; Yang, Zhuo-Shun; Zou, Dan-Dan; Wang, Bin; Warnock, Garth L; Dai, Long-Jun; Luo, Jie

    2015-12-29

    Chimeric antigen receptor (CAR)-based T-cell adoptive immunotherapy is a distinctively promising therapy for cancer. The engineering of CARs into T cells provides T cells with tumor-targeting capabilities and intensifies their cytotoxic activity through stimulated cell expansion and enhanced cytokine production. As a novel and potent therapeutic modality, there exists some uncontrollable processes which are the potential sources of adverse events. As an extension of this impactful modality, CAR-T cell-derived exosomes may substitute CAR-T cells to act as ultimate attackers, thereby overcoming some limitations. Exosomes retain most characteristics of parent cells and play an essential role in intercellular communications via transmitting their cargo to recipient cells. The application of CAR-T cell-derived exosomes will make this cell-based therapy more clinically controllable as it also provides a cell-free platform to diversify anticancer mediators, which responds effectively to the complexity and volatility of cancer. It is believed that the appropriate application of both cellular and exosomal platforms will make this effective treatment more practicable.

  3. Proinflammatory effect in whole blood by free soluble bacterial components released from planktonic and biofilm cells

    Directory of Open Access Journals (Sweden)

    Thay Bernard

    2008-11-01

    Full Text Available Abstract Background Aggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressive forms of periodontitis. Increasing evidence points to a link between periodontitis and cardiovascular diseases, however, the underlying mechanisms are poorly understood. This study investigated the pathogenic potential of free-soluble surface material, released from live planktonic and biofilm A. actinomycetemcomitans cells. Results By employing an ex vivo insert model (filter pore size 20 nm we demonstrated that the A. actinomycetemcomitans strain D7S and its derivatives, in both planktonic and in biofilm life-form, released free-soluble surface material independent of outer membrane vesicles. This material clearly enhanced the production of several proinflammatory cytokines (IL-1β, TNF-α, IL-6, IL-8, MIP-1β in human whole blood, as evidenced by using a cytokine antibody array and dissociation-enhanced-lanthanide-fluorescent-immunoassay. In agreement with this, quantitative real-time PCR indicated a concomitant increase in transcription of each of these cytokine genes. Experiments in which the LPS activity was blocked with polymyxin B showed that the stimulatory effect was only partly LPS-dependent, suggesting the involvement of additional free-soluble factors. Consistent with this, MALDI-TOF-MS and immunoblotting revealed release of GroEL-like protein in free-soluble form. Conversely, the immunomodulatory toxins, cytolethal distending toxin and leukotoxin, and peptidoglycan-associated lipoprotein, appeared to be less important, as evidenced by studying strain D7S cdt/ltx double, and pal single mutants. In addition to A. actinomycetemcomitans a non-oral species, Escherichia coli strain IHE3034, tested in the same ex vivo model also released free-soluble surface material with proinflammatory activity. Conclusion A. actinomycetemcomitans, grown in biofilm and planktonic form, releases free-soluble surface material independent of outer

  4. Proinflammatory effect in whole blood by free soluble bacterial components released from planktonic and biofilm cells.

    Science.gov (United States)

    Oscarsson, Jan; Karched, Maribasappa; Thay, Bernard; Chen, Casey; Asikainen, Sirkka

    2008-11-27

    Aggregatibacter actinomycetemcomitans is an oral bacterium associated with aggressive forms of periodontitis. Increasing evidence points to a link between periodontitis and cardiovascular diseases, however, the underlying mechanisms are poorly understood. This study investigated the pathogenic potential of free-soluble surface material, released from live planktonic and biofilm A. actinomycetemcomitans cells. By employing an ex vivo insert model (filter pore size 20 nm) we demonstrated that the A. actinomycetemcomitans strain D7S and its derivatives, in both planktonic and in biofilm life-form, released free-soluble surface material independent of outer membrane vesicles. This material clearly enhanced the production of several proinflammatory cytokines (IL-1 beta, TNF-alpha, IL-6, IL-8, MIP-1 beta) in human whole blood, as evidenced by using a cytokine antibody array and dissociation-enhanced-lanthanide-fluorescent-immunoassay. In agreement with this, quantitative real-time PCR indicated a concomitant increase in transcription of each of these cytokine genes. Experiments in which the LPS activity was blocked with polymyxin B showed that the stimulatory effect was only partly LPS-dependent, suggesting the involvement of additional free-soluble factors. Consistent with this, MALDI-TOF-MS and immunoblotting revealed release of GroEL-like protein in free-soluble form. Conversely, the immunomodulatory toxins, cytolethal distending toxin and leukotoxin, and peptidoglycan-associated lipoprotein, appeared to be less important, as evidenced by studying strain D7S cdt/ltx double, and pal single mutants. In addition to A. actinomycetemcomitans a non-oral species, Escherichia coli strain IHE3034, tested in the same ex vivo model also released free-soluble surface material with proinflammatory activity. A. actinomycetemcomitans, grown in biofilm and planktonic form, releases free-soluble surface material independent of outer membrane vesicles, which induces proinflammatory

  5. Efficient generation of integration-free human induced pluripotent stem cells from keratinocytes by simple transfection of episomal vectors.

    Science.gov (United States)

    Piao, Yulan; Hung, Sandy Shen-Chi; Lim, Shiang Y; Wong, Raymond Ching-Bong; Ko, Minoru S H

    2014-07-01

    Keratinocytes represent an easily accessible cell source for derivation of human induced pluripotent stem (hiPS) cells, reportedly achieving higher reprogramming efficiency than fibroblasts. However, most studies utilized a retroviral or lentiviral method for reprogramming of keratinocytes, which introduces undesirable transgene integrations into the host genome. Moreover, current protocols of generating integration-free hiPS cells from keratinocytes are mostly inefficient. In this paper, we describe a more efficient, simple-to-use, and cost-effective method for generating integration-free hiPS cells from keratinocytes. Our improved method using lipid-mediated transfection achieved a reprogramming efficiency of ∼0.14% on average. Keratinocyte-derived hiPS cells showed no integration of episomal vectors, expressed stem cell-specific markers and possessed potentials to differentiate into all three germ layers by in vitro embryoid body formation as well as in vivo teratoma formation. To our knowledge, this represents the most efficient method to generate integration-free hiPS cells from keratinocytes. ©AlphaMed Press.

  6. Artificial neural network-aided image analysis system for cell counting.

    Science.gov (United States)

    Sjöström, P J; Frydel, B R; Wahlberg, L U

    1999-05-01

    In histological preparations containing debris and synthetic materials, it is difficult to automate cell counting using standard image analysis tools, i.e., systems that rely on boundary contours, histogram thresholding, etc. In an attempt to mimic manual cell recognition, an automated cell counter was constructed using a combination of artificial intelligence and standard image analysis methods. Artificial neural network (ANN) methods were applied on digitized microscopy fields without pre-ANN feature extraction. A three-layer feed-forward network with extensive weight sharing in the first hidden layer was employed and trained on 1,830 examples using the error back-propagation algorithm on a Power Macintosh 7300/180 desktop computer. The optimal number of hidden neurons was determined and the trained system was validated by comparison with blinded human counts. System performance at 50x and lO0x magnification was evaluated. The correlation index at 100x magnification neared person-to-person variability, while 50x magnification was not useful. The system was approximately six times faster than an experienced human. ANN-based automated cell counting in noisy histological preparations is feasible. Consistent histology and computer power are crucial for system performance. The system provides several benefits, such as speed of analysis and consistency, and frees up personnel for other tasks.

  7. Norepinephrine stimulates progesterone production in highly estrogenic bovine granulosa cells cultured under serum-free, chemically defined conditions.

    Science.gov (United States)

    Piccinato, Carla A; Montrezor, Luis H; Collares, Cristhianna A V; Vireque, Alessandra A; Rosa e Silva, Alzira A M

    2012-11-22

    Since noradrenergic innervation was described in the ovarian follicle, the actions of the intraovarian catecholaminergic system have been the focus of a variety of studies. We aimed to determine the gonadotropin-independent effects of the catecholamine norepinephrine (NE) in the steroid hormone profile of a serum-free granulosa cell (GC) culture system in the context of follicular development and dominance. Primary bovine GCs were cultivated in a serum-free, chemically defined culture system supplemented with 0.1% polyvinyl alcohol. The culture features were assessed by hormone measurements and ultrastructural characteristics of GCs. GCs produced increasing amounts of estradiol and pregnenolone for 144h and maintained ultrastructural features of healthy steroidogenic cells. Progesterone production was also detected, although it significantly increased only after 96h of culture. There was a highly significant positive correlation between estradiol and pregnenolone production in high E2-producing cultures. The effects of NE were further evaluated in a dose-response study. The highest tested concentration of NE (10 (-7) M) resulted in a significant increase in progesterone production, but not in estradiol or pregnenolone production. The specificity of NE effects on progesterone production was further investigated by incubating GCs with propranolol (10 (-8) M), a non-selective beta-adrenergic antagonist. The present culture system represents a robust model to study the impact of intrafollicular factors, such as catecholamines, in ovarian steroidogenesis and follicular development. The results of noradrenergic effects in the steroidogenesis of GC have implications on physiological follicular fate and on certain pathological ovarian conditions such as cyst formation and anovulation.

  8. Hand-made cloned goat (Capra hircus) embryos—a comparison of different donor cells and culture systems.

    Science.gov (United States)

    Akshey, Yogesh S; Malakar, Dhruba; De, Arun K; Jena, Manoj K; Garg, Shweta; Dutta, Rahul; Pawar, Sachin Kumar; Mukesh, Manisha

    2010-10-01

    Nuclear transfer is a very effective method for propagation of valuable, extinct, and endangered animals. Hand-made cloning (HMC) is an efficient alternative to the conventional micromanipulator-based technique in some domestic species. The present study was carried out for the selection of suitable somatic cells as a nuclear donor and development of an optimum culture system for in vitro culture of zona-free goat cloned embryos. Cleavage and blastocyst rates were observed 72.06 ± 2.94% and 0% for fresh cumulus cells, 81.95 ± 3.40% and 12.74 ± 2.12% for cultured cumulus cells, and 92.94 ± 0.91% and 23.78 ± 3.33% for fetal fibroblast cells, respectively. There was a significant (p cloned embryos and donor cells. In conclusion, the present study describes that the fetal fibroblast cell is a suitable candidate as nuclear donor, and the flat surface culture system is suitable for zona-free blastocyst development by the hand-made cloning technique in the goat.

  9. Isolation and characterization of Wharton’s jelly-derived multipotent mesenchymal stromal cells obtained from bovine umbilical cord and maintained in a defined serum-free three-dimensional system

    Directory of Open Access Journals (Sweden)

    Cardoso Tereza C

    2012-05-01

    Full Text Available Abstract Background The possibility for isolating bovine mesenchymal multipotent cells (MSCs from fetal adnexa is an interesting prospect because of the potential for these cells to be used for biotechnological applications. Bone marrow and adipose tissue are the most common sources of MSCs derived from adult animals. However, little knowledge exists about the characteristics of these progenitors cells in the bovine species. Traditionally most cell cultures are developed in two dimensional (2D environments. In mammalian tissue, cells connect not only to each other, but also support structures called the extracellular matrix (ECM. The three-dimensional (3D cultures may play a potential role in cell biotechnology, especially in tissue therapy. In this study, bovine-derived umbilical cord Wharton’s jelly (UC-WJ cells were isolated, characterized and maintained under 3D-free serum condition as an alternative of stem cell source for future cell banking. Results Bovine-derived UC-WJ cells, collected individually from 5 different umbilical cords sources, were successfully cultured under serum-free conditions and were capable to support 60 consecutive passages using commercial Stemline® mesenchymal stem cells expansion medium. Moreover, the UC-WJ cells were differentiated into osteocytes, chondrocytes, adipocytes and neural-like cells and cultured separately. Additionally, the genes that are considered important embryonic, POU5F1 and ITSN1, and mesenchymal cell markers, CD105+, CD29+, CD73+ and CD90+ in MSCs were also expressed in five bovine-derived UC-WJ cultures. Morphology of proliferating cells typically appeared fibroblast-like spindle shape presenting the same viability and number. These characteristics were not affected during passages. There were 60 chromosomes at the metaphase, with acrocentric morphology and intense telomerase activity. Moreover, the proliferative capacity of T cells in response to a mitogen stimulus was suppressed when

  10. Cell-free DNA levels and correlation to stage and outcome following treatment of locally advanced rectal cancer.

    Science.gov (United States)

    Boysen, Anders Kindberg; Wettergren, Yvonne; Sorensen, Boe Sandahl; Taflin, Helena; Gustavson, Bengt; Spindler, Karen-Lise Garm

    2017-11-01

    Accurate staging of rectal cancer remains essential for optimal patient selection for combined modality treatment, including radiotherapy, chemotherapy and surgery. We aimed at examining the correlation of cell free DNA with the pathologic stage and subsequent risk of recurrence for patients with locally advanced rectal cancer undergoing preoperative chemoradiation. We examined 75 patients with locally advanced rectal cancer receiving preoperative chemoradiation. Blood samples for translational use were drawn prior to rectal surgery. The level of cell free DNA was quantified by digital droplet PCR and expressed as copy number of beta 2 microglobulin. We found a median level of cell free DNA in the AJCC stages I-III of 3100, 8300, and 10,700 copies/mL respectively. For patients with 12 sampled lymph nodes or above, the median level of cell free DNA were 2400 copies/mL and 4400 copies/mL (p = 0.04) for node negative and node positive disease respectively. The median follow-up was 39 months and 11 recurrences were detected (15%). The median level for patients with recurrent disease was 13,000 copies/mL compared to 5200 copies/mL for non-recurrent patients (p = 0.08). We have demonstrated a correlation between the level of total cell free DNA and the pathologic stage and nodal involvement. Furthermore, we have found a trend towards a correlation with the risk of recurrence following resection of localized rectal cancer.

  11. Cell-free translational screening of an expression sequence tag library of Clonorchis sinensis for novel antigen discovery.

    Science.gov (United States)

    Kasi, Devi; Catherine, Christy; Lee, Seung-Won; Lee, Kyung-Ho; Kim, Yu Jung; Ro Lee, Myeong; Ju, Jung Won; Kim, Dong-Myung

    2017-05-01

    The rapidly evolving cloning and sequencing technologies have enabled understanding of genomic structure of parasite genomes, opening up new ways of combatting parasite-related diseases. To make the most of the exponentially accumulating genomic data, however, it is crucial to analyze the proteins encoded by these genomic sequences. In this study, we adopted an engineered cell-free protein synthesis system for large-scale expression screening of an expression sequence tag (EST) library of Clonorchis sinensis to identify potential antigens that can be used for diagnosis and treatment of clonorchiasis. To allow high-throughput expression and identification of individual genes comprising the library, a cell-free synthesis reaction was designed such that both the template DNA and the expressed proteins were co-immobilized on the same microbeads, leading to microbead-based linkage of the genotype and phenotype. This reaction configuration allowed streamlined expression, recovery, and analysis of proteins. This approach enabled us to identify 21 antigenic proteins. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:832-837, 2017. © 2017 American Institute of Chemical Engineers.

  12. Automated Micro-Object Detection for Mobile Diagnostics Using Lens-Free Imaging Technology

    Directory of Open Access Journals (Sweden)

    Mohendra Roy

    2016-05-01

    Full Text Available Lens-free imaging technology has been extensively used recently for microparticle and biological cell analysis because of its high throughput, low cost, and simple and compact arrangement. However, this technology still lacks a dedicated and automated detection system. In this paper, we describe a custom-developed automated micro-object detection method for a lens-free imaging system. In our previous work (Roy et al., we developed a lens-free imaging system using low-cost components. This system was used to generate and capture the diffraction patterns of micro-objects and a global threshold was used to locate the diffraction patterns. In this work we used the same setup to develop an improved automated detection and analysis algorithm based on adaptive threshold and clustering of signals. For this purpose images from the lens-free system were then used to understand the features and characteristics of the diffraction patterns of several types of samples. On the basis of this information, we custom-developed an automated algorithm for the lens-free imaging system. Next, all the lens-free images were processed using this custom-developed automated algorithm. The performance of this approach was evaluated by comparing the counting results with standard optical microscope results. We evaluated the counting results for polystyrene microbeads, red blood cells, and HepG2, HeLa, and MCF7 cells. The comparison shows good agreement between the systems, with a correlation coefficient of 0.91 and linearity slope of 0.877. We also evaluated the automated size profiles of the microparticle samples. This Wi-Fi-enabled lens-free imaging system, along with the dedicated software, possesses great potential for telemedicine applications in resource-limited settings.

  13. A simple, xeno-free method for oligodendrocyte generation from human neural stem cells derived from umbilical cord: engagement of gelatinases in cell commitment and differentiation.

    Science.gov (United States)

    Sypecka, Joanna; Ziemka-Nalecz, Małgorzata; Dragun-Szymczak, Patrycja; Zalewska, Teresa

    2017-05-01

    Oligodendrocyte progenitors (OPCs) are ranked among the most likely candidates for cell-based strategies aimed at treating neurodegenerative diseases accompanied by dys/demyelination of the central nervous system (CNS). In this regard, different sources of stem cells are being tested to elaborate xeno-free protocols for efficient generation of OPCs for clinical applications. In the present study, neural stem cells of human umbilical cord blood (HUCB-NSCs) have been used to derive OPCs and subsequently to differentiate them into mature, GalC-expressing oligodendrocytes. Applied components of the extracellular matrix (ECM) and the analogues of physiological substances known to increase glial commitment of neural stem cells have been shown to significantly increase the yield of the resulting OPC fraction. The efficiency of ECM components in promoting oligodendrocyte commitment and differentiation prompted us to investigate the potential role of gelatinases in those processes. Subsequently, endogenous and ECM metalloproteinases (MMPs) activity has been compared with that detected in primary cultures of rat oligodendrocytes in vitro, as well as in rat brains in vivo. The data indicate that gelatinases are engaged in gliogenesis both in vitro and in vivo, although differently, which presumably results from distinct extracellular conditions. In conclusion, the study presents an efficient xeno-free method of deriving oligodendrocyte from HUCB-NSCs and analyses the engagement of MMP-2/MMP-9 in the processes of cell commitment and maturation. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  14. Free and Cell Wall-Bound Polyamines under Long-Term Water Stress Applied at Different Growth Stages of ×Triticosecale Wittm.

    Directory of Open Access Journals (Sweden)

    Tomasz Hura

    Full Text Available Long-stemmed and semi-dwarf cultivars of triticale were exposed to water stress at tillering, heading and anthesis stage. Quantitative determination of free and cell wall-bound polyamines, i.e. agmatine, cadaverine, putrescine, spermidine and spermine, was supplemented with an analysis of quantitative relationships between free and cell wall-bound polyamines.The content of free and cell wall-bound polyamines varied depending on the development stage, both under optimal and water stress conditions. Drought-induced increase in free agmatine content was observed at all developmental stages in long-stemmed cultivar. A depletion of spermidine and putrescine was also reported in this cultivar, and spermidine was less abundant in semi-dwarf cultivar exposed to drought stress at the three analyzed developmental stages. Changes in the content of the other free polyamines did not follow a steady pattern reflecting the developmental stages. On the contrary, the content of cell wall-bound polyamines gradually increased from tillering, through heading and until anthesis period.Water stress seemed to induce a progressive decrease in the content of free polyamines and an accumulation of cell wall-bound polyamines.

  15. Growth of single T cells and single thymocytes in a high cloning efficiency filler-cell free microculture system.

    Science.gov (United States)

    Chen, W F; Ewing, T; Scollay, R; Shortman, K

    1988-01-01

    A high cloning-efficiency microculture system is described in which single T cells, stimulated to divide by phorbol ester and calcium ionophore, grow rapidly under the influence of purified growth factors in the absence of other cells. The kinetics of clonal growth has been monitored over a five day period by phase-contrast microscopy. Mature peripheral T cells, and mature subpopulations from the thymus, responded with a cloning efficiency over 80%; they required IL-2 as a minimum but several other factors enhanced growth. Ly2+L3T4- thymocytes (mean doubling time 10.4 hr) grew more rapidly than Ly2-L3T4+ thymocytes (mean doubling time 15.2 hr). Early (Ly2-L3T4-) thymocytes responded with a cloning efficiency of 60%; their efficient growth was dependent on both IL-1 and IL-2. The typical Ly2+L3T4+ cortical thymocyte did not grow under these conditions.

  16. Bioethanol production from starchy biomass by direct fermentation using saccharomyces diastaticus in batch free and immobilized cell systems

    Energy Technology Data Exchange (ETDEWEB)

    Kilonzo, P.M.; Margaritis, A. [University of Western Ontario, London, ON (Canada). Dept. of Chemical and Biochemical Engineering; Yu, J.; Ye, Q. [East China Univ. of Science and Technology, Shanghai (China). Biochemical Engineering Research Inst. and State Key Lab

    2006-07-01

    The feasibility of using amylolytic yeasts for the direct fermentation of starchy biomass to ethanol was discussed. Although amylolytic yeasts such as Saccharomycopsis, Lipomyces, and Schwaniomyces secrete both {alpha}-amylase and glucoamylase enzymes that synergistically enhance starch degradation, they are not suitable for industrial bio-ethanol production because of low tolerance for ethanol and slow fermentation rate. For that reason, this study examined the direct ethanol fermentation of soluble starch or dextrin with the amylolytic yeast Saccharomyces diastaticus in batch free and immobilized cells systems. Saccharomyces diastaticus secretes glucoamylase and can therefore assimilate and ferment starch and starch-like biomass. The main focus of the study was on parameters leading to higher ethanol yields from high concentration of dextrin and soluble starch using batch cultures. A natural attachment method was proposed in which polyurethane foam sheets were used as the carrier for amylolytic yeasts immobilization in ethanol fermentations. The support was chosen because it was inexpensive, autoclavable, pliable and could be tailored to suit process requirements regarding net surface charge, shape and size. It was found that Saccharomyces diastaticus was very efficient in terms of fermentation of high initial concentrations of dextrin or soluble starch. Higher concentrations of ethanol were produced. In batch fermentations, the cells fermented high dextrin concentrations more efficiently. In particular, in batch fermentation, more than 92 g-L of ethanol was produced from 240 g-L of dextrin, at conversion efficiency of 90 per cent. The conversion efficiency decreased to 60 per cent but a higher final ethanol concentration of 147 g/L was attained with a medium containing 500 g/L of dextrin. In an immobilized cell bioreactor, Saccharomyces diastaticus produced 83 g/L of ethanol from 240 g/L of dextrin, corresponding to ethanol volumetric productivity of 9.1 g

  17. Derivation of xeno-free and GMP-grade human embryonic stem cells--platforms for future clinical applications.

    Directory of Open Access Journals (Sweden)

    Shelly E Tannenbaum

    Full Text Available Clinically compliant human embryonic stem cells (hESCs should be developed in adherence to ethical standards, without risk of contamination by adventitious agents. Here we developed for the first time animal-component free and good manufacturing practice (GMP-compliant hESCs. After vendor and raw material qualification, we derived xeno-free, GMP-grade feeders from umbilical cord tissue, and utilized them within a novel, xeno-free hESC culture system. We derived and characterized three hESC lines in adherence to regulations for embryo procurement, and good tissue, manufacturing and laboratory practices. To minimize freezing and thawing, we continuously expanded the lines from initial outgrowths and samples were cryopreserved as early stocks and banks. Batch release criteria included DNA-fingerprinting and HLA-typing for identity, characterization of pluripotency-associated marker expression, proliferation, karyotyping and differentiation in-vitro and in-vivo. These hESCs may be valuable for regenerative therapy. The ethical, scientific and regulatory methodology presented here may serve for development of additional clinical-grade hESCs.

  18. Chrome - Free Aluminum Coating System

    Science.gov (United States)

    Bailey, John H.; Gugel, Jeffrey D.

    2010-01-01

    This slide presentation concerns the program to qualify a chrome free coating for aluminum. The program was required due to findings by OSHA and EPA, that hexavalent chromium, used to mitigate corrosion in aerospace aluminum alloys, poses hazards for personnel. This qualification consisted of over 4,000 tests. The tests revealed that a move away from Cr+6, required a system rather than individual components and that the maximum corrosion protection required pretreatment, primer and topcoat.

  19. Toward microfluidic sperm refinement: continuous flow label-free analysis and sorting of sperm cells

    NARCIS (Netherlands)

    de Wagenaar, B.; Dekker, Stefan; van den Berg, Albert; Segerink, Loes Irene

    2015-01-01

    This manuscript reports upon the development of a microfluidic setup to detect and sort sperm cells from polystyrene beads label-free and non-invasively. Detection is performed by impedance analysis. When sperm cells passed the microelectrodes, the recorded impedance (19.6 ± 5.7 Ω) was higher

  20. Xeno-Free Strategies for Safe Human Mesenchymal Stem/Stromal Cell Expansion: Supplements and Coatings

    Directory of Open Access Journals (Sweden)

    M. Cimino

    2017-01-01

    Full Text Available Human mesenchymal stem/stromal cells (hMSCs have generated great interest in regenerative medicine mainly due to their multidifferentiation potential and immunomodulatory role. Although hMSC can be obtained from different tissues, the number of available cells is always low for clinical applications, thus requiring in vitro expansion. Most of the current protocols for hMSC expansion make use of fetal bovine serum (FBS as a nutrient-rich supplement. However, regulatory guidelines encourage novel xeno-free alternatives to define safer and standardized protocols for hMSC expansion that preserve their intrinsic therapeutic potential. Since hMSCs are adherent cells, the attachment surface and cell-adhesive components also play a crucial role on their successful expansion. This review focuses on the advantages/disadvantages of FBS-free media and surfaces/coatings that avoid the use of animal serum, overcoming ethical issues and improving the expansion of hMSC for clinical applications in a safe and reproducible way.

  1. Environmentally Friendly Plasma-Treated PEDOT:PSS as Electrodes for ITO-Free Perovskite Solar Cells.

    Science.gov (United States)

    Vaagensmith, Bjorn; Reza, Khan Mamun; Hasan, Md Nazmul; Elbohy, Hytham; Adhikari, Nirmal; Dubey, Ashish; Kantack, Nick; Gaml, Eman; Qiao, Qiquan

    2017-10-18

    Solution processed poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) transparent electrodes (TEs) offer great potential as a low cost alternative to expensive indium tin oxide (ITO). However, strong acids are typically used for enhancing the conductivity of PEDOT:PSS TEs, which produce processing complexity and environmental issues. This work presents an environmentally friendly acid free approach to enhance the conductivity of PEDOT:PSS using a light oxygen plasma treatment, in addition to solvent blend additives and post treatments. The plasma treatment was found to significantly reduce the sheet resistance of PEDOT:PSS TEs from 85 to as low as 15 Ω sq -1 , which translates to the highest reported conductivity of 5012 S/cm for PEDOT:PSS TEs. The plasma treated PEDOT:PSS TE resulted in an ITO-free perovskite solar cell efficiency of 10.5%, which is the highest reported efficiency for ITO-free perovskite solar cells with a PEDOT:PSS electrode that excludes the use of acid treatments. This research presents the first demonstration of this technology. Moreover, the PEDOT:PSS TEs enabled better charge extraction from the perovskite solar cells and reduced hysteresis in the current density-voltage (J-V) curves.

  2. Increased cell-free DNA concentrations in patients with obstructive sleep apnea.

    Science.gov (United States)

    Shin, Chol; Kim, Jin K; Kim, Je H; Jung, Ki H; Cho, Kyung J; Lee, Chang K; Lee, Seung G

    2008-12-01

    Blood concentrations of cell-free DNA, which is considered to be released during apoptosis, are elevated under some pathological conditions such as cardiovascular disease and cancer. The association between obstructive sleep apnea (OSA) and cell-free DNA concentrations has not been reported so far. The purpose of the present study was to examine the association between OSA and plasma DNA concentrations. A case-control study was conducted using a total of 164 men aged 39-67 years, who were free of coronary heart disease and cancer. Laboratory-based overnight polysomnography was performed for all participants. On the basis of polysomnography, patients with an apnea-hypopnea index (AHI) = 5-30 events/h were defined as having mild-moderate OSA (n = 33) and those with >30 events/h were defined as having severe OSA (n = 49). All 82 controls had AHI DNA concentrations from all participants were analyzed for the beta-globin gene using fluorescence-based real-time polymerase chain reaction. Patients with severe OSA had significantly higher plasma DNA concentrations than persons with mild-moderate OSA and those without OSA (P DNA concentration (P DNA concentrations (>8 microg/L) had approximately fourfold higher odds of OSA than those with low DNA levels. Further data are warranted to confirm the association for men and to evaluate the association for women.

  3. Combining tissue repair and tissue engineering ; bioactivating implantable cell-free vascular scaffolds

    NARCIS (Netherlands)

    Muylaert, D.E.P.; Fledderus, J.O.; Bouten, C.V.C.; Dankers, P.Y.W.; Verhaar, M.C.

    2014-01-01

    Synthetic replacement grafts for heart valves and small-diameter blood vessels such as coronary arteries have the potential to circumvent many of the limitations of currently available autologous grafting materials. Cell-free material incorporating biologically active compounds may guide the

  4. Active Stat3 is required for survival of human squamous cell carcinoma cells in serum-free conditions

    Directory of Open Access Journals (Sweden)

    DiGiovanni John

    2006-04-01

    Full Text Available Abstract Background Squamous cell carcinoma (SCC of the skin is the most aggressive form of non-melanoma skin cancer (NMSC, and is the single most commonly diagnosed cancer in the U.S., with over one million new cases reported each year. Recent studies have revealed an oncogenic role of activated signal transducer and activator of transcription 3 (Stat3 in many human tumors, especially in those of epithelial origin, including skin SCC. Stat3 is a mediator of numerous growth factor and cytokine signaling pathways, all of which activate it through phosphorylation of tyrosine 705. Results To further address the role of Stat3 in skin SCC tumorigenesis, we have analyzed a panel of human skin-derived cell lines ranging from normal human epidermal keratinocytes (NHEK, to non-tumorigenic transformed skin cells (HaCaT, to highly tumorigenic cells (SRB1-m7 and SRB12-p9 and observed a positive correlation between Stat3 phosphorylation and SCC malignancy. We next determined the role of Stat3 activity in cell proliferation and viability under serum-free culture conditions. This was accomplished by suppressing Stat3 activity in the SRB12-p9 cells through stable expression of a dominant negative acting form of Stat3β, which contains a tyrosine 705 to phenylalanine mutation (S3DN. The S3DN cells behaved similar to parental SRB12-p9 cells when cultured in optimal growth conditions, in the presence of 10% fetal calf serum. However, unlike the SRB12-p9 cells, S3DN cells underwent apoptotic cell death when cultured in serum-free medium (SFM. This was evidenced by multiple criteria, including accumulation of sub-G1 particles, induced PARP cleavage, and acquisition of the characteristic morphological changes associated with apoptosis. Conclusion This study provides direct evidence for a role for Stat3 in maintaining cell survival in the conditions of exogenous growth factor deprivation produced by culture in SFM. We also propose that delivery of the S3DN gene or

  5. Human dental pulp stem cells cultured in serum-free supplemented medium

    Directory of Open Access Journals (Sweden)

    Virginie eBonnamain

    2013-12-01

    Full Text Available Growing evidence show that human dental pulp stem cells (DPSCs could provide a source of adult stem cells for the treatment of neurodegenerative pathologies. In this study, DPSCs were expanded and cultured with a protocol generally used for the culture of neural stem/progenitor cells.Methodology: DPSC cultures were established from third molars. The pulp tissue was enzymatically digested and cultured in serum-supplemented basal medium for 12 hours. Adherent (ADH and non-adherent (non-ADH cell populations were separated according to their differential adhesion to plastic and then cultured in serum-free defined N2 medium with epidermal growth factor (EGF and basic fibroblast growth factor (bFGF. Both ADH and non-ADH populations were analyzed by FACS and/or PCR.Results: FACS analysis of ADH-DPSCs revealed the expression of the mesenchymal cell marker CD90, the neuronal marker CD56, the transferrin receptor CD71, and the chemokine receptor CXCR3, whereas hematopoietic stem cells markers CD45, CD133 and CD34 were not expressed. ADH-DPSCs expressed transcripts coding for the Nestin gene, whereas expression levels of genes coding for the neuronal markers β-III tubulin and NF-M, and the oligodendrocyte marker PLP-1 were donor dependent. ADH-DPSCs did not express the transcripts for GFAP, an astrocyte marker. Cells of the non-ADH population that grew as spheroids expressed Nestin, β-III tubulin, NF-M and PLP-1 transcripts. DPSCs migrated out of the spheroids exhibited an odontoblast-like morphology and expressed a higher level of DSPP and osteocalcin transcripts than ADH-DPSCs. Conclusion: Collectively, these data indicate that human DPSCs can be expended and cultured in serum-free supplemented medium with EGF and bFGF. ADH-DPSCs and non-ADH populations contained neuronal and/or oligodendrocyte precursors at different stages of commitment and interestingly, cells from spheroid structures seem to be more engaged into the odontoblastic lineage than the

  6. Machining and metrology systems for free-form laser printer mirrors

    Indian Academy of Sciences (India)

    R. Narasimhan (Krishtel eMaging) 1461 1996 Oct 15 13:05:22

    optical systems were designed and manufactured for the individual laser wavelength to be ... The design objective in utilizing a free-form mirror has resulted in a corresponding ... Since mass production of these free-form mirrors is required, the ...

  7. Maternal plasma levels of cell-free β-HCG mRNA as a prenatal diagnostic indicator of placenta accrete.

    Science.gov (United States)

    Zhou, J; Li, J; Yan, P; Ye, Y H; Peng, W; Wang, S; Wang, X Tong

    2014-09-01

    Several biomarkers, including maternal serum creatinine kinase and α-fetoprotein, have been described as potential tools for the diagnosis of placental abnormalities. This study aimed to determine whether maternal plasma mRNA levels of the β subunit of human chorionic gonadotropin (β-HCG) could predict placenta accreta prenatally. Sixty-eight singleton pregnant women with prior cesarean deliveries (CDs) were classified into three groups: normal placentation (35 women, control group); placenta previa alone (21 women, placenta previa group); and both placenta previa and placenta accreta (12 women, placenta previa/accreta group). Maternal plasma concentrations of cell-free β-HCG mRNA were measured by real-time reverse-transcription polymerase chain reaction and were expressed as multiples of the median (MoM). Cell-free β-HCG mRNA concentrations (MoM, range) were significantly higher in women with placenta accreta (3.65, 2.78-7.19) than in women with placenta previa (0.94, 0.00-2.97) or normal placentation (1.00, 0.00-2.69) (Steel-Dwass test, P accreta group, the concentration of cell-free β-HCG mRNA was significantly higher among women who underwent CDs with hysterectomy (4.41, 3.49-7.19) than among women whose CDs did not result in hysterectomy (3.20, 2.78-3.70) (Mann-Whitney U test, P = 0.012). An increased level of cell-free β-HCG mRNA in the maternal plasma of women with placenta accreta may arise from direct uteroplacental transfer of cell-free placental mRNA molecules. The concentration of cell-free β-HCG mRNA in maternal plasma may be applicable to the prenatal diagnosis of placenta accreta, especially to identify women with placenta accreta likely to require hysterectomy. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Surface free energy predominates in cell adhesion to hydroxyapatite through wettability.

    Science.gov (United States)

    Nakamura, Miho; Hori, Naoko; Ando, Hiroshi; Namba, Saki; Toyama, Takeshi; Nishimiya, Nobuyuki; Yamashita, Kimihiro

    2016-05-01

    The initial adhesion of cells to biomaterials is critical in the regulation of subsequent cell behaviors. The purpose of this study was to investigate a mechanism through which the surface wettability of biomaterials can be improved and determine the effects of biomaterial surface characteristics on cellular behaviors. We investigated the surface characteristics of various types of hydroxyapatite after sintering in different atmospheres and examined the effects of various surface characteristics on cell adhesion to study cell-biomaterial interactions. Sintering atmosphere affects the polarization capacity of hydroxyapatite by changing hydroxide ion content and grain size. Compared with hydroxyapatite sintered in air, hydroxyapatite sintered in saturated water vapor had a higher polarization capacity that increased surface free energy and improved wettability, which in turn accelerated cell adhesion. We determined the optimal conditions of hydroxyapatite polarization for the improvement of surface wettability and acceleration of cell adhesion. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Biosynthesis of 12α-and 13-hydroxylated gibberellins in a cell-free system from Cucurbita maxima endosperm and the identification of new endogenous gibberellins.

    Science.gov (United States)

    Lange, T; Hedden, P; Graebe, J E

    1993-03-01

    Gibberellin (GA) biosynthesis in cell-free systems from Cucurbita maxima L. endosperm was reinvestigated using incubation conditions different from those employed in previous work. The metabolism of GA12 yielded GA13, GA43 and 12α-hydroxyGA43 as major products, GA4, GA37, GA39, GA46 and four unidentified compounds as minor products. The intermediates GA15, GA24 and GA25 accumulated at low protein concentrations. The structure of the previously uncharacterised 12α-hydroxyGA43 was inferred from its mass spectrum and by its formation from both GA39 and GA43. Gibberellin A39 and 12α-hydroxyGA43 were formed by a soluble 12α-hydroxylase that had not been detected before. Gibberellin A12-aldehyde was metabolised to essentially the same products as GA12 but with less efficiency. A new 13-hydroxylation pathway was found. Gibberellin A53, formed from GA12 by a microsomal oxidase, was converted by soluble 2-oxoglutarate-dependent oxidases to GA1 GA23, GA28, GA44, and putative 2β-hydroxyGA28. Minor products were GA19, GA20, GA38 and three unidentified GAs. Microsomal 13-hydroxylation (the formation of GA53) was suppressed by the cofactors for 2-oxoglutarate-dependent enzymes. Reinvestigation of the endogenous GAs confirmed the significance of the new metabolic products. In addition to the endogenous GAs reported by Blechschmidt et al. (1984, Phytochemistry 23, 553-558), GA1, GA8, GA25, GA28, GA36, GA48 and 12α-hydroxyGA43 were identified by full-scan capillary gas chromatography-mass spectrometry and Kovats retention indices. Thus both the 12α-hydroxylation and the 13-hydroxylation pathways found in the cell-free system operate also in vivo, giving rise to 12α-hydroxyGA43 and GA1 (or GA8), respectively, as their end products. Evidence for endogenous GA20 and GA24 was also obtained but it was less conclusive due to interference.

  10. Norepinephrine stimulates progesterone production in highly estrogenic bovine granulosa cells cultured under serum-free, chemically defined conditions

    Directory of Open Access Journals (Sweden)

    Piccinato Carla A

    2012-11-01

    Full Text Available Abstract Background Since noradrenergic innervation was described in the ovarian follicle, the actions of the intraovarian catecholaminergic system have been the focus of a variety of studies. We aimed to determine the gonadotropin-independent effects of the catecholamine norepinephrine (NE in the steroid hormone profile of a serum-free granulosa cell (GC culture system in the context of follicular development and dominance. Methods Primary bovine GCs were cultivated in a serum-free, chemically defined culture system supplemented with 0.1% polyvinyl alcohol. The culture features were assessed by hormone measurements and ultrastructural characteristics of GCs. Results GCs produced increasing amounts of estradiol and pregnenolone for 144h and maintained ultrastructural features of healthy steroidogenic cells. Progesterone production was also detected, although it significantly increased only after 96h of culture. There was a highly significant positive correlation between estradiol and pregnenolone production in high E2-producing cultures. The effects of NE were further evaluated in a dose–response study. The highest tested concentration of NE (10 (−7 M resulted in a significant increase in progesterone production, but not in estradiol or pregnenolone production. The specificity of NE effects on progesterone productio n was further investigated by incubating GCs with propranolol (10 (−8 M, a non-selective beta-adrenergic antagonist. Conclusions The present culture system represents a robust model to study the impact of intrafollicular factors, such as catecholamines, in ovarian steroidogenesis and follicular development. The results of noradrenergic effects in the steroidogenesis of GC have implications on physiological follicular fate and on certain pathological ovarian conditions such as cyst formation and anovulation.

  11. A defined and xeno-free culture method enabling the establishment of clinical-grade human embryonic, induced pluripotent and adipose stem cells.

    Directory of Open Access Journals (Sweden)

    Kristiina Rajala

    2010-04-01

    Full Text Available The growth of stem cells in in vitro conditions requires optimal balance between signals mediating cell survival, proliferation, and self-renewal. For clinical application of stem cells, the use of completely defined conditions and elimination of all animal-derived materials from the establishment, culture, and differentiation processes is desirable.Here, we report the development of a fully defined xeno-free medium (RegES, capable of supporting the expansion of human embryonic stem cells (hESC, induced pluripotent stem cells (iPSC and adipose stem cells (ASC. We describe the use of the xeno-free medium in the derivation and long-term (>80 passages culture of three pluripotent karyotypically normal hESC lines: Regea 06/015, Regea 07/046, and Regea 08/013. Cardiomyocytes and neural cells differentiated from these cells exhibit features characteristic to these cell types. The same formulation of the xeno-free medium is capable of supporting the undifferentiated growth of iPSCs on human feeder cells. The characteristics of the pluripotent hESC and iPSC lines are comparable to lines derived and cultured in standard undefined culture conditions. In the culture of ASCs, the xeno-free medium provided significantly higher proliferation rates than ASCs cultured in medium containing allogeneic human serum (HS, while maintaining the differentiation potential and characteristic surface marker expression profile of ASCs, although significant differences in the surface marker expression of ASCs cultured in HS and RegES media were revealed.Our results demonstrate that human ESCs, iPSCs and ASCs can be maintained in the same defined xeno-free medium formulation for a prolonged period of time while maintaining their characteristics, demonstrating the applicability of the simplified xeno-free medium formulation for the production of clinical-grade stem cells. The basic xeno-free formulation described herein has the potential to be further optimized for specific

  12. VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

    International Nuclear Information System (INIS)

    Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica; Gonzalez Espinosa, Claudia

    2010-01-01

    Research highlights: → Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. → CoCl 2 -induced VEGF secretion in mast cells occurs by a Ca 2+ -insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. → Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits FcεRI-dependent anaphylactic degranulation in mast cells. → Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl 2 ) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl 2 promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl 2 -induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl 2 -induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl 2 in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals-dependent Fyn kinase activation.

  13. VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

    Energy Technology Data Exchange (ETDEWEB)

    Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico); Gonzalez Espinosa, Claudia, E-mail: cgonzal@cinvestav.mx [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico)

    2010-10-15

    Research highlights: {yields} Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. {yields} CoCl{sub 2}-induced VEGF secretion in mast cells occurs by a Ca{sup 2+}-insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. {yields} Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits Fc{epsilon}RI-dependent anaphylactic degranulation in mast cells. {yields} Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl{sub 2}) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl{sub 2} promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl{sub 2}-induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl{sub 2}-induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl{sub 2} in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals

  14. DMSO-free cryopreservation of adipose-derived mesenchymal stromal cells: expansion medium affects post-thaw survival

    Czech Academy of Sciences Publication Activity Database

    Rogulska, O.; Petrenko, Yuriy; Petrenko, A.

    2017-01-01

    Roč. 69, č. 2 (2017), s. 265-276 ISSN 0920-9069 Institutional support: RVO:68378041 Keywords : human adiposederived mesenchymalstromal cells * DMSO-free cryopreservation * plateletlysate Subject RIV: FP - Other Medical Disciplines OBOR OECD: Cell biology Impact factor: 1.857, year: 2016

  15. Students learn systems-based care and facilitate system change as stakeholders in a free clinic experience.

    Science.gov (United States)

    Colbert, Colleen Y; Ogden, Paul E; Lowe, Darla; Moffitt, Michael J

    2010-10-01

    Systems-based practice (SBP) is rarely taught or evaluated during medical school, yet is one of the required competencies once students enter residency. We believe Texas A&M College of Medicine students learn about systems issues informally, as they care for patients at a free clinic in Temple, TX. The mandatory free clinic rotation is part of the Internal Medicine clerkship and does not include formal instruction in SBP. During 2008-2009, a sample of students (n = 31) on the IMED clerkship's free clinic rotation participated in a program evaluation/study regarding their experiences. Focus groups (M = 5 students/group) were held at the end of each outpatient rotation. Students were asked: "Are you aware of any system issues which can affect either the delivery of or access to care at the free clinic?" Data saturation was reached after six focus groups, when investigators noted a repetition of responses. Based upon investigator consensus opinion, data collection was discontinued. Based upon a content analysis, six themes were identified: access to specialists, including OB-GYN, was limited; cost containment; lack of resources affects delivery of care; delays in care due to lack of insurance; understanding of larger healthcare system and free clinic role; and delays in tests due to language barriers. Medical students were able to learn about SBP issues during free clinic rotations. Students experienced how SBP issues affected the health care of uninsured individuals. We believe these findings may be transferable to medical schools with mandatory free clinic rotations.

  16. The thioredoxin system in breast cancer cell invasion and migration

    Directory of Open Access Journals (Sweden)

    Maneet Bhatia

    2016-08-01

    Full Text Available Metastasis is the most life threatening aspect of breast cancer. It is a multi-step process involving invasion and migration of primary tumor cells with a subsequent colonization of these cells at a secondary location. The aim of the present study was to investigate the role of thioredoxin (Trx1 in the invasion and migration of breast cancer cells and to assess the strength of the association between high levels of Trx1 and thioredoxin reductase (TrxR1 expression with breast cancer patient survival. Our results indicate that the expression of both Trx1 and TrxR1 are statistically significantly increased in breast cancer patient cells compared with paired normal breast tissue from the same patient. Over-expression of Trx1 in MDA-MB-231 breast cancer cell lines enhanced cell invasion in in vitro assays while expression of a redox inactive mutant form of Trx1 (designated 1SS or the antisense mRNA inhibited cell invasion. Addition of exogenous Trx1 also enhanced cell invasion, while addition of a specific monoclonal antibody that inhibits Trx1 redox function decreased cell invasion. Over-expression of intracellular Trx1 did not increase cell migration but expression of intracellular 1SS inhibited migration. Addition of exogenous Trx1 enhanced cell migration while 1SS had no effect. Treatment with auranofin inhibited TrxR activity, cell migration and clonogenic activity of MDA-MB-231 cells, while increasing reactive oxygen species (ROS levels. Analysis of 25 independent cohorts with 5910 patients showed that Trx1 and TrxR1 were both associated with a poor patient prognosis in terms of overall survival, distant metastasis free survival and disease free survival. Therefore, targeting the Trx system with auranofin or other specific inhibitors may provide improved breast cancer patient outcomes through inhibition of cancer invasion and migration.

  17. Toll-Like Receptor-Mediated Free Radical Generation in Clonorchis sinensis Excretory-Secretory Product-Treated Cholangiocarcinoma Cells.

    Science.gov (United States)

    Bahk, Young Yil; Pak, Jhang Ho

    2016-10-01

    Clonorchiasis, caused by direct contact with Clonorchis sinensis worms and their excretory-secretory products (ESPs), is associated with chronic inflammation, malignant changes in bile ducts, and even cholangiocarcinogenesis. Our previous report revealed that intracellular free radicals enzymatically generated by C. sinensis ESPs cause NF-κB-mediated inflammation in human cholangiocarcinoma cells (HuCCT1). Therefore, the present study was conducted to examine the role of upstream Toll-like receptors (TLRs) on the initial host innate immune responses to infection. We found that treatment of HuCCT1 cells with native ESPs induced changes in TLR mRNA levels in a time-dependent manner, concomitant with the generation of free radicals. ESP-mediated free radical generation was markedly attenuated by preincubation of the cells with TLR1-4-neutralizing antibodies, indicating that at least TLR1 through 4 participate in stimulation of the host innate immune responses. These findings indicate that free radicals triggered by ESPs are critically involved in TLR signal transduction. Continuous signaling by this pathway may function in initiating C. sinensis infection-associated inflammation cascades, a detrimental event leading to progression to more severe hepatobiliary diseases.

  18. Integrating Cell Phone Imaging with Magnetic Levitation (i-LEV) for Label-Free Blood Analysis at the Point-of-Living.

    Science.gov (United States)

    Baday, Murat; Calamak, Semih; Durmus, Naside Gozde; Davis, Ronald W; Steinmetz, Lars M; Demirci, Utkan

    2016-03-02

    There is an emerging need for portable, robust, inexpensive, and easy-to-use disease diagnosis and prognosis monitoring platforms to share health information at the point-of-living, including clinical and home settings. Recent advances in digital health technologies have improved early diagnosis, drug treatment, and personalized medicine. Smartphones with high-resolution cameras and high data processing power enable intriguing biomedical applications when integrated with diagnostic devices. Further, these devices have immense potential to contribute to public health in resource-limited settings where there is a particular need for portable, rapid, label-free, easy-to-use, and affordable biomedical devices to diagnose and continuously monitor patients for precision medicine, especially those suffering from rare diseases, such as sickle cell anemia, thalassemia, and chronic fatigue syndrome. Here, a magnetic levitation-based diagnosis system is presented in which different cell types (i.e., white and red blood cells) are levitated in a magnetic gradient and separated due to their unique densities. Moreover, an easy-to-use, smartphone incorporated levitation system for cell analysis is introduced. Using our portable imaging magnetic levitation (i-LEV) system, it is shown that white and red blood cells can be identified and cell numbers can be quantified without using any labels. In addition, cells levitated in i-LEV can be distinguished at single-cell resolution, potentially enabling diagnosis and monitoring, as well as clinical and research applications. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Bone Marrow Mesenchymal Stem Cells Enhance the Differentiation of Human Switched Memory B Lymphocytes into Plasma Cells in Serum-Free Medium

    Directory of Open Access Journals (Sweden)

    Guillaume Bonnaure

    2016-01-01

    Full Text Available The differentiation of human B lymphocytes into plasma cells is one of the most stirring questions with regard to adaptive immunity. However, the terminal differentiation and survival of plasma cells are still topics with much to be discovered, especially when targeting switched memory B lymphocytes. Plasma cells can migrate to the bone marrow in response to a CXCL12 gradient and survive for several years while secreting antibodies. In this study, we aimed to get closer to niches favoring plasma cell survival. We tested low oxygen concentrations and coculture with mesenchymal stem cells (MSC from human bone marrow. Besides, all cultures were performed using an animal protein-free medium. Overall, our model enables the generation of high proportions of CD38+CD138+CD31+ plasma cells (≥50% when CD40-activated switched memory B lymphocytes were cultured in direct contact with mesenchymal stem cells. In these cultures, the secretion of CXCL12 and TGF-β, usually found in the bone marrow, was linked to the presence of MSC. The level of oxygen appeared less impactful than the contact with MSC. This study shows for the first time that expanded switched memory B lymphocytes can be differentiated into plasma cells using exclusively a serum-free medium.

  20. Effect of kinetic properties of extraction systems on separation of some elements by liquid chromatography method with free fixed phase

    International Nuclear Information System (INIS)

    Fedotov, P.S.; Maryutina, T.A.; Pichugin, A.A.; Spivakov, B.Ya.

    1993-01-01

    Effect of kinetic properties of a series of extraction systems on the separation of certain elements by the method of liquid chromatography with free fixed phase is considered. Chromatographic behaviour of europium 3 and iron 3 ions when using systems based on di-2-ethylhexylphosphovers acid and tetraphenylmethylenediphosphine dioxide is investigated. Kinetic properties of the extraction systems used are studied by diffusion cell method with mixing, europium 3 and iron 3 mass transfer coefficients are determined

  1. Task path planning, scheduling and learning for free-ranging robot systems

    Science.gov (United States)

    Wakefield, G. Steve

    1987-01-01

    The development of robotics applications for space operations is often restricted by the limited movement available to guided robots. Free ranging robots can offer greater flexibility than physically guided robots in these applications. Presented here is an object oriented approach to path planning and task scheduling for free-ranging robots that allows the dynamic determination of paths based on the current environment. The system also provides task learning for repetitive jobs. This approach provides a basis for the design of free-ranging robot systems which are adaptable to various environments and tasks.

  2. A practical implementation of free viewpoint video system for soccer games

    Science.gov (United States)

    Suenaga, Ryo; Suzuki, Kazuyoshi; Tezuka, Tomoyuki; Panahpour Tehrani, Mehrdad; Takahashi, Keita; Fujii, Toshiaki

    2015-03-01

    In this paper, we present a free viewpoint video generation system with billboard representation for soccer games. Free viewpoint video generation is a technology that enables users to watch 3-D objects from their desired viewpoints. Practical implementation of free viewpoint video for sports events is highly demanded. However, a commercially acceptable system has not yet been developed. The main obstacles are insufficient user-end quality of the synthesized images and highly complex procedures that sometimes require manual operations. In this work, we aim to develop a commercially acceptable free viewpoint video system with a billboard representation. A supposed scenario is that soccer games during the day can be broadcasted in 3-D, even in the evening of the same day. Our work is still ongoing. However, we have already developed several techniques to support our goal. First, we captured an actual soccer game at an official stadium where we used 20 full-HD professional cameras. Second, we have implemented several tools for free viewpoint video generation as follow. In order to facilitate free viewpoint video generation, all cameras should be calibrated. We calibrated all cameras using checker board images and feature points on the field (cross points of the soccer field lines). We extract each player region from captured images manually. The background region is estimated by observing chrominance changes of each pixel in temporal domain (automatically). Additionally, we have developed a user interface for visualizing free viewpoint video generation using a graphic library (OpenGL), which is suitable for not only commercialized TV sets but also devices such as smartphones. However, practical system has not yet been completed and our study is still ongoing.

  3. Quantitative Label-Free Cell Proliferation Tracking with a Versatile Electrochemical Impedance Detection Platform

    DEFF Research Database (Denmark)

    Caviglia, Claudia; Carminati, M; Heiskanen, Arto

    2012-01-01

    optimal detection strategies. Electrochemical Impedance Spectroscopy (EIS) has been used to monitor and compare adhesion of different cell lines. HeLa cells and 3T3 fibroblasts have been cultured for 12 hours on interdigitated electrode arrays integrated into a tailor-made cell culture platform. Both......Since the use of impedance measurements for label-free monitoring of cells has become widespread but still the choice of sensing configuration is not unique though crucial for a quantitative interpretation of data, we demonstrate the application of a novel custom multipotentiostat platform to study...... vertical and coplanar interdigitated sensing configuration approaches have been used and compared on the same cell populations....

  4. A control system for a free electron laser experiment

    International Nuclear Information System (INIS)

    Giove, D.

    1992-01-01

    The general layout of a control and data acquisition system for a Free Electron Laser experiment will be discussed. Some general considerations about the requirements and the architecture of the whole system will be developed. (author)

  5. A novel CuI-based iodine-free gel electrolyte for dye-sensitized solar cells

    International Nuclear Information System (INIS)

    Chen Junnian; Xia Jiangbin; Fan Ke; Peng Tianyou

    2011-01-01

    Highlights: → A novel CuI-based iodine-free gel electrolyte for DSSC is firstly prepared. → Such CuI-based electrolyte has relative high conductivity and stability. → Addition amount of LiClO 4 and PEO in the electrolyte is optimized. → Cell performance is improved by 116.2% compared with the cell without LiClO 4 . - Abstract: A novel CuI-based iodine-free gel electrolyte using polyethylene oxide (PEO, MW = 100,000) as plasticizer and lithium perchlorate (LiClO 4 ) as salt additive was developed for dye-sensitized solar cells (DSSCs). Such CuI-based gel electrolyte can avoid the problems caused by liquid iodine electrolyte and has relative high conductivity and stability. The effects of PEO and LiClO 4 concentrations on the viscosity and ionic conductivity of the mentioned iodine-free electrolyte, as well as the performance of the corresponding quasi solid-state DSSCs were investigated comparatively. Experimental results indicate that the performance of DSSCs can be dramatically improved by adding LiClO 4 and PEO, and there are interactions (Li + -O coordination) between LiClO 4 and PEO, these Li + -O coordination interactions have important influence on the structure, morphology and ionic conductivity of the present CuI-based electrolyte. Addition of PEO into the electrolyte can inhibit the rapid crystal growth of CuI, and enhance the ion and hole transportation property owing to its long helix chain structure. The optimal efficiency (2.81%) was obtained for the quasi solid-state DSSC fabricated with CuI-based electrolyte containing 3 wt% LiClO 4 and 20 wt% PEO under AM 1.5 G (1 sun) light illumination, with a 116.2% improvement in the efficiency compared with the cell without addition of LiClO 4 , indicating the promising application in solar cells of the present CuI-based iodine-free electrolyte.

  6. Method for Estimating Optimum Free Resonant Frequencies in Overcoupled WPT System

    Directory of Open Access Journals (Sweden)

    Dong-Wook Seo

    2017-01-01

    Full Text Available In our previous work, we proposed the method to maximize the output power even in the overcoupled state of the wireless power transfer (WPT system by controlling free resonant frequencies and derived closed-form expression for optimum free resonant frequencies of the primary and secondary resonators. In this paper, we propose the mutual coupling approach to derive the optimum free resonant frequencies and show the measured power transfer efficiency (PTE using the transmission efficiency as well as the system energy efficiency. The results of the proposed approach exactly coincide with those of the previous work, and the fabricated prototype achieves the transmission efficiency of about 80% by tuning the free resonant frequencies to the optimum values in the overcoupled state.

  7. Development of fuel cell systems for aircraft applications based on synthetic fuels

    Energy Technology Data Exchange (ETDEWEB)

    Pasel, J.; Samsun, R.C.; Doell, C.; Peters, R.; Stolten, D. [Forschungszentrum Juelich GmbH (Germany)

    2010-07-01

    At present, in the aviation sector considerable scientific project work deals with the development of fuel cell systems based on synthetic fuels to be integrated in future aircraft. The benefits of fuel cell systems in aircraft are various. They offer the possibility to simplify the aircraft layout. Important systems, i.e. the gas turbine powered auxiliary power unit (APU) for electricity supply, the fuel tank inserting system and the water tank, can be substituted by one single system, the fuel cell system. Additionally, the energy demand for ice protection can be covered assisted by fuel cell systems. These measures reduce the consumption of jet fuel, increase aircraft efficiency and allow the operation at low emissions. Additionally, the costs for aircraft related investments, for aircraft maintenance and operation can be reduced. On the background of regular discussions about environmental concerns (global warming) of kerosene Jet A-1 and its availability, which might be restricted in a few years, the aircraft industry is keen to employ synthetic, sulfur-free fuels such as Fischer-Tropsch fuels. These comprise Bio-To-Liquid and Gas-To-Liquid fuels. Within this field of research the Institute of Energy Research (IEF-3) in Juelich develops complete and compact fuel cell systems based on the autothermal reforming of these kinds of fuels in cooperation with industry. This paper reports about this work. (orig.)

  8. High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients.

    Science.gov (United States)

    Kukita, Yoji; Matoba, Ryo; Uchida, Junji; Hamakawa, Takuya; Doki, Yuichiro; Imamura, Fumio; Kato, Kikuya

    2015-08-01

    Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  9. Use of a cocktail of spin traps for fingerprinting large range of free radicals in biological systems.

    Science.gov (United States)

    Marchand, Valérie; Charlier, Nicolas; Verrax, Julien; Buc-Calderon, Pedro; Levêque, Philippe; Gallez, Bernard

    2017-01-01

    It is well established that the formation of radical species centered on various atoms is involved in the mechanism leading to the development of several diseases or to the appearance of deleterious effects of toxic molecules. The detection of free radical is possible using Electron Paramagnetic Resonance (EPR) spectroscopy and the spin trapping technique. The classical EPR spin-trapping technique can be considered as a "hypothesis-driven" approach because it requires an a priori assumption regarding the nature of the free radical in order to select the most appropriate spin-trap. We here describe a "data-driven" approach using EPR and a cocktail of spin-traps. The rationale for using this cocktail was that it would cover a wide range of biologically relevant free radicals and have a large range of hydrophilicity and lipophilicity in order to trap free radicals produced in different cellular compartments. As a proof-of-concept, we validated the ability of the system to measure a large variety of free radicals (O-, N-, C-, or S- centered) in well characterized conditions, and we illustrated the ability of the technique to unambiguously detect free radical production in cells exposed to chemicals known to be radical-mediated toxic agents.

  10. Lipid nanocarriers based on natural oils with high activity against oxygen free radicals and tumor cell proliferation

    International Nuclear Information System (INIS)

    Lacatusu, I.; Badea, N.; Badea, G.; Oprea, O.; Mihaila, M.A.; Kaya, D.A.; Stan, R.; Meghea, A.

    2015-01-01

    The development of nano-dosage forms of phytochemicals represents a significant progress of the scientific approach in the biomedical research. The aim of this study was to assess the effectiveness of lipid nanocarriers based on natural oils (grape seed oil, fish oil and laurel leaf oil) in counteracting free radicals and combating certain tumor cells. No drug was encapsulated in the nanocarriers. The cytotoxic effect exerted by bioactive nanocarriers against two tumor cells, MDA-MB 231 and HeLa cell lines, and two normal cells, L929 and B16 cell lines, was measured using the MTT assay, while oxidative damage was assessed by measuring the total antioxidant activity using chemiluminescence analysis. The best performance was obtained for nanocarriers based on an association of grape seed and laurel leaf oils, with a capacity to scavenge about 98% oxygen free radicals. A dose of nanocarriers of 5 mg·mL −1 has led to a drastic decrease in tumor cell proliferation even in the absence of an antitumor drug (e.g. about 50% viability for MDA-MB 231 cell line and 60% viability for HeLa cell line). A comparative survival profile of normal and tumor cells, which were exposed to an effective dose of 2.5 mg·mL −1 lipid nanocarriers, has revealed a death rate of 20% for normal B16 cells and of 40% death rate for MDA-MB 231 and HeLa tumor cells. The results in this study imply that lipid nanocarriers based on grape seed oil in association with laurel leaf oil could be a candidate to reduce the delivery system toxicity and may significantly improve the therapeutic efficacy of antitumor drugs in clinical applications. - Highlights: • Functional lipid nanocarriers with unique features and broad spectrum effectiveness • Lipid nanocarriers based on laureal leaf oil (LLO) and grape seed oil (GSO) • Antioxidant activity has reached 98% for nanocarriers containing 25% GSO and 2% LLO. • LLO exerts a significant cytotoxic effect against HeLa and MDA-MB 231 tumor cells. • 50

  11. Lipid nanocarriers based on natural oils with high activity against oxygen free radicals and tumor cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Lacatusu, I.; Badea, N.; Badea, G.; Oprea, O. [University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Polizu Street No 1, 011061 Bucharest (Romania); Mihaila, M.A. [Institute of Virusology “Stefan S. Nicolau”, Center of Immunology, Bravu Road, No. 285, 030304 Bucharest (Romania); Kaya, D.A. [Department of Field Crops, Faculty of Agriculture, Mustafa Kemal University, 31030 Antakya, Hatay (Turkey); Stan, R., E-mail: rl_stan2000@yahoo.com [University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Polizu Street No 1, 011061 Bucharest (Romania); Meghea, A. [University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Polizu Street No 1, 011061 Bucharest (Romania)

    2015-11-01

    The development of nano-dosage forms of phytochemicals represents a significant progress of the scientific approach in the biomedical research. The aim of this study was to assess the effectiveness of lipid nanocarriers based on natural oils (grape seed oil, fish oil and laurel leaf oil) in counteracting free radicals and combating certain tumor cells. No drug was encapsulated in the nanocarriers. The cytotoxic effect exerted by bioactive nanocarriers against two tumor cells, MDA-MB 231 and HeLa cell lines, and two normal cells, L929 and B16 cell lines, was measured using the MTT assay, while oxidative damage was assessed by measuring the total antioxidant activity using chemiluminescence analysis. The best performance was obtained for nanocarriers based on an association of grape seed and laurel leaf oils, with a capacity to scavenge about 98% oxygen free radicals. A dose of nanocarriers of 5 mg·mL{sup −1} has led to a drastic decrease in tumor cell proliferation even in the absence of an antitumor drug (e.g. about 50% viability for MDA-MB 231 cell line and 60% viability for HeLa cell line). A comparative survival profile of normal and tumor cells, which were exposed to an effective dose of 2.5 mg·mL{sup −1} lipid nanocarriers, has revealed a death rate of 20% for normal B16 cells and of 40% death rate for MDA-MB 231 and HeLa tumor cells. The results in this study imply that lipid nanocarriers based on grape seed oil in association with laurel leaf oil could be a candidate to reduce the delivery system toxicity and may significantly improve the therapeutic efficacy of antitumor drugs in clinical applications. - Highlights: • Functional lipid nanocarriers with unique features and broad spectrum effectiveness • Lipid nanocarriers based on laureal leaf oil (LLO) and grape seed oil (GSO) • Antioxidant activity has reached 98% for nanocarriers containing 25% GSO and 2% LLO. • LLO exerts a significant cytotoxic effect against HeLa and MDA-MB 231 tumor

  12. The Free Software Movement and the GNU/Linux Operating System

    CERN Multimedia

    CERN. Geneva

    2003-01-01

    Richard Stallman will speak about the purpose, goals, philosophy, methods, status, and future prospects of the GNU operating system, which in combination with the kernel Linux is now used by an estimated 17 to 20 million users world wide.BiographyRichard Stallman is the founder of the Gnu Project, launched in 1984 to develop the free operating system GNU (an acronym for ''GNU's Not Unix''), and thereby give computer users the freedom that most of them have lost. GNU is free software: everyone is free to copy it and redistribute it, as well as to make changes either large or small. Today, Linux-based variants of the GNU system, based on the kernel Linux developed by Linus Torvalds, are in widespread use. There are estimated to be some 20 million users of GNU/Linux systems today. Richard Stallman is the principal author of the GNU Compiler Collection, a portable optimizing compiler which was designed to support diverse architectures and multiple languages. The compiler now supports over 30 different architect...

  13. Students Learn Systems-Based Care and Facilitate System Change as Stakeholders in a Free Clinic Experience

    Science.gov (United States)

    Colbert, Colleen Y.; Ogden, Paul E.; Lowe, Darla; Moffitt, Michael J.

    2010-01-01

    Systems-based practice (SBP) is rarely taught or evaluated during medical school, yet is one of the required competencies once students enter residency. We believe Texas A&M College of Medicine students learn about systems issues informally, as they care for patients at a free clinic in Temple, TX. The mandatory free clinic rotation is part of…

  14. Single molecular image of cytosolic free Ca2+ of skeletal muscle cells in rats pre- and post-exercise-induced fatigue

    Science.gov (United States)

    Liu, Yi; Zhang, Heming; Zhao, Yanping; Liu, Zhiming

    2009-08-01

    A growing body of literature indicated the cytosolic free Ca2+ concentration of skeletal muscle cells changes significantly during exercise-induced fatigue. But it is confusing whether cytosolic free Ca2+ concentration increase or decrease. Furthermore, current researches mainly adopt muscle tissue homogenate as experiment material, but the studies based on cellular and subcellular level is seldom. This study is aimed to establish rat skeletal muscle cell model of exercise-induced fatigue, and confirm the change of cytosolic free Ca2+ concentration of skeletal muscle cells in rats preand post- exercise-induced fatigue. In this research, six male Wistar rats were randomly divided into two groups: control group (n=3) and exercise-induced fatigue group (n=3). The former group were allowed to freely move and the latter were forced to loaded swimming to exhaustive. Three days later, all the rats were sacrificed, the muscle tissue from the same site of skeletal muscle were taken out and digested to cells. After primary culture of the two kinds of skeletal muscle cells from tissue, a fluorescent dye-Fluo-3 AM was used to label the cytosolic free Ca2+. The fluorescent of Ca2+ was recorded by confocal laser scanning microscopy. The results indicated that, the Ca2+ fluorescence intensity of cells from the rat of exercise-induced fatigue group was significantly higher than those in control group. In conclusion, cytosolic free Ca2+ concentration of skeletal muscle cells has a close relation with exercise-induced fatigue, and the increase of cytosolic free Ca2+ concentration may be one of the important factors of exercise-induced fatigue.

  15. β-cell serotonin production is associated with female sex, old age, and diabetes-free condition.

    Science.gov (United States)

    Kim, Yeong Gi; Moon, Joon Ho; Kim, Kyuho; Kim, Hyeongseok; Kim, Juok; Jeong, Ji-Seon; Lee, Junguee; Kang, Shinae; Park, Joon Seong; Kim, Hail

    2017-11-25

    Serotonin is known to be present in pancreatic β-cells and to play several physiological roles, including insulin secretion, β-cell proliferation, and paracrine inhibition of α-cells. However, the serotonin production of different cell lines and islets has not been compared based on age, sex, and diabetes related conditions. Here, we directly compared the serotonin concentrations in βTC and MIN6 cell lines, as well as in islets from mice using ultra-performance liquid chromatography tandem mass spectrometry. The average serotonin concentration was 5-10 ng/mg protein in the islets of male and non-pregnant female mice. The serotonin level was higher in females than males at 8 weeks, although there was no difference at 1 year. Furthermore, we observed serotonin by immunofluorescence staining in the pancreatic tissues of mice and human. Serotonin was detected by immunofluorescence staining in a portion of β-cells from islets of old female mice, but not of male or young female mice. A similar pattern was observed in human pancreas as well. In humans, serotonin production in β-cells was associated with a diabetes-free condition. Thus, serotonin production in β-cells was associated with old age, female sex, and diabetes-free condition. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Free-piston Stirling engine system considerations for various space power applications

    International Nuclear Information System (INIS)

    Dochat, G.R.; Dhar, M.

    1991-01-01

    The U.S. Government is evaluating power requirements for future space applications. As power requirements increase solar or nuclear dynamic systems become increasingly attractive. Free-Piston Stirling Engines (FPSE) have the potential to provide high reliability, long life, and efficient operation. Therefore, they are excellent candidates for the dynamic power conversion module of a space-based, power-generating system. FPSE can be coupled with many potential heat sources (radioisotope, solar, or nuclear reactor), various heat input systems (pumped loop, heat pipe), heat rejection (pumped loop or heat pipe), and various power management and distribution systems (AC, DC, high or low voltage, and fixed or variable load). This paper will review potential space missions that can be met using free-piston Stirling engines and discusses options of various system integration approaches. Currently free-piston Stirling engine technology for space power applications is being developed under contract with NASA-Lewis Research Center. This paper will also briefly outline the program and recent progress

  17. Optimizing the method for generation of integration-free induced pluripotent stem cells from human peripheral blood.

    Science.gov (United States)

    Gu, Haihui; Huang, Xia; Xu, Jing; Song, Lili; Liu, Shuping; Zhang, Xiao-Bing; Yuan, Weiping; Li, Yanxin

    2018-06-15

    Generation of induced pluripotent stem cells (iPSCs) from human peripheral blood provides a convenient and low-invasive way to obtain patient-specific iPSCs. The episomal vector is one of the best approaches for reprogramming somatic cells to pluripotent status because of its simplicity and affordability. However, the efficiency of episomal vector reprogramming of adult peripheral blood cells is relatively low compared with cord blood and bone marrow cells. In the present study, integration-free human iPSCs derived from peripheral blood were established via episomal technology. We optimized mononuclear cell isolation and cultivation, episomal vector promoters, and a combination of transcriptional factors to improve reprogramming efficiency. Here, we improved the generation efficiency of integration-free iPSCs from human peripheral blood mononuclear cells by optimizing the method of isolating mononuclear cells from peripheral blood, by modifying the integration of culture medium, and by adjusting the duration of culture time and the combination of different episomal vectors. With this optimized protocol, a valuable asset for banking patient-specific iPSCs has been established.

  18. Free cholesterol accumulation impairs antioxidant activities and aggravates apoptotic cell death in menadione-induced oxidative injury.

    Science.gov (United States)

    Lee, Waisin; Xu, Mingjing; Li, Yue; Gu, Yong; Chen, Jianping; Wong, Derek; Fung, Peter C W; Shen, Jiangang

    2011-10-01

    Although the relationship between hypercholesterolemia and oxidative stress has been extensively investigated, direct evidence regarding to the roles of cholesterol accumulation in the generations of reactive oxygen species (ROS) and apoptotic cell death under oxidative stress is lack. In this study, we investigated productions of superoxide anions (O(2)(-)) and nitric oxide (NO), and apoptotic cell death in wild type Chinese hamster ovary (CHO) cells and cholesterol accumulated CHO cells genetically and chemically. Oxidative stress was induced by menadione challenge. The results revealed that abundance of free cholesterol (FC) promoted menadione-induced O(2)(-) and NO productions. FC accumulation down-regulated eNOS expression but up-regulated NADPH oxidases, and inhibited the activities of superoxide dismutase (SOD) and catalase. Treatment of menadione increased the expressions of iNOS and qp91 phox, enhanced the activities of SOD and catalase in the wild-type CHO cells but inhibited the activity of glutathione peroxidase in the cholesterol accumulated CHO cells. Moreover, FC abundance promoted apoptotic cell death in these cells. Taken together, those results suggest that free cholesterol accumulation aggravates menadione-induced oxidative stress and exacerbates apoptotic cell death. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. Coping with complexity: machine learning optimization of cell-free protein synthesis.

    Science.gov (United States)

    Caschera, Filippo; Bedau, Mark A; Buchanan, Andrew; Cawse, James; de Lucrezia, Davide; Gazzola, Gianluca; Hanczyc, Martin M; Packard, Norman H

    2011-09-01

    Biological systems contain complex metabolic pathways with many nonlinearities and synergies that make them difficult to predict from first principles. Protein synthesis is a canonical example of such a pathway. Here we show how cell-free protein synthesis may be improved through a series of iterated high-throughput experiments guided by a machine-learning algorithm implementing a form of evolutionary design of experiments (Evo-DoE). The algorithm predicts fruitful experiments from statistical models of the previous experimental results, combined with stochastic exploration of the experimental space. The desired experimental response, or evolutionary fitness, was defined as the yield of the target product, and new experimental conditions were discovered to have ∼ 350% greater yield than the standard. An analysis of the best experimental conditions discovered indicates that there are two distinct classes of kinetics, thus showing how our evolutionary design of experiments is capable of significant innovation, as well as gradual improvement. Copyright © 2011 Wiley Periodicals, Inc.

  20. In vitro analysis of scaffold-free prevascularized microtissue spheroids containing human dental pulp cells and endothelial cells.

    Science.gov (United States)

    Dissanayaka, Waruna Lakmal; Zhu, Lifang; Hargreaves, Kenneth M; Jin, Lijian; Zhang, Chengfei

    2015-05-01

    Scaffolds often fail to mimic essential functions of the physiologic extracellular matrix (ECM) that regulates cell-cell communication in tissue microenvironments. The development of scaffold-free microtissues containing stem cell-derived ECM may serve as a successful alternative to the use of artificial scaffolds. The current study aimed to fabricate 3-dimensional microtissue spheroids of dental pulp cells (DPCs) prevascularized by human umbilical vein endothelial cells (HUVECs) and to characterize these scaffold-free spheroids for the in vitro formation of pulplike tissue constructs. Three-dimensional microtissue spheroids of DPC alone and DPC-HUVEC co-cultures were fabricated using agarose micro-molds. Cellular organization within the spheroids and cell viability (live/dead assay) were assessed at days 1, 7, and 14. Microtissue spheroids were allowed to self-assemble into macrotissues, induced for odontogenic differentiation (21 days), and examined for expression levels of osteo/odontogenic markers: alkaline phosphatase, bone sialoprotein and RUNX2 (Real-time PCR), mineralization (von-Kossa), and prevascularisation (immunohistochemistry for CD31). The DPC microtissue microenvironment supported HUVEC survival and capillary network formation in the absence of a scaffolding material and external angiogenic stimulation. Immunohistochemical staining for CD31 showed the capillary network formed by HUVECs did sustain-for a prolonged period-even after the microtissues transformed into a macrotissue. Induced, prevascularized macrotissues showed enhanced differentiation capacity compared with DPC alone macrotissues, as shown by higher osteo/odontogenic gene expression levels and mineralization. These findings provide insight into the complex intercellular cross talk occurring between DPCs and HUVECs in the context of angiogenesis and pulp regeneration and highlight the significance of developing a favorable 3-dimensional microenvironment that can, in turn, contribute

  1. Precise Morphology Control and Continuous Fabrication of Perovskite Solar Cells Using Droplet-Controllable Electrospray Coating System.

    Science.gov (United States)

    Hong, Seung Chan; Lee, Gunhee; Ha, Kyungyeon; Yoon, Jungjin; Ahn, Namyoung; Cho, Woohyung; Park, Mincheol; Choi, Mansoo

    2017-03-08

    Herein, we developed a novel electrospray coating system for continuous fabrication of perovskite solar cells with high performance. Our system can systemically control the size of CH 3 NH 3 PbI 3 precursor droplets by modulating the applied electrical potential, shown to be a crucial factor for the formation of perovskite films. As a result, we have obtained pinhole-free and large grain-sized perovskite solar cells, yielding the best PCE of 13.27% with little photocurrent hysteresis. Furthermore, the average PCE through the continuous coating process was 11.56 ± 0.52%. Our system demonstrates not only the high reproducibility but also a new way to commercialize high-quality perovskite solar cells.

  2. Chemiluminescent Diagnostics of Free-Radical Processes in an Abiotic System and in Liver Cells in the Presence of Nanoparticles Based on Rare-Earth Elements nReVO4:Eu3+ (Re = Gd, Y, La) and CeO2

    Science.gov (United States)

    Averchenko, E. A.; Kavok, N. S.; Klochkov, V. K.; Malyukin, Yu. V.

    2014-11-01

    We have used luminol-dependent chemiluminescence with Fenton's reagent to study the effect of nanoparticles based on rare-earth elements of different sizes and shapes on free-radical processes in abiotic and biotic cell-free systems, and also in isolated cells in vitro. We have estimated the effects of rare-earth orthovanadate nanoparticles of spherical (GdYVO4:Eu3+, 1-2 nm), spindle-shaped (GdVO4:Eu3+, 25 ×8 nm), and rod-shaped (LaVO4:Eu3+, 57 × (6-8) nm) nanoparticles and spherical CeO2 nanoparticles (sizes 1-2 nm and 8-10 nm). We have shown that in contrast to the abiotic system, in which all types of nanoparticles exhibit antiradical activity, in the presence of biological material, extra-small spherical (1-2 nm) nanoparticles of both types exhibit pro-oxidant activity, and also enhance pro-oxidant induced oxidative stress (for the pro-oxidants hydrogen peroxide and tert-butyl hydroperoxide). The effect of rare-earth orthovanadate spindle and rod shaped nanoparticles in this system was neutral; a moderate antioxidant effect was exhibited by 8-10 nm CeO2 nanoparticles.

  3. Recurrent squamous cell carcinoma of the scalp treated with serial free flaps

    DEFF Research Database (Denmark)

    Ikander, Peder; Sørensen, Jens Ahm

    2015-01-01

    was seen with recurrent squamous cell carcinoma of the scalp. The lesions were of full thickness, about 10-15 cm in diameter and included the calvarial bone and the dura layer. The reconstruction process included split-thickness skin grafting, local flaps, and three free microvascular flaps; two latissimus...

  4. Ethanol tolerant precious metal free cathode catalyst for alkaline direct ethanol fuel cells

    International Nuclear Information System (INIS)

    Grimmer, Ilena; Zorn, Paul; Weinberger, Stephan; Grimmer, Christoph; Pichler, Birgit; Cermenek, Bernd; Gebetsroither, Florian; Schenk, Alexander; Mautner, Franz-Andreas

    2017-01-01

    Highlights: • Selective ORR catalysts are presented for alkaline direct ethanol fuel cells. • Perovskite based cathode catalysts show high tolerance toward ethanol. • A membrane-free alkaline direct ethanol fuel cell is presented. - Abstract: La 0.7 Sr 0.3 (Fe 0.2 Co 0.8 )O 3 and La 0.7 Sr 0.3 MnO 3 −based cathode catalysts are synthesized by the sol-gel method. These perovskite cathode catalysts are tested in half cell configuration and compared to MnO 2 as reference material in alkaline direct ethanol fuel cells (ADEFCs). The best performing cathode is tested in single cell setup using a standard carbon supported Pt 0.4 Ru 0.2 based anode. A backside Luggin capillary is used in order to register the anode potential during all measurements. Characteristic processes of the electrodes are investigated using electrochemical impedance spectroscopy. Physical characterizations of the perovskite based cathode catalysts are performed with a scanning electron microscope (SEM) and by X-ray diffraction showing phase pure materials. In half cell setup, La 0.7 Sr 0.3 MnO 3 shows the highest tolerance toward ethanol with a performance of 614 mA cm −2 at 0.65 V vs. RHE in 6 M KOH and 1 M EtOH at RT. This catalyst outperforms the state-of-the-art precious metal-free MnO 2 catalyst in presence of ethanol. In fuel cell setup, the peak power density is 27.6 mW cm −2 at a cell voltage of 0.345 V and a cathode potential of 0.873 V vs. RHE.

  5. Possibility of hydrogen supply by shared residential fuel cell systems for fuel cell vehicles

    Directory of Open Access Journals (Sweden)

    Ono Yusuke

    2017-01-01

    Full Text Available Residential polymer electrolyte fuel cells cogeneration systems (residential PEFC systems produce hydrogen from city gas by internal gas-reformer, and generate electricity, the hot water at the same time. From the viewpoint of the operation, it is known that residential PEFC systems do not continuously work but stop for long time, because the systems generate enough hot water for short operation time. In other words, currently residential PEFC systems are dominated by the amount of hot water demand. This study focuses on the idle time of residential PEFC systems. Since their gas-reformers are free, the systems have potential to produce hydrogen during the partial load operations. The authors expect that residential PEFC systems can take a role to supply hydrogen for fuel cell vehicles (FCVs before hydrogen fueling stations are distributed enough. From this perspective, the objective of this study is to evaluate the hydrogen production potential of residential PEFC systems. A residential PEFC system was modeled by the mixed integer linear programming to optimize the operation including hydrogen supply for FCV. The objective function represents annual system cost to be minimized with the constraints of energy balance. It should be noted that the partial load characteristics of the gas-reformer and the fuel cell stack are taken into account to derive the optimal operation. The model was employed to estimate the possible amount of hydrogen supply by a residential PEFC system. The results indicated that the system could satisfy at least hydrogen demand for transportation of 8000 km which is as far as the average annual mileage of a passenger car in Japan. Furthermore, hydrogen production by sharing a residential PEFC system with two households is more effective to reduce primary energy consumption with hydrogen supply for FCV than the case of introducing PEFC in each household.

  6. Approaches to systems biology. Four methods to study single-cell gene expression, cell motility, antibody reactivity, and respiratory metabolism

    DEFF Research Database (Denmark)

    Hagedorn, Peter

    To understand how complex systems, such as cells, function, comprehensive Measurements of their constituent parts must be made. This can be achieved by combining methods that are each optimized to measure specific parts of the system. Four such methods,each covering a different area, are presented...... from such measurements allows models of the system to be developed and tested. For each of the methods, such analysis and modelling approaches have beenapplied and are presented: Differentially regulated genes are identified and classified according to function; cell-specfic motility models...... are developed that can distinguish between different surfaces; a method for selecting repertoires of antigens thatseparate mice based on their response to treatment is developed; and the observed concentrations of free and bound NADH is used to build and test a basic model of respiratory metabolism...

  7. The healing of bony defects by cell-free collagen-based scaffolds compared to stem cell-seeded tissue engineered constructs.

    LENUS (Irish Health Repository)

    Lyons, Frank G

    2010-12-01

    One of the key challenges in tissue engineering is to understand the host response to scaffolds and engineered constructs. We present a study in which two collagen-based scaffolds developed for bone repair: a collagen-glycosaminoglycan (CG) and biomimetic collagen-calcium phosphate (CCP) scaffold, are evaluated in rat cranial defects, both cell-free and when cultured with MSCs prior to implantation. The results demonstrate that both cell-free scaffolds showed excellent healing relative to the empty defect controls and somewhat surprisingly, to the tissue engineered (MSC-seeded) constructs. Immunological analysis of the healing response showed higher M1 macrophage activity in the cell-seeded scaffolds. However, when the M2 macrophage response was analysed, both groups (MSC-seeded and non-seeded scaffolds) showed significant activity of these cells which are associated with an immunomodulatory and tissue remodelling response. Interestingly, the location of this response was confined to the construct periphery, where a capsule had formed, in the MSC-seeded groups as opposed to areas of new bone formation in the non-seeded groups. This suggests that matrix deposited by MSCs during in vitro culture may adversely affect healing by acting as a barrier to macrophage-led remodelling when implanted in vivo. This study thus improves our understanding of host response in bone tissue engineering.

  8. Prospective guidance in a free-swimming cell.

    Science.gov (United States)

    Delafield-Butt, Jonathan T; Pepping, Gert-Jan; McCaig, Colin D; Lee, David N

    2012-07-01

    A systems theory of movement control in animals is presented in this article and applied to explaining the controlled behaviour of the single-celled Paramecium caudatum in an electric field. The theory-General Tau Theory-is founded on three basic principles: (i) all purposive movement entails prospectively controlling the closure of action-gaps (e.g. a distance gap when reaching, or an angle gap when steering); (ii) the sole informational variable required for controlling gaps is the relative rate of change of the gap (the time derivative of the gap size divided by the size), which can be directly sensed; and (iii) a coordinated movement is achieved by keeping the relative rates of change of gaps in a constant ratio. The theory is supported by studies of controlled movement in mammals, birds and insects. We now show for the first time that it is also supported by single-celled paramecia steering to the cathode in a bi-polar electric field. General Tau Theory is deployed to explain this guided steering by the cell. This article presents the first computational model of prospective perceptual control in a non-neural, single-celled system.

  9. Oil-Free Shaft Support System Rotordynamics: Past, Present, and Future Challenges and Opportunities

    Science.gov (United States)

    DellaCorte, Christopher

    2011-01-01

    Recent breakthroughs in Oil-Free technologies have enabled new high-speed rotor systems and turbomachinery. Such technologies can include compliant-surface gas bearings, magnetic bearings, and advanced solid lubricants and tribo-materials. This presentation briefly reviews critical technology developments and the current state-of-the-art, emerging Oil-Free rotor systems and discusses obstacles preventing more widespread use. Key examples of "best practices" for deploying Oil-Free technologies will be presented and remaining major technical questions surrounding Oil-Free technologies will be brought forward.

  10. Non-invasive in-cell determination of free cytosolic [NAD+]/[NADH] ratios using hyperpolarized glucose show large variations in metabolic phenotypes

    DEFF Research Database (Denmark)

    Christensen, Caspar Elo; Karlsson, Magnus; Winther, Jakob R.

    2014-01-01

    Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyse substrate oxidation and as such it plays a key role in various biological processes such as aging, cell...... death and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD+]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labelled metabolic bioprobe of free cytosolic [NAD+]/[NADH] by combining...... a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD+]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/ [lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD...

  11. Structural study of the membrane protein MscL using cell-free expression and solid-state NMR

    Science.gov (United States)

    Abdine, Alaa; Verhoeven, Michiel A.; Park, Kyu-Ho; Ghazi, Alexandre; Guittet, Eric; Berrier, Catherine; Van Heijenoort, Carine; Warschawski, Dror E.

    2010-05-01

    High-resolution structures of membrane proteins have so far been obtained mostly by X-ray crystallography, on samples where the protein is surrounded by detergent. Recent developments of solid-state NMR have opened the way to a new approach for the study of integral membrane proteins inside a membrane. At the same time, the extension of cell-free expression to the production of membrane proteins allows for the production of proteins tailor made for NMR. We present here an in situ solid-state NMR study of a membrane protein selectively labeled through the use of cell-free expression. The sample consists of MscL (mechano-sensitive channel of large conductance), a 75 kDa pentameric α-helical ion channel from Escherichia coli, reconstituted in a hydrated lipid bilayer. Compared to a uniformly labeled protein sample, the spectral crowding is greatly reduced in the cell-free expressed protein sample. This approach may be a decisive step required for spectral assignment and structure determination of membrane proteins by solid-state NMR.

  12. Using label-free screening technology to improve efficiency in drug discovery.

    Science.gov (United States)

    Halai, Reena; Cooper, Matthew A

    2012-02-01

    Screening assays have traditionally utilized reporter labels to quantify biological responses relevant to the disease state of interest. However, there are limitations associated with the use of labels that may be overcome with temporal measurements possible with label-free. This review comprises general and system-specific information from literature searches using PubMed, published books and the authors' personal experience. This review highlights the label-free approaches in the context of various applications. The authors also note technical issues relevant to the development of label-free assays and their application to HTS. The limitations associated with the use of transfected cell lines and the use of label-based assays are gradually being realized. As such, greater emphasis is being placed on label-free biophysical techniques using native cell lines. The introduction of 96- and 384-well plate label-free systems is helping to broker a wider acceptance of these approaches in high-throughput screening. However, potential users of the technologies remain skeptical, primarily because the physical basis of the signals generated, and their contextual relevance to cell biology and signal transduction, has not been fully elucidated. Until this is done, these new technology platforms are more likely to complement, rather than replace, traditional screening platforms.

  13. A novel cobalt-free layered GdBaFe{sub 2}O{sub 5+{delta}} cathode for proton conducting solid oxide fuel cells

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Hanping; Xue, Xingjian [Department of Mechanical Engineering, University of South Carolina, Columbia, SC 29208 (United States)

    2010-07-01

    While cobalt-containing perovskite-type cathode materials facilitate the activation of oxygen reduction, they also suffer from problems like poor chemical stability in CO{sub 2} and high thermal expansion coefficients. In this research, a cobalt-free layered GdBaFe{sub 2}O{sub 5+{delta}} (GBF) perovskite was developed as a cathode material for protonic ceramic membrane fuel cells (PCMFCs) based on proton conducting electrolyte of stable BaZr{sub 0.1}Ce{sub 0.7}Y{sub 0.2}O{sub 3-{delta}} (BZCY7). The button cells of Ni-BZCY7 vertical stroke BZCY7 vertical stroke GBF were fabricated and characterized using complex impedance technique from 600 to 700 C. An open-circuit potential of 1.007 V, maximum power density of 417 mW cm{sup -2}, and a low electrode polarization resistance of 0.18 {omega} cm{sup 2} were achieved at 700 C. The results indicate that layered GBF perovskite is a good candidate for cobalt-free cathode material, while the developed Ni-BZCY7 vertical stroke BZCY7 vertical stroke GBF cell is a promising functional material system for solid oxide fuel cells. (author)

  14. A Traceless Selection: Counter-selection System That Allows Efficient Generation of Transposon and CRISPR-modified T-cell Products

    Directory of Open Access Journals (Sweden)

    Riccardo Mezzadra

    2016-01-01

    Full Text Available Recent years have seen major breakthroughs in genome-engineering systems, such as transposon-mediated gene delivery systems and CRISPR-Cas9-mediated genome-editing tools. In these systems, transient expression of auxiliary genes is responsible for permanent genomic modification. For both systems, it would be valuable to select for cells that are likely to undergo stable genome modification. Importantly, in particular for clinical applications of genome-engineered cell products, it will also be of importance to remove those cells that, due to random vector integration, display an unwanted stable expression of the auxiliary gene. Here, we develop a traceless selection system that on the one hand allows efficient enrichment of modified cells, and on the other hand can be used to select against cells that retain expression of the auxiliary gene. The value of this system to produce highly enriched-auxiliary gene-free cell products is demonstrated.

  15. Serum-free keloid fibroblast cell culture: an in vitro model for the study of aberrant wound healing.

    Science.gov (United States)

    Koch, R J; Goode, R L; Simpson, G T

    1997-04-01

    The purpose of this study was to develop an in vitro serum-free keloid fibroblast model. Keloid formation remains a problem for every surgeon. Prior evaluations of fibroblast characteristics in vitro, especially those of growth factor measurement, have been confounded by the presence of serum-containing tissue culture media. The serum itself contains growth factors, yet has been a "necessary evil" to sustain cell growth. The design of this study is laboratory-based and uses keloid fibroblasts obtained from five patients undergoing facial (ear lobule) keloid removal in a university-affiliated clinic. Keloid fibroblasts were established in primary cell culture and then propagated in a serum-free environment. The main outcome measures included sustained keloid fibroblast growth and viability, which was comparable to serum-based models. The keloid fibroblast cell cultures exhibited logarithmic growth, sustained a high cellular viability, maintained a monolayer, and displayed contact inhibition. Demonstrating model consistency, there was no statistically significant difference between the mean cell counts of the five keloid fibroblast cell lines at each experimental time point. The in vitro growth of keloid fibroblasts in a serum-free model has not been done previous to this study. The results of this study indicate that the proliferative characteristics described are comparable to those of serum-based models. The described model will facilitate the evaluation of potential wound healing modulators, and cellular effects and collagen modifications of laser resurfacing techniques, and may serve as a harvest source for contaminant-free fibroblast autoimplants. Perhaps its greatest utility will be in the evaluation of endogenous and exogenous growth factors.

  16. Stability of cell-free DNA from maternal plasma isolated following a single centrifugation step.

    Science.gov (United States)

    Barrett, Angela N; Thadani, Henna A; Laureano-Asibal, Cecille; Ponnusamy, Sukumar; Choolani, Mahesh

    2014-12-01

    Cell-free fetal DNA can be used for prenatal testing with no procedure-related risk to the fetus. However, yield of fetal DNA is low compared with maternal cell-free DNA fragments, resulting in technical challenges for some downstream applications. To maximize the fetal fraction, careful blood processing procedures are essential. We demonstrate that fetal fraction can be preserved using a single centrifugation step followed by postage of plasma to the laboratory for further processing. Digital PCR was used to quantify copies of total, maternal, and fetal DNA present in single-spun plasma at time points over a two-week period, compared with immediately processed double-spun plasma, with storage at room temperature, 4°C, and -80°C representing different postage scenarios. There was no significant change in total, maternal, or fetal DNA copy numbers when single-spun plasma samples were stored for up to 1 week at room temperature and 2 weeks at -80°C compared with plasma processed within 4 h. Following storage at 4°C no change in composition of cell-free DNA was observed. Single-spun plasma can be transported at room temperature if the journey is expected to take one week or less; shipping on dry ice is preferable for longer journeys. © 2014 John Wiley & Sons, Ltd.

  17. Final design of a free-piston hydraulic advanced Stirling conversion system

    Science.gov (United States)

    Wallace, D. A.; Noble, J. E.; Emigh, S. G.; Ross, B. A.; Lehmann, G. A.

    1991-01-01

    Under the US Department of Energy's (DOEs) Solar Thermal Technology Program, Sandia National Laboratories is evaluating heat engines for solar distributed receiver systems. The final design is described of an engineering prototype advanced Stirling conversion system (ASCS) with a free-piston hydraulic engine output capable of delivering about 25 kW of electric power to a utility grid. The free-piston Stirling engine has the potential for a highly reliable engine with long life because it has only a few moving parts, has noncontacting bearings, and can be hermetically sealed. The ASCS is designed to deliver maximum power per year over a range of solar input with a design life of 30 years (60,000 h). The system includes a liquid Nak pool boiler heat transport system and a free-piston Stirling engine with high-pressure hydraulic output, coupled with a bent axis variable displacement hydraulic motor and a rotary induction generator.

  18. Final design of a free-piston hydraulic advanced Stirling conversion system

    Science.gov (United States)

    Wallace, D. A.; Noble, J. E.; Emigh, S. G.; Ross, B. A.; Lehmann, G. A.

    Under the US Department of Energy's (DOEs) Solar Thermal Technology Program, Sandia National Laboratories is evaluating heat engines for solar distributed receiver systems. The final design is described of an engineering prototype advanced Stirling conversion system (ASCS) with a free-piston hydraulic engine output capable of delivering about 25 kW of electric power to a utility grid. The free-piston Stirling engine has the potential for a highly reliable engine with long life because it has only a few moving parts, has noncontacting bearings, and can be hermetically sealed. The ASCS is designed to deliver maximum power per year over a range of solar input with a design life of 30 years (60,000 h). The system includes a liquid Nak pool boiler heat transport system and a free-piston Stirling engine with high-pressure hydraulic output, coupled with a bent axis variable displacement hydraulic motor and a rotary induction generator.

  19. Label-Free Imaging and Biochemical Characterization of Bovine Sperm Cells

    Science.gov (United States)

    Ferrara, Maria Antonietta; Di Caprio, Giuseppe; Managò, Stefano; De Angelis, Annalisa; Sirleto, Luigi; Coppola, Giuseppe; De Luca, Anna Chiara

    2015-01-01

    A full label-free morphological and biochemical characterization is desirable to select spermatozoa during preparation for artificial insemination. In order to study these fundamental parameters, we take advantage of two attractive techniques: digital holography (DH) and Raman spectroscopy (RS). DH presents new opportunities for studying morphological aspect of cells and tissues non-invasively, quantitatively and without the need for staining or tagging, while RS is a very specific technique allowing the biochemical analysis of cellular components with a spatial resolution in the sub-micrometer range. In this paper, morphological and biochemical bovine sperm cell alterations were studied using these techniques. In addition, a complementary DH and RS study was performed to identify X- and Y-chromosome-bearing sperm cells. We demonstrate that the two techniques together are a powerful and highly efficient tool elucidating some important criterions for sperm morphological selection and sex-identification, overcoming many of the limitations associated with existing protocols. PMID:25836358

  20. Environmentally friendly, oil-free free piston engine. Displacement engines in distributed energy systems. Research funding decision. Subproject: Free piston engine. Final report; Ympaeistoeystaevaellinen, oeljytoen vapaamaentaemoottori. Syrjaeytysmoottoriprosessit hajautetussa energiahuollossa. Lineaarimoottori-osaprojekti. Loppuraportti

    Energy Technology Data Exchange (ETDEWEB)

    Larjola, J.; Honkatukia, J.; Sallinen, P.

    2005-07-01

    A free piston engine suitable for small-scale energy production in distributed energy systems was preliminarily designed in this project, including a manufacturing survey as well. The properties of the engine were simulated using a simulation program developed in this project, and the results were utilized in preliminary constructional design. The engine simulation program was developed by combining and modifying the source codes of the simulation and calculation programs obtained from Helsinki University of Technology, Tampere University of Technology, and Lappeenranta University of Technology. Because of the contact-free labyrinth seal used in the piston, the efficiency of the motor is about 5 percentage points lower than the efficiency of a conventional motor with oil-lubricated piston rings. On the other hand, the lack of bearing losses, and the lack of losses associated with a crankshaft system and a gearbox, as well as the lack of lubrication oil expenses, compensates this effect. As a net result, it can be estimated, that the operating expenses of this new motor could be about one percentage point lower than with a conventional motor; that is, the new motor would be slightly better than the conventional one. An oil-free free piston engine is particularly suitable for distributed energy systems using natural gas, biogas, or liquid fuel made from biomass. Because it is completely oil-free, it is very environmentally friendly, and its exhaust gases are completely free of oil residuals which are causing problems in normal gas motors. In principle the oil-free free piston engine could be used also in road vehicles which are provided with an electric power transmission system. This could enable a complete oil-free traffic system, where DME (dimethyl ether) or alcohol produced from domestic biomass would be used as a fuel. The distribution of this kind of a fuel would be easier with the present service station network than the distribution of hydrogen. Because this

  1. The microalga Chlamydomonas reinhardtii CW-15 as a solar cell for hydrogen peroxide photoproduction. Comparison between free and immobilized cells and thylakoids for energy conversion efficiency

    Energy Technology Data Exchange (ETDEWEB)

    Scholz, W.; Galvan, F.; Rosa, F.F. de la [Instituto de Bioquimica Vegetal y Fotosintesis, Universidad de Sevilla y CSIC, Sevilla (Spain)

    1995-11-28

    Immobilized cells and thylakoid vesicles of the microalga Chlamydomonas reinhardtii CW-15 have been developed as a solar cell because of their capabilities of producing hydrogen peroxide. This compound is an efficient and clean fuel used for rocket propulsion, motors and for heating. Hydrogen peroxide is produced by the photosystem in a catalyst cycle in which a redox mediator (methyl viologen) is reduced by electrons obtained from water by the photosynthetic apparatus of the microalga and it is re-oxidized by the oxygen dissolved in the solution. The photoproduction has been investigated using a discontinuous system with whole cells, or thylakoid vesicles, free or immobilized on alginate. The stimulation by azide as an inhibitor of catalase has also been analyzed. Under determined optimum conditions, the photoproduction by Ca-alginate entrapped cells, with a rate of 33 {mu}mol H{sub 2}O{sub 2}/mg Chl.h, was maintained for several hours with an energy conversion efficiency of 0.25%

  2. ElectroCat: DOE's approach to PGM-free catalyst and electrode R&D

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, Simon T.; Wilson, Adria R.; Zelenay, Piotr; Myers, Deborah J.; More, Karren L.; Neyerlin, K. C.; Papageorgopoulos, Dimitrios

    2018-06-01

    The successful development of high-performance, durable platinum group metal-free (PGM-free) electrocatalysts and electrodes for polymer electrolyte membrane fuel cells (PEMFCs) will ultimately improve the cost-competiveness of fuel cells in a wide range of applications. This is considered to be a critical development especially for automotive fuel cell applications in order to bring the system cost of an automotive fuel cell system down to the $30/kW cost target set by the U.S. Department of Energy (DOE). The platinum group metal (PGM) electrocatalysts are a major contributor to the system cost. Addressing the technical challenges to PGM-free electrocatalyst and electrode development, therefore, represents one of DOE's most pressing research and development (R&D) priorities. ElectroCat was formed by the DOE as part of the Energy Materials Network (EMN) in early 2016, and shares with other EMN consortia the goal of decreasing the time to market for advanced materials related to clean energy technologies, in the context of increasing U.S. fuel cell electric vehicle (FCEV) manufacturing competitiveness. To accomplish this, the consortium performs core research and development and provides universities and companies streamlined access to the unique, world-class set of tools and expertise relevant to early-stage applied PGM-free catalyst R&D of the member national laboratories. Moreover, ElectroCat fosters a systematic methodology by which prospective catalysts and electrodes are prepared and analyzed rapidly and comprehensively using high-throughput, combinatorial methods. Catalyst discovery is augmented by theory as well as foundational electrocatalysis and materials knowledge at the participating national laboratories. Furthermore, ElectroCat has developed a data sharing framework, requisite of all EMN consortia, for disseminating its findings to the public via a searchable database, to further expedite incorporation of PGM-free electrocatalysts into next

  3. The cell-free integration of a polytopic mitochondrial membrane protein into liposomes occurs cotranslationally and in a lipid-dependent manner.

    Directory of Open Access Journals (Sweden)

    Ashley R Long

    Full Text Available The ADP/ATP Carrier (AAC is the most abundant transporter of the mitochondrial inner membrane. The central role that this transporter plays in cellular energy production highlights the importance of understanding its structure, function, and the basis of its pathologies. As a means of preparing proteoliposomes for the study of membrane proteins, several groups have explored the use of cell-free translation systems to facilitate membrane protein integration directly into preformed unilamellar vesicles without the use of surfactants. Using AAC as a model, we report for the first time the detergent-free reconstitution of a mitochondrial inner membrane protein into liposomes using a wheat germ-based in vitro translation system. Using a host of independent approaches, we demonstrate the efficient integration of AAC into vesicles with an inner membrane-mimetic lipid composition and, more importantly, that the integrated AAC is functionally active in transport. By adding liposomes at different stages of the translation reaction, we show that this direct integration is obligatorily cotranslational, and by synthesizing stable ribosome-bound nascent chain intermediates, we show that the nascent AAC polypeptide interacts with lipid vesicles while ribosome-bound. Finally, we show that the presence of the phospholipid cardiolipin in the liposomes specifically enhances AAC translation rate as well as the efficiency of vesicle association and integration. In light of these results, the possible mechanisms of liposome-assisted membrane protein integration during cell-free translation are discussed with respect to the mode of integration and the role of specific lipids.

  4. Free and Licensed Operating Systems: Direct Comparison

    Directory of Open Access Journals (Sweden)

    Samy Hagman

    2015-06-01

    Full Text Available When developers comparing open with proprietary code, which should be a civilized debate often degenerates into a flame war. This paper presents a report on code quality metrics that have been collected from four major operating systems on an industrial scale is as follows: FreeBSD, Linux, OpenSolaris and Windows Research Kernel (WRK. This article is not a mysterious crime and it can be concluded as the main finding no significant differences in the quality of the code of these systems.

  5. Estimating cell capacity for multi-cell electrical energy system

    Science.gov (United States)

    Hashemi, Iman Ahari

    A Multi-Cell Electrical Energy System is a set of batteries that are connected in series. The series batteries provide the required voltage necessary for the contraption. After using the energy that is provided by the batteries, some cells within the system tend to have a lower voltage than the other cells. Also, other factors, such as the number of times a battery has been charged or discharged, how long it has been within the system and many other factors, result in some cells having a lesser capacity compared to the other cells within the system. The outcome is that it lowers the required capacity that the electrical energy system is required to provide. By having an unknown cell capacity within the system, it is unknown how much of a charge can be provided to the system so that the cells are not overcharged or undercharged. Therefore, it is necessary to know the cells capacity within the system. Hence, if we were dealing with a single cell, the capacity could be obtained by a full charge and discharge of the cell. In a series system that contains multiple cells a full charging or discharging cannot happen as it might result in deteriorating the structure of some cells within the system. Hence, to find the capacity of a single cell within an electrical energy system it is required to obtain a method that can estimate the value of each cell within the electrical energy system. To approach this method an electrical energy system is required. The electrical energy system consists of rechargeable non-equal capacity batteries to provide the required energy to the system, a battery management system (BMS) board to monitor the cells voltages, an Arduino board that provides the required communication to BMS board, and the PC, and a software that is able to deliver the required data obtained from the Arduino board to the PC. The outcome, estimating the capacity of a cell within a multi-cell system, can be used in many battery related technologies to obtain unknown

  6. Stress Management in Cyst-Forming Free-Living Protists: Programmed Cell Death and/or Encystment

    Science.gov (United States)

    Khan, Naveed Ahmed; Iqbal, Junaid

    2015-01-01

    In the face of harsh conditions and given a choice, a cell may (i) undergo programmed cell death, (ii) transform into a cancer cell, or (iii) enclose itself into a cyst form. In metazoans, the available evidence suggests that cellular machinery exists only to execute or avoid programmed cell death, while the ability to form a cyst was either lost or never developed. For cyst-forming free-living protists, here we pose the question whether the ability to encyst was gained at the expense of the programmed cell death or both functions coexist to counter unfavorable environmental conditions with mutually exclusive phenotypes. PMID:25648302

  7. Stress Management in Cyst-Forming Free-Living Protists: Programmed Cell Death and/or Encystment

    Directory of Open Access Journals (Sweden)

    Naveed Ahmed Khan

    2015-01-01

    Full Text Available In the face of harsh conditions and given a choice, a cell may (i undergo programmed cell death, (ii transform into a cancer cell, or (iii enclose itself into a cyst form. In metazoans, the available evidence suggests that cellular machinery exists only to execute or avoid programmed cell death, while the ability to form a cyst was either lost or never developed. For cyst-forming free-living protists, here we pose the question whether the ability to encyst was gained at the expense of the programmed cell death or both functions coexist to counter unfavorable environmental conditions with mutually exclusive phenotypes.

  8. Percutaneous Mitral Valve Repair in Mitral Regurgitation Reduces Cell-Free Hemoglobin and Improves Endothelial Function.

    Directory of Open Access Journals (Sweden)

    Christos Rammos

    Full Text Available Endothelial dysfunction is predictive for cardiovascular events and may be caused by decreased bioavailability of nitric oxide (NO. NO is scavenged by cell-free hemoglobin with reduction of bioavailable NO up to 70% subsequently deteriorating vascular function. While patients with mitral regurgitation (MR suffer from an impaired prognosis, mechanisms relating to coexistent vascular dysfunctions have not been described yet. Therapy of MR using a percutaneous mitral valve repair (PMVR approach has been shown to lead to significant clinical benefits. We here sought to investigate the role of endothelial function in MR and the potential impact of PMVR.Twenty-seven patients with moderate-to-severe MR treated with the MitraClip® device were enrolled in an open-label single-center observational study. Patients underwent clinical assessment, conventional echocardiography, and determination of endothelial function by measuring flow-mediated dilation (FMD of the brachial artery using high-resolution ultrasound at baseline and at 3-month follow-up. Patients with MR demonstrated decompartmentalized hemoglobin and reduced endothelial function (cell-free plasma hemoglobin in heme 28.9±3.8 μM, FMD 3.9±0.9%. Three months post-procedure, PMVR improved ejection fraction (from 41±3% to 46±3%, p = 0.03 and NYHA functional class (from 3.0±0.1 to 1.9±1.7, p<0.001. PMVR was associated with a decrease in cell free plasma hemoglobin (22.3±2.4 μM, p = 0.02 and improved endothelial functions (FMD 4.8±1.0%, p<0.0001.We demonstrate here that plasma from patients with MR contains significant amounts of cell-free hemoglobin, which is accompanied by endothelial dysfunction. PMVR therapy is associated with an improved hemoglobin decompartmentalization and vascular function.

  9. Application of confocal Raman micro-spectroscopy for label-free monitoring of oxidative stress in living bronchial cells

    Science.gov (United States)

    Surmacki, Jakub M.; Quirós Gonzalez, Isabel; Bohndiek, Sarah E.

    2018-02-01

    Oxidative stress in cancer is implicated in tumor progression, being associated with increased therapy resistance and metastasis. Conventional approaches for monitoring oxidative stress in tissue such as high-performance liquid chromatography and immunohistochemistry are bulk measurements and destroy the sample, meaning that longitudinal monitoring of cancer cell heterogeneity remains elusive. Raman spectroscopy has the potential to overcome this challenge, providing a chemically specific, label free readout from single living cells. Here, we applied a standardized protocol for label-free confocal Raman micro-spectroscopy in living cells to monitor oxidative stress in bronchial cells. We used a quartz substrate in a commercial cell chamber contained within a microscope incubator providing culture media for cell maintenance. We studied the effect of a potent reactive oxygen species inducer, tert-butyl hydroperoxide (TBHP), and antioxidant, N-acetyl-L-cysteine (NAC) on living cells from a human bronchial epithelial cells (HBEC). We found that the Raman bands corresponding to nucleic acids, proteins and lipids were significantly different (pmicro-spectroscopy may be able to monitor the biological impact of oxidative and reductive processes in cells, hence enabling longitudinal studies of oxidative stress in therapy resistance and metastasis at the single cell level.

  10. Serum-Free Media and the Immunoregulatory Properties of Mesenchymal Stem Cells In Vivo and In Vitro

    OpenAIRE

    Mei Wu; Zhi-Bo Han; Jun Feng Liu; You Wei Wang; Jian Zhong Zhang; Chun Tuan Li; Peng Liang Xin; Zhong Chao Han; Xiong Peng Zhu

    2014-01-01

    Background: Mesenchymal stem cells are capable of self-renewal and multi-lineage differentiation. They are used extensively to treat several diseases. Traditionally, mesenchymal stem cells are cultured in serum-containing media, typically supplemented with fetal bovine serum (FBS). However, the variability of FBS is likely to skew experimental results. Although serum-free media used to expand mesenchymal stem cells has facilitated remarkable achievements, immunomodulation of these cells in un...

  11. Flexible inverted polymer solar cells with an indium-free tri-layer cathode

    International Nuclear Information System (INIS)

    El Hajj, Ahmad; Lucas, Bruno; Schirr-Bonnans, Martin; Ratier, Bernard; Kraft, Thomas M.; Torchio, Philippe

    2014-01-01

    Indium tin oxide (ITO)-free inverted polymer solar cells (PSCs) have been fabricated without the need of an additional electron transport layer. The indium-free transparent electrode consists of a tri-layer stack ZnO (30 nm)/Ag (14 nm)/ZnO (30 nm) deposited on glass and plastic substrates via ion-beam sputtering. The tri-layer electrodes exhibit similar physical properties to its ITO counterpart, specifically yielding high transmittance and low resistivity (76.5% T at 550 nm, R sq of 8 Ω/◻) on plastic substrates. The novel tri-layer electrode allows for the fabrication of inverted PSCs without the additional ZnO interfacial layer commonly deposited between ITO and the photoactive layer. This allows for the preparation of thinner plastic solar cells using less material than conventional architectures. Initial studies involving the newly realized architecture (tri-layer electrode/P3HT:PCBM/PEDOT:PSS/Ag) have shown great promise for the transition from ITO to other viable electrodes in organic electronics

  12. Free volume and relaxation dynamics of polymeric systems

    International Nuclear Information System (INIS)

    Bartos, J.; Kristiak, J.

    1999-01-01

    In this contribution use of positron annihilation spectroscopy (PALS) for the study of free volume and relaxation dynamics of some polymeric systems (1,4-polybutadiene, cis-1,4-polyisoprene, polyisobutylene, trans-1,4-polychloropropene, atactic polypropylene and 1,2-polybutadiene) is discussed

  13. Fuel cell-based cogeneration system covering data centers’ energy needs

    International Nuclear Information System (INIS)

    Guizzi, Giuseppe Leo; Manno, Michele

    2012-01-01

    The Information and Communication Technology industry has gone in the recent years through a dramatic expansion, driven by many new online (local and remote) applications and services. Such growth has obviously triggered an equally remarkable growth in energy consumption by data centers, which require huge amounts of power not only for IT devices, but also for power distribution units and for air-conditioning systems needed to cool the IT equipment. This paper is dedicated to the economic and energy performance assessment of a cogeneration system based on a natural gas membrane steam reformer producing a pure hydrogen flow for electric power generation in a polymer electrolyte membrane fuel cell. Heat is recovered from both the reforming unit and the fuel cell in order to supply the needs of an office building located near the data center. In this case, the cooling energy needs of the data center are covered by means of a vapor-compression chiller equipped with a free-cooling unit. Since the fuel cell’s output is direct current rather than alternate current, the possibility of further improving data centers’ energy efficiency adopting DC-powered data center equipment is also discussed. -- Highlights: ► Data centers' energy needs are discussed and possible savings from advanced energy management techniques are estimated. ► The thermal energy requirements of an office building close to the data center are added to the energy scenario. ► Significant energy and cost savings can be obtained by means of free-cooling, high-voltage direct current, and a cogeneration facility. ► The cogeneration system is based on a natural gas membrane reformer and a PEM fuel cell. ► Energy flows in the membrane reformer are analyzed and an optimal value of steam-to-carbon ratio is found in order to minimize the required membrane area.

  14. Easy xeno-free and feeder-free method for isolating and growing limbal stromal and epithelial stem cells of the human cornea.

    Directory of Open Access Journals (Sweden)

    Djida Ghoubay-Benallaoua

    Full Text Available Epithelial and stromal stem cells are required to maintain corneal transparency. The aim of the study was to develop a new method to isolate and grow both corneal stromal (SSC and epithelial limbal (LSC stem cells from small human limbal biopsies under culture conditions in accordance with safety requirements mandatory for clinical use in humans. Superficial limbal explants were retrieved from human donor corneo-scleral rims. Human limbal cells were dissociated by digestion with collagenase A, either after epithelial scraping or with no scraping. Isolated cells were cultured with Essential 8 medium (E8, E8 supplemented with EGF (E8+ or Green's medium with 3T3 feeder-layers. Cells were characterized by immunostaining, RT-qPCR, colony forming efficiency, sphere formation, population doubling, second harmonic generation microscopy and differentiation potentials. LSC were obtained from unscraped explants in E8, E8+ and Green's media and were characterized by colony formation and expression of PAX6, ΔNP63α, Bmi1, ABCG2, SOX9, CK14, CK15 and vimentin, with a few cells positive for CK3. LSC underwent 28 population doublings still forming colonies. SSC were obtained from both scraped and unscraped explants in E8 and E8+ media and were characterized by sphere formation, expression of PAX6, SOX2, BMI1, NESTIN, ABCG2, KERATOCAN, VIMENTIN, SOX9, SOX10 and HNK1, production of collagen fibrils and differentiation into keratocytes, fibroblasts, myofibroblasts, neurons, adipocytes, chondrocytes and osteocytes. SSC underwent 48 population doublings still forming spheres, Thus, this new method allows both SSC and LSC to be isolated from small superficial limbal biopsies and to be primary cultured in feeder-free and xeno-free conditions, which will be useful for clinical purposes.

  15. Effects of cell suspension and cell·free culture filtrate of Pseudomonas aeruginosa in the control of root rot-root kont disease complex of tomato (Lycopersicon esculentum Mill.

    Directory of Open Access Journals (Sweden)

    I. A. Siddiqui

    2013-12-01

    Full Text Available The plant growth-promoting rhizobacterium Pseudomonas aeruginosa strain IE-6 was tested for antagonistic activity towards Meloidogyne javanica, the root-knot nematode and soilbome root-infecting fungi viz., Macrophomina phaseolina, Fusarium solani and Rhizoctonia solani under laboratory and greenhouse conditions. Cell-free culture filtrate of the bacterium caused significant reduction in egg hatching of M.javanica and inhibited radial growth of fungi in vitro. Cell-free culture filtrate also caused lyses in mycelium of F.solani. Under greenhouse conditions, soil drenches with the aqueous cell suspension or cell-free culture resulted in a considerable reduction in nematode population densities in soil and subsequent root-knot development due to M.javanica. In addition to nematode control, rhizobacterium application also inhibited root-infection caused by soilborne root~infecting fungi with significant enhancement of growth of tomato seedlings.

  16. Comparison of real-time reverse transcriptase polymerase chain reaction of peripheral blood mononuclear cells, serum and cell-free body cavity effusion for the diagnosis of feline infectious peritonitis.

    Science.gov (United States)

    Doenges, Stephanie J; Weber, Karin; Dorsch, Roswitha; Fux, Robert; Hartmann, Katrin

    2017-04-01

    Objectives Diagnosis of feline infectious peritonitis (FIP) remains challenging, especially in cats without effusions. The objective of this study was to evaluate the sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction (RT-PCR) detecting feline coronavirus (FCoV) RNA in peripheral blood mononuclear cells (PBMCs) and serum in comparison with the same real-time RT-PCR in cell-free body cavity effusion. Methods This prospective case-control study included 92 cats. Forty-three cats had a definitive diagnosis of FIP, established either by histopathological examination (n = 28) or by positive immunofluorescence staining of FCoV antigen in macrophages of effusions (n = 11), or by both methods (n = 4). Forty-nine control cats had other diseases but similar clinical signs. Real-time RT-PCR was performed on PBMCs of 37 cats (21 cats with FIP, 16 controls), on serum of 51 cats (26 cats with FIP, 25 controls) and on cell-free body cavity effusion of 69 cats (36 cats with FIP, 33 controls). Sensitivity, specificity, positive and negative predictive value, including 95% confidence intervals (CI), were calculated. Results Real-time RT-PCR of PBMCs, serum and cell-free body cavity effusion showed a specificity of 100% (95% CI 79.4-100% in PBMCs, 86.3-100% in serum, 89.4-100% in cell-free body cavity effusion) and a sensitivity of 28.6% (95% CI 11.3-52.2%) in PBMCs, 15.4% (95% CI 4.4-34.9%) in serum and 88.9% (95% CI 73.9-96.9%) in cell-free body cavity effusion to diagnose FIP. Conclusions and relevance Although it is known that RT-PCR can often provide false-positive results in healthy cats, this real-time RT-PCR was shown to be a specific tool for the diagnosis of FIP when applied in a clinical setting. Sensitivity in cell-free body cavity effusion was high but low in PBMCs and serum. PBMC samples showed a higher sensitivity than serum samples, and are therefore a better choice if no effusion is present.

  17. Comparison of intracellular accumulation and cytotoxicity of free mTHPC and mTHPC-loaded PLGA nanoparticles in human colon carcinoma cells

    International Nuclear Information System (INIS)

    Loew, Karin; Wagner, Sylvia; Briesen, Hagen von; Knobloch, Thomas; Wiehe, Arno; Engel, Andrea; Langer, Klaus

    2011-01-01

    The second generation photosensitizer mTHPC was approved by the European Medicines Agency (EMA) for the palliative treatment of advanced head and neck cancer in October 2001. It is known that mTHPC possesses a significant phototoxicity against a variety of human cancer cells in vitro but also exhibits dark toxicity and can cause adverse effects (especially skin photosensitization). Due to its poor water solubility, the administration of hydrophobic photosensitizer still presents several difficulties. To overcome the administration problems, the use of nanoparticles as drug carrier systems is much investigated. Nanoparticles based on poly(lactic-co-glycolic acid) (PLGA) have been extensively studied as delivery systems into tumours due to their biocompatibility and biodegradability. The goal of this study was the comparison of free mTHPC and mTHPC-loaded PLGA nanoparticles concerning cytotoxicity and intracellular accumulation in human colon carcinoma cells (HT29). The nanoparticles delivered the photosensitizer to the colon carcinoma cells and enabled drug release without losing its activity. The cytotoxicity assays showed a time- and concentration-dependent decrease in cell proliferation and viability after illumination. However, first and foremost mTHPC lost its dark toxic effects using the PLGA nanoparticles as a drug carrier system. Therefore, PLGA nanoparticles are a promising drug carrier system for the hydrophobic photosensitizer mTHPC.

  18. Comparison of intracellular accumulation and cytotoxicity of free mTHPC and mTHPC-loaded PLGA nanoparticles in human colon carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Loew, Karin; Wagner, Sylvia; Briesen, Hagen von [Fraunhofer-Institute for Biomedical Engineering, D-66386 Strasse Ingbert (Germany); Knobloch, Thomas [Institute of Pharmaceutical Technology, Biocenter of Goethe-University, D-60438 Frankfurt (Germany); Wiehe, Arno [Biolitec AG, D-07745 Jena (Germany); Engel, Andrea; Langer, Klaus, E-mail: hagen.briesen@ibmt.fraunhofer.de [Institute of Pharmaceutical Technology and Biopharmacy, University of Muenster, D-48149 Muenster (Germany)

    2011-06-17

    The second generation photosensitizer mTHPC was approved by the European Medicines Agency (EMA) for the palliative treatment of advanced head and neck cancer in October 2001. It is known that mTHPC possesses a significant phototoxicity against a variety of human cancer cells in vitro but also exhibits dark toxicity and can cause adverse effects (especially skin photosensitization). Due to its poor water solubility, the administration of hydrophobic photosensitizer still presents several difficulties. To overcome the administration problems, the use of nanoparticles as drug carrier systems is much investigated. Nanoparticles based on poly(lactic-co-glycolic acid) (PLGA) have been extensively studied as delivery systems into tumours due to their biocompatibility and biodegradability. The goal of this study was the comparison of free mTHPC and mTHPC-loaded PLGA nanoparticles concerning cytotoxicity and intracellular accumulation in human colon carcinoma cells (HT29). The nanoparticles delivered the photosensitizer to the colon carcinoma cells and enabled drug release without losing its activity. The cytotoxicity assays showed a time- and concentration-dependent decrease in cell proliferation and viability after illumination. However, first and foremost mTHPC lost its dark toxic effects using the PLGA nanoparticles as a drug carrier system. Therefore, PLGA nanoparticles are a promising drug carrier system for the hydrophobic photosensitizer mTHPC.

  19. A chemo-mechanical free-energy-based approach to model durotaxis and extracellular stiffness-dependent contraction and polarization of cells.

    Science.gov (United States)

    Shenoy, Vivek B; Wang, Hailong; Wang, Xiao

    2016-02-06

    We propose a chemo-mechanical model based on stress-dependent recruitment of myosin motors to describe how the contractility, polarization and strain in cells vary with the stiffness of their surroundings and their shape. A contractility tensor, which depends on the distribution of myosin motors, is introduced to describe the chemical free energy of the cell due to myosin recruitment. We explicitly include the contributions to the free energy that arise from mechanosensitive signalling pathways (such as the SFX, Rho-Rock and MLCK pathways) through chemo-mechanical coupling parameters. Taking the variations of the total free energy, which consists of the chemical and mechanical components, in accordance with the second law of thermodynamics provides equations for the temporal evolution of the active stress and the contractility tensor. Following this approach, we are able to recover the well-known Hill relation for active stresses, based on the fundamental principles of irreversible thermodynamics rather than phenomenology. We have numerically implemented our free energy-based approach to model spatial distribution of strain and contractility in (i) cells supported by flexible microposts, (ii) cells on two-dimensional substrates, and (iii) cells in three-dimensional matrices. We demonstrate how the polarization of the cells and the orientation of stress fibres can be deduced from the eigenvalues and eigenvectors of the contractility tensor. Our calculations suggest that the chemical free energy of the cell decreases with the stiffness of the extracellular environment as the cytoskeleton polarizes in response to stress-dependent recruitment of molecular motors. The mechanical energy, which includes the strain energy and motor potential energy, however, increases with stiffness, but the overall energy is lower for cells in stiffer environments. This provides a thermodynamic basis for durotaxis, whereby cells preferentially migrate towards stiffer regions of the

  20. N-Terminal Prodomain of Pfs230 Synthesized Using a Cell-Free System Is Sufficient To Induce Complement-Dependent Malaria Transmission-Blocking Activity▿

    Science.gov (United States)

    Tachibana, Mayumi; Wu, Yimin; Iriko, Hideyuki; Muratova, Olga; MacDonald, Nicholas J.; Sattabongkot, Jetsumon; Takeo, Satoru; Otsuki, Hitoshi; Torii, Motomi; Tsuboi, Takafumi

    2011-01-01

    The aim of a malaria transmission-blocking vaccine is to block the development of malaria parasites in the mosquito and thus prevent subsequent infection of the human host. Previous studies have demonstrated that the gametocyte/gamete surface protein Pfs230 can induce transmission-blocking immunity and have evaluated Escherichia coli-produced Pfs230 as a transmission-blocking vaccine candidate. In this study, we used the wheat germ cell-free expression system to produce N-terminal fragments of Pfs230 and evaluated the transmission-blocking activity of antisera raised against the recombinant Pfs230 protein. The rabbit antisera reacted to the surface of cultured gametocytes and gametes of the Plasmodium falciparum NF54 line, recognized the 360-kDa form of parasite-produced Pfs230 by Western blot assay, and reduced the infectivity of NF54 parasites to Anopheles stefensi mosquitoes in the presence of complement in a standard membrane feeding assay. Thus, our data demonstrate that the N-terminal pro domain of Pfs230 is sufficient to induce complement-dependent transmission-blocking activity against P. falciparum. PMID:21715579

  1. N-terminal prodomain of Pfs230 synthesized using a cell-free system is sufficient to induce complement-dependent malaria transmission-blocking activity.

    Science.gov (United States)

    Tachibana, Mayumi; Wu, Yimin; Iriko, Hideyuki; Muratova, Olga; MacDonald, Nicholas J; Sattabongkot, Jetsumon; Takeo, Satoru; Otsuki, Hitoshi; Torii, Motomi; Tsuboi, Takafumi

    2011-08-01

    The aim of a malaria transmission-blocking vaccine is to block the development of malaria parasites in the mosquito and thus prevent subsequent infection of the human host. Previous studies have demonstrated that the gametocyte/gamete surface protein Pfs230 can induce transmission-blocking immunity and have evaluated Escherichia coli-produced Pfs230 as a transmission-blocking vaccine candidate. In this study, we used the wheat germ cell-free expression system to produce N-terminal fragments of Pfs230 and evaluated the transmission-blocking activity of antisera raised against the recombinant Pfs230 protein. The rabbit antisera reacted to the surface of cultured gametocytes and gametes of the Plasmodium falciparum NF54 line, recognized the 360-kDa form of parasite-produced Pfs230 by Western blot assay, and reduced the infectivity of NF54 parasites to Anopheles stefensi mosquitoes in the presence of complement in a standard membrane feeding assay. Thus, our data demonstrate that the N-terminal pro domain of Pfs230 is sufficient to induce complement-dependent transmission-blocking activity against P. falciparum.

  2. Purification and characterization of enterovirus 71 viral particles produced from vero cells grown in a serum-free microcarrier bioreactor system.

    Directory of Open Access Journals (Sweden)

    Chia-Chyi Liu

    Full Text Available BACKGROUND: Enterovirus 71 (EV71 infections manifest most commonly as a childhood exanthema known as hand-foot-and-mouth disease (HFMD and can cause neurological disease during acute infection. PRINCIPAL FINDING: In this study, we describe the production, purification and characterization of EV71 virus produced from Vero cells grown in a five-liter serum-free bioreactor system containing 5 g/L Cytodex 1 microcarrier. The viral titer was >10(6 TCID(50/mL by 6 days post infection when a MOI of 10(-5 was used at the initial infection. Two EV71 virus fractions were separated and detected when the harvested EV71 virus concentrate was purified by sucrose gradient zonal ultracentrifugation. The EV71 viral particles detected in the 24-28% sucrose fractions had an icosahedral structure 30-31 nm in diameter and had low viral infectivity and RNA content. Three major viral proteins (VP0, VP1 and VP3 were observed by SDS-PAGE. The EV71 viral particles detected in the fractions containing 35-38% sucrose were 33-35 nm in size, had high viral infectivity and RNA content, and were composed of four viral proteins (VP1, VP2, VP3 and VP4, as shown by SDS-PAGE analyses. The two virus fractions were formalin-inactivated and induced high virus neutralizing antibody responses in mouse immunogenicity studies. Both mouse antisera recognized the immunodominant linear neutralization epitope of VP1 (residues 211-225. CONCLUSION: These results provide important information for cell-based EV71 vaccine development, particularly for the preparation of working standards for viral antigen quantification.

  3. Capture of free-floating planets by planetary systems

    Science.gov (United States)

    Goulinski, Nadav; Ribak, Erez N.

    2018-01-01

    Evidence of exoplanets with orbits that are misaligned with the spin of the host star may suggest that not all bound planets were born in the protoplanetary disc of their current planetary system. Observations have shown that free-floating Jupiter-mass objects can exceed the number of stars in our Galaxy, implying that capture scenarios may not be so rare. To address this issue, we construct a three-dimensional simulation of a three-body scattering between a free-floating planet and a star accompanied by a Jupiter-mass bound planet. We distinguish between three different possible scattering outcomes, where the free-floating planet may get weakly captured after the brief interaction with the binary, remain unbound or 'kick out' the bound planet and replace it. The simulation was performed for different masses of the free-floating planets and stars, as well as different impact parameters, inclination angles and approach velocities. The outcome statistics are used to construct an analytical approximation of the cross-section for capturing a free-floating planet by fitting their dependence on the tested variables. The analytically approximated cross-section is used to predict the capture rate for these kinds of objects, and to estimate that about 1 per cent of all stars are expected to experience a temporary capture of a free-floating planet during their lifetime. Finally, we propose additional physical processes that may increase the capture statistics and whose contribution should be considered in future simulations in order to determine the fate of the temporarily captured planets.

  4. Murine pluripotent stem cells derived scaffold-free cartilage grafts from a micro-cavitary hydrogel platform.

    Science.gov (United States)

    He, Pengfei; Fu, Jiayin; Wang, Dong-An

    2016-04-15

    By means of appropriate cell type and scaffold, tissue-engineering approaches aim to construct grafts for cartilage repair. Pluripotent stem cells especially induced pluripotent stem cells (iPSCs) are of promising cell candidates due to the pluripotent plasticity and abundant cell source. We explored three dimensional (3D) culture and chondrogenesis of murine iPSCs (miPSCs) on an alginate-based micro-cavity hydrogel (MCG) platform in pursuit of fabricating synthetic-scaffold-free cartilage grafts. Murine embryonic stem cells (mESCs) were employed in parallel as the control. Chondrogenesis was fulfilled using a consecutive protocol via mesoderm differentiation followed by chondrogenic differentiation; subsequently, miPSC and mESC-seeded constructs were further respectively cultured in chondrocyte culture (CC) medium. Alginate phase in the constructs was then removed to generate a graft only comprised of induced chondrocytic cells and cartilaginous extracellular matrix (ECMs). We found that from the mESC-seeded constructs, formation of intact grafts could be achieved in greater sizes with relatively fewer chondrocytic cells and abundant ECMs; from miPSC-seeded constructs, relatively smaller sized cartilaginous grafts could be formed by cells with chondrocytic phenotype wrapped by abundant and better assembled collagen type II. This study demonstrated successful creation of pluripotent stem cells-derived cartilage/chondroid graft from a 3D MCG interim platform. By the support of materials and methodologies established from this study, particularly given the autologous availability of iPSCs, engineered autologous cartilage engraftment may be potentially fulfilled without relying on the limited and invasive autologous chondrocytes acquisition. In this study, we explored chondrogenic differentiation of pluripotent stem cells on a 3D micro-cavitary hydrogel interim platform and creation of pluripotent stem cells-derived cartilage/chondroid graft via a consecutive

  5. Tail-extension following the termination codon is critical for release of the nascent chain from membrane-bound ribosomes in a reticulocyte lysate cell-free system.

    Science.gov (United States)

    Takahara, Michiyo; Sakaue, Haruka; Onishi, Yukiko; Yamagishi, Marifu; Kida, Yuichiro; Sakaguchi, Masao

    2013-01-11

    Nascent chain release from membrane-bound ribosomes by the termination codon was investigated using a cell-free translation system from rabbit supplemented with rough microsomal membrane vesicles. Chain release was extremely slow when mRNA ended with only the termination codon. Tail extension after the termination codon enhanced the release of the nascent chain. Release reached plateau levels with tail extension of 10 bases. This requirement was observed with all termination codons: TAA, TGA and TAG. Rapid release was also achieved by puromycin even in the absence of the extension. Efficient translation termination cannot be achieved in the presence of only a termination codon on the mRNA. Tail extension might be required for correct positioning of the termination codon in the ribosome and/or efficient recognition by release factors. Copyright © 2012. Published by Elsevier Inc.

  6. β Style Free-Piston Stirling Engine Control System Research

    Directory of Open Access Journals (Sweden)

    Xu Jian

    2016-01-01

    Full Text Available For the Free-Piston Stirling Engines (FPSE control system, a three -phase bridge circuit is reused as the system output about rectifier and start inverter. When FPSE system is in the power stage, the double closed loop control strategy and optimization algorithm of PI control parameters is adopted to ensure the highest system transmission efficiency under the requirements of the system output power and guarantee the stability of the running system. The simulation results prove the effectiveness of the above research content.

  7. Chasing Salmonella Typhimurium in free range egg production system.

    Science.gov (United States)

    Chousalkar, Kapil; Gole, Vaibhav; Caraguel, Charles; Rault, Jean-Loup

    2016-08-30

    Free range production systems are becoming a major source of egg production in Australia and worldwide. This study investigated shedding and ecology of Salmonella Typhimurium and Salmonella species in a free range layer flock, wild birds and foxes in the vicinity of the free range farm in different seasons. Shedding of Salmonella was significantly higher in summer. Within the shed, overall, Salmonella prevalence was highest in dust. Corticosterone level in faeces was highest in spring and lowest in winter. There was no direct association between the Salmonella shedding (MPN/gm) and corticosterone levels in faeces. Salmonella Typhimurium MLVA types isolated from fox and wild birds were similar to MLVA types isolated from layer flock and reported during human food borne illness. Wild birds and foxes appear to play an important role in S. Typhimurium ecology and food safety. Environmental factors could play a role in evolution of S. Typhimurium in free range environment. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

  8. Cell-Free DNA and Active Rejection in Kidney Allografts.

    Science.gov (United States)

    Bloom, Roy D; Bromberg, Jonathan S; Poggio, Emilio D; Bunnapradist, Suphamai; Langone, Anthony J; Sood, Puneet; Matas, Arthur J; Mehta, Shikha; Mannon, Roslyn B; Sharfuddin, Asif; Fischbach, Bernard; Narayanan, Mohanram; Jordan, Stanley C; Cohen, David; Weir, Matthew R; Hiller, David; Prasad, Preethi; Woodward, Robert N; Grskovic, Marica; Sninsky, John J; Yee, James P; Brennan, Daniel C

    2017-07-01

    Histologic analysis of the allograft biopsy specimen is the standard method used to differentiate rejection from other injury in kidney transplants. Donor-derived cell-free DNA (dd-cfDNA) is a noninvasive test of allograft injury that may enable more frequent, quantitative, and safer assessment of allograft rejection and injury status. To investigate this possibility, we prospectively collected blood specimens at scheduled intervals and at the time of clinically indicated biopsies. In 102 kidney recipients, we measured plasma levels of dd-cfDNA and correlated the levels with allograft rejection status ascertained by histology in 107 biopsy specimens. The dd-cfDNA level discriminated between biopsy specimens showing any rejection (T cell-mediated rejection or antibody-mediated rejection [ABMR]) and controls (no rejection histologically), P rejection at a cutoff of 1.0% dd-cfDNA were 61% and 84%, respectively. The AUC for discriminating ABMR from samples without ABMR was 0.87 (95% CI, 0.75 to 0.97). Positive and negative predictive values for ABMR at a cutoff of 1.0% dd-cfDNA were 44% and 96%, respectively. Median dd-cfDNA was 2.9% (ABMR), 1.2% (T cell-mediated types ≥IB), 0.2% (T cell-mediated type IA), and 0.3% in controls ( P =0.05 for T cell-mediated rejection types ≥IB versus controls). Thus, dd-cfDNA may be used to assess allograft rejection and injury; dd-cfDNA levels rejection (T cell-mediated type ≥IB or ABMR) and levels >1% indicate a probability of active rejection. Copyright © 2017 by the American Society of Nephrology.

  9. Integration of CpG-free DNA induces de novo methylation of CpG islands in pluripotent stem cells

    KAUST Repository

    Takahashi, Yuta; Wu, Jun; Suzuki, Keiichiro; Martinez-Redondo, Paloma; Li, Mo; Liao, Hsin-Kai; Wu, Min-Zu; Herná ndez-Bení tez, Reyna; Hishida, Tomoaki; Shokhirev, Maxim Nikolaievich; Esteban, Concepcion Rodriguez; Sancho-Martinez, Ignacio; Belmonte, Juan Carlos Izpisua

    2017-01-01

    that insertion of CpG-free DNA into targeted CGIs induces de novo methylation of the entire CGI in human pluripotent stem cells (PSCs). The methylation status is stably maintained even after CpG-free DNA removal, extensive passaging, and differentiation

  10. Free-piston Stirling Engine system considerations for various space power applications

    Science.gov (United States)

    Dochat, George R.; Dhar, Manmohan

    1991-01-01

    Free-Piston Stirling Engines (FPSE) have the potential to provide high reliability, long life, and efficient operation. Therefore, they are excellent candidates for the dynamic power conversion module of a space-based, power-generating system. FPSE can be coupled with many potential heat sources (radioisotope, solar, or nuclear reactor), various heat input systems (pumped loop, heat pipe), heat rejection (pumped loop or heat pipe), and various power management and distribution systems (ac, dc, high or low voltage, and fixed or variable load). This paper reviews potential space missions that can be met using free-piston Stirling engines and discusses options of various system integration approaches. This paper briefly outlines the program and recent progress.

  11. PRODUCTIVITY OF LAYERS AND EGG QUALITY IN FREE RANGE AND CAGE SYSTEM OF HOUSING

    Directory of Open Access Journals (Sweden)

    Đ. Senčić

    2006-12-01

    Full Text Available The research was conducted with two groups of Lohmann Brown hybrid layers. Production of eggs lasted for 52 weeks. A control group of layers was kept in the conventional housing system, that is, in cages, while experimental group was kept in the free range system. Layers from the free range system, compared to those kept in cages, laid fewer eggs, (266:295, they consumed more feed on daily basis (129 g : 115 g, more feed per kilogram of egg weight (2.83 kg : 2.35 kg, they had higher mortality rate (6.80 % : 5.50 % and lower end of lay body weight (1.95 kg : 2.10 kg. Eggs from free range layers, compared to those from the cages system, had significantly (P0.05 were determined between the free range and the cages system of housing hens. Considering somewhat lower productivity and higher mortality rate of hens, higher feed consumption per kilogram of egg mass, but also better quality of eggs, profitability of egg production in the free range system will depend, to the maximum extent, on market evaluation of the production.

  12. Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei, E-mail: biehzw@nus.edu.sg [Optical Bioimaging Laboratory, Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, Singapore 117576 (Singapore)

    2014-09-08

    We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.

  13. Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

    International Nuclear Information System (INIS)

    Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei

    2014-01-01

    We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.

  14. Achievements and Problems of Reform of Investment Management System for Shanghai Pilot Free Trade Zone

    Institute of Scientific and Technical Information of China (English)

    Chengrong; PAN

    2014-01-01

    The establishment of Shanghai Pilot Free Trade Zone provides many possibilities for China’s economic construction. This paper made a comparative analysis on investment management system of Shanghai Pilot Free Trade Zone and traditional investment management system,discussed achievements and problems of reform of investment management system of Shanghai Pilot Free Trade Zone,and finally came up with pertinent policy recommendations.

  15. Development of a NO/x/-free combustion system

    Science.gov (United States)

    Sadakata, M.; Furusawa, T.; Kunii, D.; Imagawa, M.; Nawada, M.

    1980-04-01

    The development of a NO(x)-free combustion-heating system realizing both pollution control and energy savings is described. An experiment was carried out by using a small model plant. The system consists of a combustion furnace and a new-type multifunctional heat exchanger. The heat exchanger is a rotary continuous type designed for soot collection and for catalytic combustion of CO and H2 as well as for preheating combustion air.

  16. In vitro protein expression: an emerging alternative to cell-based approaches.

    Science.gov (United States)

    He, Mingyue

    2011-04-30

    Protein expression remains a bottleneck in the production of proteins. Owing to several advantages, cell-free translation is emerging as an alternative to cell-based methods for the generation of proteins. Recent advances have led to many novel applications of cell-free systems in biotechnology, proteomics and fundamental biological research. This special issue of New Biotechnology describes recent advances in cell-free protein expression systems and their applications. Copyright © 2010 Elsevier B.V. All rights reserved.

  17. Fibronectin-synthesizing activity of free and membrane-bound polyribosomes from human embryonic fibroblasts and chick embryos

    International Nuclear Information System (INIS)

    Belkin, V.M.; Volodarskaya, S.M.

    1986-01-01

    The fibronectin-synthesizing activity of membrane-bound and free polyribosomes in a cell-free system was studied using immunochemical methods. It was found that fibronectin biosynthesis on membrane-bound polyribosomes from human embryonic fibroblasts accounts for 4.9% and those from 10-day-old chick embryos for 1.1% of the total amount of newly synthesized proteins, whereas on free polyribosomes it is 1.0 and 0.3%, respectively. Fibronectin monomers with a molecular weight of 220,000 were found only in the material of the cell-free system containing heavy fractions of membrane-bound polyribosomes newly synthesized in the presence of spermidine. Thus, it was shown that fibronectin is synthesized primarily on membrane-bound polyribosomes

  18. Emergence of scale-free characteristics in socio-ecological systems with bounded rationality.

    Science.gov (United States)

    Kasthurirathna, Dharshana; Piraveenan, Mahendra

    2015-06-11

    Socio-ecological systems are increasingly modelled by games played on complex networks. While the concept of Nash equilibrium assumes perfect rationality, in reality players display heterogeneous bounded rationality. Here we present a topological model of bounded rationality in socio-ecological systems, using the rationality parameter of the Quantal Response Equilibrium. We argue that system rationality could be measured by the average Kullback--Leibler divergence between Nash and Quantal Response Equilibria, and that the convergence towards Nash equilibria on average corresponds to increased system rationality. Using this model, we show that when a randomly connected socio-ecological system is topologically optimised to converge towards Nash equilibria, scale-free and small world features emerge. Therefore, optimising system rationality is an evolutionary reason for the emergence of scale-free and small-world features in socio-ecological systems. Further, we show that in games where multiple equilibria are possible, the correlation between the scale-freeness of the system and the fraction of links with multiple equilibria goes through a rapid transition when the average system rationality increases. Our results explain the influence of the topological structure of socio-ecological systems in shaping their collective cognitive behaviour, and provide an explanation for the prevalence of scale-free and small-world characteristics in such systems.

  19. Dimethylfumarate induces cell cycle arrest and apoptosis via regulating intracellular redox systems in HeLa cells.

    Science.gov (United States)

    Han, Guocan; Zhou, Qiang

    2016-12-01

    Dimethylfumarate (DMF) is cytotoxic to several kinds of cells and serves as an anti-tumor drug. This study was designed to investigate the effects and underlying mechanism of DMF on cervical cancer cells. HeLa cells were cultured and treated with 0, 50, 100, 150, and 200 μM DMF, respectively. After 24 h, cell growth was evaluated using Cell Counting Kit-8 (CCK-8) assay and the cell cycle was examined using flow cytometry. In addition, cell apoptosis was detected by Annexin V/propidium iodide (PI) staining and the expressions of caspase-3 and poly-ADP-ribose polymerase (PARP) were detected using western blotting. The redox-related factors were then assessed. Furthermore, all of the indicators were detected in HeLa cells after combined treatment of DMF and N-acetyl-L-cysteine (NAC, an oxygen-free radical scavenger). The cell number and cell growth of HeLa were obviously inhibited by DMF in a dose-dependent manner, as the cell cycle was arrested at G0/G1 phase (P HeLa cells were markedly increased, and the expression levels of caspase-3 and PARP were significantly increased in a DMF concentration-dependent way (P cell proliferation and apoptosis of HeLa cells was mainly related to the intracellular redox systems by depletion of intracellular GSH.

  20. Cell-Free DNA in Metastatic Colorectal Cancer: A Systematic Review and Meta-Analysis.

    Science.gov (United States)

    Spindler, Karen-Lise G; Boysen, Anders K; Pallisgård, Niels; Johansen, Julia S; Tabernero, Josep; Sørensen, Morten M; Jensen, Benny V; Hansen, Torben F; Sefrioui, David; Andersen, Rikke F; Brandslund, Ivan; Jakobsen, Anders

    2017-09-01

    systemic therapy. Small fragments of circulating cell-free DNA (cfDNA) can be measured in a simple blood sample. This report presents the first meta-analysis of the prognostic value of total cfDNA measurement in patients with metastatic colorectal cancer. Data from 1,076 patients confirmed that patients with the lowest pre-treatment levels of cfDNA had a significantly higher chance of longer survival than those with higher levels. Cell-free DNA analysis can also be used for detection of tumor-specific mutations, and hold potential as a valuable tool in colorectal cancer treatment. © AlphaMed Press 2017.

  1. Newly-derived neuroblastoma cell lines propagated in serum-free media recapitulate the genotype and phenotype of primary neuroblastoma tumours

    NARCIS (Netherlands)

    Bate-Eya, Laurel T.; Ebus, Marli E.; Koster, Jan; den Hartog, Ilona J. M.; Zwijnenburg, Danny A.; Schild, Linda; van der Ploeg, Ida; Dolman, M. Emmy M.; Caron, Huib N.; Versteeg, Rogier; Molenaar, Jan J.

    2014-01-01

    Recently protocols have been devised for the culturing of cell lines from fresh tumours under serum-free conditions in defined neural stem cell medium. These cells, frequently called tumour initiating cells (TICs) closely retained characteristics of the tumours of origin. We report the isolation of

  2. In vitro effects of preservative-free and preserved prostaglandin analogs on primary cultured human conjunctival fibroblast cells.

    Science.gov (United States)

    Kim, Eun Joo; Kim, Yeoun-Hee; Kang, Sun-Hee; Lee, Kyoo Won; Park, Young Jeung

    2013-12-01

    Long-term use of topical medication is needed for glaucoma treatment. One of the most commonly prescribed classes of hypotensive agents are prostaglandin analogs (PGs) used as both first-line monotherapy; as well as in combination therapy with other hypotensive agents. Several side effects of eye drops can be caused by preservatives. The purpose of this study was to evaluate the effects of PGs with varying concentrations of benzalkonium chloride (BAC), alternative preservatives, or no preservatives on human conjunctival fibroblast cells. Primary human conjunctival fibroblast cells were used in these experiments. Cells were exposed to the following drugs: BAC at different concentrations, bimatoprost 0.01% (with BAC 0.02%), latanoprost 0.005% (with BAC 0.02%), tafluprost 0.0015% with/without 0.001% BAC and travoprost 0.004% (with 0.001% Polyquad) for 15 and 30 minutes. Cell cytotoxicity was evaluated by phase-contrast microscopy to monitor morphological changes of cells, Counting Kit-8 (CCK-8) assay to cell viability, and fluorescent activated cell sorting (FACS) analysis to measure apoptosis. BAC caused cell shrinkage and detachment from the plate in a dose-dependent manner. Morphological changes were observed in cells treated with bimatoprost 0.01% and latanoprost 0.005%. However, mild cell shrinkage was noted in cells treated with tafluprost 0.0015%, while a non-toxic effect was noted with travoprost 0.004% and preservative-free tafluprost 0.0015%. CCK-8 assay and FACS analysis showed all groups had a significantly decreased cell viability and higher apoptosis rate compared with the control group. However, travoprost 0.004% and preservative-free tafluprost 0.0015% showed lower cytotoxicity and apoptosis rate than other drugs. This in vitro study revealed that BAC-induced cytotoxicity is dose-dependent, although it is important to emphasize that the clinical significance of toxicity differences observed among the different PGs formulations has not yet been firmly

  3. The response analysis of fractional-order stochastic system via generalized cell mapping method.

    Science.gov (United States)

    Wang, Liang; Xue, Lili; Sun, Chunyan; Yue, Xiaole; Xu, Wei

    2018-01-01

    This paper is concerned with the response of a fractional-order stochastic system. The short memory principle is introduced to ensure that the response of the system is a Markov process. The generalized cell mapping method is applied to display the global dynamics of the noise-free system, such as attractors, basins of attraction, basin boundary, saddle, and invariant manifolds. The stochastic generalized cell mapping method is employed to obtain the evolutionary process of probability density functions of the response. The fractional-order ϕ 6 oscillator and the fractional-order smooth and discontinuous oscillator are taken as examples to give the implementations of our strategies. Studies have shown that the evolutionary direction of the probability density function of the fractional-order stochastic system is consistent with the unstable manifold. The effectiveness of the method is confirmed using Monte Carlo results.

  4. Free vibration of complex systems of shells of revolution

    International Nuclear Information System (INIS)

    Markov, P.

    1987-01-01

    Simplified relations are presented for shells of revolution and the finite difference energy method is described as is its numerical application to the problems of the mechanics of the shells of revolution of a complex and branched meridian, used in the BOSOR4 program. Also presented are two examples of calculating the free vibration of systems of shells of revolution using the said program. Both problems stemmed from the needs of SKODA, Energeticke Strojirenstvi. The first concerns the free vibration of the system of WWER-440 reactor vessels, approximating its internals. The second concerns the eigenfrequencies and corresponding shapes of the vibrations of the DK3 diagnostic assembly which was designed and manufactured for improved knowledge of events taking place in the reactor core during different operating modes. (author). 7 figs., 2 tabs., 7 refs

  5. Development of special analytical system for determination of free acid

    International Nuclear Information System (INIS)

    Zhang Lihua; Wu Jizong; Liu Huanliang; Liu Quanwei; Fan Dejun; Su Tao

    2008-01-01

    The determination of free-acid plays an important role in spent fuel reprocessing analysis. Its work accounts for about 30% of all analytical work in reprocessing. It is necessary to study and develop a special fast analytical system for determination of free acid. The special analytical system is particularly applicable to determination of free acid in high-level radioactive environment, which is composed of an optical fiber spectrophotometer and an automatic sample-in device. Small sample-in volume needed, fast procedure, easy operation and physical protection are its advantages. All kinds of performance and parameters satisfy the requirements of spent fuel reprocessing control analysis. For long-distance determination, the optical fiber spectrophotometer is connected with an 4.5 meters long optical fiber. To resolve the change of 0.1 mol/L acidity, the measuring optical path is 2 cm. Mass of 10-20 μm in diameter optical fibers are assembled. The optical fiber probe is composed of a reflecting mirror and a concave mirror on the top of optical fibers. To eliminate the interference of external light, a stainless steel measuring chamber is used. The automatic sample-in device is composed of state valve, quantifying pump and pipe. The sample-in precision of 15 μl and 35 μl quantifying loops is better than 0.5%. The special analytical system takes less than 7 minutes to complete one measurement. The linear range is 0.5 mol/L-3.5 mol/L. The relative standard deviation is better than 2.0% when the concentration of the free acid is about 2.0 mol/L. For samples in different medium, the results are comparable with the method of pH titration of determining the free acid in reprocessing. (authors)

  6. Achievements and Problems of Reform of Investment Management System for Shanghai Pilot Free Trade Zone

    OpenAIRE

    PAN, Chengrong

    2014-01-01

    The establishment of Shanghai Pilot Free Trade Zone provides many possibilities for China’s economic construction. This paper made a comparative analysis on investment management system of Shanghai Pilot Free Trade Zone and traditional investment management system, discussed achievements and problems of reform of investment management system of Shanghai Pilot Free Trade Zone, and finally came up with pertinent policy recommendations.

  7. Cell-free placental DNA beyond Down syndrome: Lessons learned from fetal RHD genotyping

    NARCIS (Netherlands)

    Thurik, F.F.

    2016-01-01

    In this thesis research is presented on cell-free fetal DNA (cffDNA), which is present in plasma and serum of pregnant women. This fetal DNA can be used for fetal genotyping, but may also give indirect information on pregnancy and pregnancy outcome. The research consists of two sections. In the

  8. Advanced diffusion system for low contamination in-line rapid thermal processing of silicon solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Biro, D.; Preu, R.; Schultz, O.; Peters, S.; Huljic, D.M.; Zickermann, D.; Schindler, R.; Luedemann, R.; Willeke, G. [Fraunhofer Institute for Solar Energy Systems ISE, Freiburg (Germany)

    2002-10-01

    A novel diffusion system for in-line rapid thermal diffusion is presented. The lamp-heated furnace has a low thermal mass and a metal free transport system based on the walking beam principle. The furnace has been used to process first solar cells with lightly and highly doped emitters respectively. Solar cells with shallow lightly doped emitters show that the emitters processed in the new device can be well passivated. Shallow emitters with sheet resistances of up to 40/sq. have been contacted successfully by means of screen printing and firing through a SiN{sub x} antireflection coating. (author)

  9. Free radicals in health and disease

    International Nuclear Information System (INIS)

    Gonet, B.

    1993-01-01

    Free radicals appear in the cells as the result of exogenic factors (ionizing radiation, UV) or reactions naturally occurring in the cell. Free radical reactions may cause destruction of macromolecules (DNA, lipids, proteins). Free radical pathology is important in many diseases and aging processes in organisms

  10. Automated processing of label-free Raman microscope images of macrophage cells with standardized regression for high-throughput analysis.

    Science.gov (United States)

    Milewski, Robert J; Kumagai, Yutaro; Fujita, Katsumasa; Standley, Daron M; Smith, Nicholas I

    2010-11-19

    Macrophages represent the front lines of our immune system; they recognize and engulf pathogens or foreign particles thus initiating the immune response. Imaging macrophages presents unique challenges, as most optical techniques require labeling or staining of the cellular compartments in order to resolve organelles, and such stains or labels have the potential to perturb the cell, particularly in cases where incomplete information exists regarding the precise cellular reaction under observation. Label-free imaging techniques such as Raman microscopy are thus valuable tools for studying the transformations that occur in immune cells upon activation, both on the molecular and organelle levels. Due to extremely low signal levels, however, Raman microscopy requires sophisticated image processing techniques for noise reduction and signal extraction. To date, efficient, automated algorithms for resolving sub-cellular features in noisy, multi-dimensional image sets have not been explored extensively. We show that hybrid z-score normalization and standard regression (Z-LSR) can highlight the spectral differences within the cell and provide image contrast dependent on spectral content. In contrast to typical Raman imaging processing methods using multivariate analysis, such as single value decomposition (SVD), our implementation of the Z-LSR method can operate nearly in real-time. In spite of its computational simplicity, Z-LSR can automatically remove background and bias in the signal, improve the resolution of spatially distributed spectral differences and enable sub-cellular features to be resolved in Raman microscopy images of mouse macrophage cells. Significantly, the Z-LSR processed images automatically exhibited subcellular architectures whereas SVD, in general, requires human assistance in selecting the components of interest. The computational efficiency of Z-LSR enables automated resolution of sub-cellular features in large Raman microscopy data sets without

  11. Mechanisms of collective cell movement lacking a leading or free front edge in vivo.

    Science.gov (United States)

    Uechi, Hiroyuki; Kuranaga, Erina

    2017-08-01

    Collective cell movement is one of the strategies for achieving the complex shapes of tissues and organs. In this process, multiple cells within a group held together by cell-cell adhesion acquire mobility and move together in the same direction. In some well-studied models of collective cell movement, the mobility depends strongly on traction generated at the leading edge by cells located at the front. However, recent advances in live-imaging techniques have led to the discovery of other types of collective cell movement lacking a leading edge or even a free edge at the front, in a diverse array of morphological events, including tubule elongation, epithelial sheet extension, and tissue rotation. We herein review some of the developmental events that are organized by collective cell movement and attempt to elucidate the underlying cellular and molecular mechanisms, which include membrane protrusions, guidance cues, cell intercalation, and planer cell polarity, or chirality pathways.

  12. Aberration-free FTIR spectroscopic imaging of live cells in microfluidic devices.

    Science.gov (United States)

    Chan, K L Andrew; Kazarian, Sergei G

    2013-07-21

    The label-free, non-destructive chemical analysis offered by FTIR spectroscopic imaging is a very attractive and potentially powerful tool for studies of live biological cells. FTIR imaging of live cells is a challenging task, due to the fact that cells are cultured in an aqueous environment. While the synchrotron facility has proven to be a valuable tool for FTIR microspectroscopic studies of single live cells, we have demonstrated that high quality infrared spectra of single live cells using an ordinary Globar source can also be obtained by adding a pair of lenses to a common transmission liquid cell. The lenses, when placed on the transmission cell window, form pseudo hemispheres which removes the refraction of light and hence improve the imaging and spectral quality of the obtained data. This study demonstrates that infrared spectra of single live cells can be obtained without the focus shifting effect at different wavenumbers, caused by the chromatic aberration. Spectra of the single cells have confirmed that the measured spectral region remains in focus across the whole range, while spectra of the single cells measured without the lenses have shown some erroneous features as a result of the shift of focus. It has also been demonstrated that the addition of lenses can be applied to the imaging of cells in microfabricated devices. We have shown that it was not possible to obtain a focused image of an isolated cell in a droplet of DPBS in oil unless the lenses are applied. The use of the approach described herein allows for well focused images of single cells in DPBS droplets to be obtained.

  13. Overcrowding drives the unjamming transition of gap-free monolayers

    Science.gov (United States)

    Lan, Ganhui; Su, Tao

    Collective cell motility plays central roles in various biological phenomena such as wound healing, cancer metastasis and embryogenesis. These are demonstrations of the unjamming transition in biology. However, contradictory to the typical density-driven jamming in particulate assemblies, cellular systems often get unjammed in highly packed, sometimes overcrowding environments. Here, we investigate monolayers' collective behaviors when cell number changes under the gap-free constraint. We report that overcrowding can unjam gap-free monolayers through increasing isotropic compression. We show that the transition boundary is determined by the isotropic compression and the cell-cell adhesion. Furthermore, we construct the free energy landscape for the T1 topological transition during monolayer rearrangement, and discover that the landscape evolves from single-barrier W shape to double-barrier M shape during the unjamming process. We also discover a distributed-to-disordered morphological transition of cells' geometry, coinciding with the unjamming transition. Our analyses reveal that the overcrowding and adhesion induced unjamming reflects the mechanical yielding of the highly deformable monolayer, suggesting an alternative mechanism that cells may robustly gain collective mobility through proliferation in confined environments, which differs from those caused by loosing up a packed particulate assembly. This work is supported by the GWU College Facilitating Funds.

  14. a Man-Portable Imu-Free Mobile Mapping System

    Science.gov (United States)

    Nüchter, A.; Borrmann, D.; Koch, P.; Kühn, M.; May, S.

    2015-08-01

    Mobile mapping systems are commonly mounted on cars, ships and robots. The data is directly geo-referenced using GPS data and expensive IMU (inertial measurement systems). Driven by the need for flexible, indoor mapping systems we present an inexpensive mobile mapping solution that can be mounted on a backpack. It combines a horizontally mounted 2D profiler with a constantly spinning 3D laser scanner. The initial system featuring a low-cost MEMS IMU was revealed and demonstrated at MoLaS: Technology Workshop Mobile Laser Scanning at Fraunhofer IPM in Freiburg in November 2014. In this paper, we present an IMU-free solution.

  15. Validation of liquid biopsy: plasma cell-free DNA testing in clinical management of advanced non-small cell lung cancer

    Directory of Open Access Journals (Sweden)

    Veldore VH

    2018-01-01

    Full Text Available Vidya H Veldore,1,* Anuradha Choughule,2,* Tejaswi Routhu,1 Nitin Mandloi,1 Vanita Noronha,2 Amit Joshi,2 Amit Dutt,3 Ravi Gupta,1 Ramprasad Vedam,1 Kumar Prabhash2 1MedGenome Labs Private Ltd,, Bangalore, India; 2Tata Memorial Centre, Parel, Mumbai, India; 3The Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Center, Kharghar, Navi Mumbai, Maharashtra, India *These authors contributed equally to this work Abstract: Plasma cell-free tumor DNA, or circulating tumor DNA (ctDNA, from liquid biopsy is a potential source of tumor genetic material, in the absence of tissue biopsy, for EGFR testing. Our validation study reiterates the clinical utility of ctDNA next generation sequencing (NGS for EGFR mutation testing in non-small cell lung cancer (NSCLC. A total of 163 NSCLC cases were included in the validation, of which 132 patients had paired tissue biopsy and ctDNA. We chose to validate ctDNA using deep sequencing with custom designed bioinformatics methods that could detect somatic mutations at allele frequencies as low as 0.01%. Benchmarking allele specific real time PCR as one of the standard methods for tissue-based EGFR mutation testing, the ctDNA NGS test was validated on all the plasma derived cell-free DNA samples. We observed a high concordance (96.96% between tissue biopsy and ctDNA for oncogenic driver mutations in Exon 19 and Exon 21 of the EGFR gene. The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of the assay were 91.1%, 100% 100%, 95.6%, and 97%, respectively. A false negative rate of 3% was observed. A subset of mutations was also verified on droplet digital PCR. Sixteen percent EGFR mutation positivity was observed in patients where only liquid biopsy was available, thus creating options for targeted therapy. This is the first and largest study from India, demonstrating successful validation of circulating cell-free DNA as a clinically

  16. Serum-free Erythroid Differentiation for Efficient Genetic Modification and High-Level Adult Hemoglobin Production.

    Science.gov (United States)

    Uchida, Naoya; Demirci, Selami; Haro-Mora, Juan J; Fujita, Atsushi; Raines, Lydia N; Hsieh, Matthew M; Tisdale, John F

    2018-06-15

    In vitro erythroid differentiation from primary human cells is valuable to develop genetic strategies for hemoglobin disorders. However, current erythroid differentiation methods are encumbered by modest transduction rates and high baseline fetal hemoglobin production. In this study, we sought to improve both genetic modification and hemoglobin production among human erythroid cells in vitro . To model therapeutic strategies, we transduced human CD34 + cells and peripheral blood mononuclear cells (PBMCs) with lentiviral vectors and compared erythropoietin-based erythroid differentiation using fetal-bovine-serum-containing media and serum-free media. We observed more efficient transduction (85%-93%) in serum-free media than serum-containing media (20%-69%), whereas the addition of knockout serum replacement (KSR) was required for serum-free media to promote efficient erythroid differentiation (96%). High-level adult hemoglobin production detectable by electrophoresis was achieved using serum-free media similar to serum-containing media. Importantly, low fetal hemoglobin production was observed in the optimized serum-free media. Using KSR-containing, serum-free erythroid differentiation media, therapeutic adult hemoglobin production was detected at protein levels with β-globin lentiviral transduction in both CD34 + cells and PBMCs from sickle cell disease subjects. Our in vitro erythroid differentiation system provides a practical evaluation platform for adult hemoglobin production among human erythroid cells following genetic manipulation.

  17. Establishing elements of a synthetic biology platform for Vaccinia virus production: BioBrick™ design, serum-free virus production and microcarrier-based cultivation of CV-1 cells.

    Science.gov (United States)

    Liu, Shuchang; Ruban, Ludmila; Wang, Yaohe; Zhou, Yuhong; Nesbeth, Darren N

    2017-02-01

    Vaccinia virus (VACV) is an established vector for vaccination and is beginning to prove effective as an oncolytic agent. Industrial production of VACV stands to benefit in future from advances made by synthetic biology in genome engineering and standardisation. The CV-1 cell line can be used for VACV propagation and has been used extensively with the CRISPR/Cas9 system for making precise edits of the VACV genome. Here we take first steps toward establishing a scalable synthetic biology platform for VACV production with CV-1 cells featuring standardised biological tools and serum free cell cultivation. We propose a new BioBrick™ plasmid backbone format for inserting transgenes into VACV. We then test the performance of CV-1 cells in propagation of a conventional recombinant Lister strain VACV, VACVL-15 RFP, in a serum-free process. CV-1 cells grown in 5% foetal bovine serum (FBS) Dulbecco's Modified Eagle Medium (DMEM) were adapted to growth in OptiPRO and VP-SFM brands of serum-free media. Specific growth rates of 0.047 h -1 and 0.044 h -1 were observed for cells adapted to OptiPRO and VP-SFM respectively, compared to 0.035 h -1 in 5% FBS DMEM. Cells adapted to OptiPRO and to 5% FBS DMEM achieved recovery ratios of over 96%, an indication of their robustness to cryopreservation. Cells adapted to VP-SFM showed a recovery ratio of 82%. Virus productivity in static culture, measured as plaque forming units (PFU) per propagator cell, was 75 PFU/cell for cells in 5% FBS DMEM. VP-SFM and OptiPRO adaptation increased VACV production to 150 PFU/cell and 350 PFU/cell respectively. Boosted PFU/cell from OptiPRO-adapted cells persisted when 5% FBS DMEM or OptiPRO medium was observed during the infection step and when titre was measured using cells adapted to 5% FBS DMEM or OptiPRO medium. Finally, OptiPRO-adapted CV-1 cells were successfully cultivated using Cytodex-1 microcarriers to inform future scale up studies.

  18. Establishing elements of a synthetic biology platform for Vaccinia virus production: BioBrick™ design, serum-free virus production and microcarrier-based cultivation of CV-1 cells

    Directory of Open Access Journals (Sweden)

    Shuchang Liu

    2017-02-01

    Full Text Available Vaccinia virus (VACV is an established vector for vaccination and is beginning to prove effective as an oncolytic agent. Industrial production of VACV stands to benefit in future from advances made by synthetic biology in genome engineering and standardisation. The CV-1 cell line can be used for VACV propagation and has been used extensively with the CRISPR/Cas9 system for making precise edits of the VACV genome. Here we take first steps toward establishing a scalable synthetic biology platform for VACV production with CV-1 cells featuring standardised biological tools and serum free cell cultivation. We propose a new BioBrick™ plasmid backbone format for inserting transgenes into VACV. We then test the performance of CV-1 cells in propagation of a conventional recombinant Lister strain VACV, VACVL-15 RFP, in a serum-free process. CV-1 cells grown in 5% foetal bovine serum (FBS Dulbecco’s Modified Eagle Medium (DMEM were adapted to growth in OptiPRO and VP-SFM brands of serum-free media. Specific growth rates of 0.047 h−1 and 0.044 h−1 were observed for cells adapted to OptiPRO and VP-SFM respectively, compared to 0.035 h−1 in 5% FBS DMEM. Cells adapted to OptiPRO and to 5% FBS DMEM achieved recovery ratios of over 96%, an indication of their robustness to cryopreservation. Cells adapted to VP-SFM showed a recovery ratio of 82%. Virus productivity in static culture, measured as plaque forming units (PFU per propagator cell, was 75 PFU/cell for cells in 5% FBS DMEM. VP-SFM and OptiPRO adaptation increased VACV production to 150 PFU/cell and 350 PFU/cell respectively. Boosted PFU/cell from OptiPRO-adapted cells persisted when 5% FBS DMEM or OptiPRO medium was observed during the infection step and when titre was measured using cells adapted to 5% FBS DMEM or OptiPRO medium. Finally, OptiPRO-adapted CV-1 cells were successfully cultivated using Cytodex-1 microcarriers to inform future scale up studies.

  19. Taurine Biosynthesis in a Fish Liver Cell Line (ZFL) Adapted to a Serum-Free Medium.

    Science.gov (United States)

    Liu, Chieh-Lun; Watson, Aaron M; Place, Allen R; Jagus, Rosemary

    2017-05-25

    Although taurine has been shown to play multiple important physiological roles in teleosts, little is known about the molecular mechanisms underlying dietary requirements. Cell lines can provide useful tools for deciphering biosynthetic pathways and their regulation. However, culture media and sera contain variable taurine levels. To provide a useful cell line for the investigation of taurine homeostasis, an adult zebrafish liver cell line (ZFL) has been adapted to a taurine-free medium by gradual accommodation to a commercially available synthetic medium, UltraMEM™-ITES. Here we show that ZFL cells are able to synthesize taurine and be maintained in medium without taurine. This has allowed for the investigation of the effects of taurine supplementation on cell growth, cellular amino acid pools, as well as the expression of the taurine biosynthetic pathway and taurine transporter genes in a defined fish cell type. After taurine supplementation, cellular taurine levels increase but hypotaurine levels stay constant, suggesting little suppression of taurine biosynthesis. Cellular methionine levels do not change after taurine addition, consistent with maintenance of taurine biosynthesis. The addition of taurine to cells grown in taurine-free medium has little effect on transcript levels of the biosynthetic pathway genes for cysteine dioxygenase (CDO), cysteine sulfinate decarboxylase (CSAD), or cysteamine dioxygenase (ADO). In contrast, supplementation with taurine causes a 30% reduction in transcript levels of the taurine transporter, TauT. This experimental approach can be tailored for the development of cell lines from aquaculture species for the elucidation of their taurine biosynthetic capacity.

  20. Analysis of single-cell differences by use of an on-chip microculture system and optical trapping.

    Science.gov (United States)

    Wakamoto, Y; Inoue, I; Moriguchi, H; Yasuda, K

    2001-09-01

    A method is described for continuous observation of isolated single cells that enables genetically identical cells to be compared; it uses an on-chip microculture system and optical tweezers. Photolithography is used to construct microchambers with 5-microm-high walls made of thick photoresist (SU-8) on the surface of a glass slide. These microchambers are connected by a channel through which cells are transported, by means of optical tweezers, from a cultivation microchamber to an analysis microchamber, or from the analysis microchamber to a waste microchamber. The microchambers are covered with a semi-permeable membrane to separate them from nutrient medium circulating through a "cover chamber" above. Differential analysis of isolated direct descendants of single cells showed that this system could be used to compare genetically identical cells under contamination-free conditions. It should thus help in the clarification of heterogeneous phenomena, for example unequal cell division and cell differentiation.

  1. Boundary-value problems with free boundaries for elliptic systems of equations

    CERN Document Server

    Monakhov, V N

    1983-01-01

    This book is concerned with certain classes of nonlinear problems for elliptic systems of partial differential equations: boundary-value problems with free boundaries. The first part has to do with the general theory of boundary-value problems for analytic functions and its applications to hydrodynamics. The second presents the theory of quasiconformal mappings, along with the theory of boundary-value problems for elliptic systems of equations and applications of it to problems in the mechanics of continuous media with free boundaries: problems in subsonic gas dynamics, filtration theory, and problems in elastico-plasticity.

  2. Production of inactivated influenza H5N1 vaccines from MDCK cells in serum-free medium.

    Directory of Open Access Journals (Sweden)

    Alan Yung-Chih Hu

    Full Text Available BACKGROUND: Highly pathogenic influenza viruses pose a constant threat which could lead to a global pandemic. Vaccination remains the principal measure to reduce morbidity and mortality from such pandemics. The availability and surging demand for pandemic vaccines needs to be addressed in the preparedness plans. This study presents an improved high-yield manufacturing process for the inactivated influenza H5N1 vaccines using Madin-Darby canine kidney (MDCK cells grown in a serum-free (SF medium microcarrier cell culture system. PRINCIPAL FINDING: The current study has evaluated the performance of cell adaptation switched from serum-containing (SC medium to several commercial SF media. The selected SF medium was further evaluated in various bioreactor culture systems for process scale-up evaluation. No significant difference was found in the cell growth in different sizes of bioreactors studied. In the 7.5 L bioreactor runs, the cell concentration reached to 2.3 × 10(6 cells/mL after 5 days. The maximum virus titers of 1024 Hemagglutinin (HA units/50 µL and 7.1 ± 0.3 × 10(8 pfu/mL were obtained after 3 days infection. The concentration of HA antigen as determined by SRID was found to be 14.1 µg/mL which was higher than those obtained from the SC medium. A mouse immunogenicity study showed that the formalin-inactivated purified SF vaccine candidate formulated with alum adjuvant could induce protective level of virus neutralization titers similar to those obtained from the SC medium. In addition, the H5N1 viruses produced from either SC or SF media showed the same antigenic reactivity with the NIBRG14 standard antisera. CONCLUSIONS: The advantages of this SF cell-based manufacturing process could reduce the animal serum contamination, the cost and lot-to-lot variation of SC medium production. This study provides useful information to manufacturers that are planning to use SF medium for cell-based influenza vaccine production.

  3. A comparison of cell-free placental messenger ribonucleic acid and color Doppler ultrasound for the prediction of placental invasion in patients with placenta accreta

    OpenAIRE

    Naghshineh, Elham; Khorvash, Elahe; Kamali, Sara

    2015-01-01

    Background: The aim of the present study was to comparison between cell-free placental messenger ribonucleic acid (mRNA) and Doppler ultrasound for the prediction of placental invasion in women with placenta accreta. Materials and Methods: In this cross-sectional study, 50 pregnant women at risk for placenta accreta underwent color Doppler and assessment of cell-free placental mRNA. Real-time reverse-transcription polymerase chain reaction was used for measurement of cell-free placental m...

  4. Protective effect of aqueous extract from Spirulina platensis against cell death induced by free radicals

    Directory of Open Access Journals (Sweden)

    Radhakrishnan Ammu

    2010-09-01

    Full Text Available Abstract Background Spirulina is a commercial alga well known to contain various antioxidants, especially phycocyanin. Apart from being sold as a nutraceutical, Spirulina is incorporated as a functional ingredient in food products and beverages. Most of the previous reports on antioxidant activity of Spirulina were based on chemical rather than cell-based assays. The primary objective of this study was to assess the antioxidant activity of aqueous extract from Spirulina based on its protective effect against cell death induced by free radicals. Methods The antioxidant activity of the cold water extract from food-grade Spirulina platensis was assessed using both chemical and cell-based assays. In the cell-based assay, mouse fibroblast cells (3T3 cells were incubated for 1 h in medium containing aqueous extract of Spirulina or vitamin C (positive control at 25, 125 and 250 μg/mL before the addition of 50 μM 1,1-diphenyl-2-picrylhydrazyl (DPPH or 3-ethylbenzothiazoline-6-sulfonic acid (ABTS. The cells were incubated for another 24 h before being assessed for cell death due to apoptosis using the Cell Death Detection ELISA Kit. Spectrophotometric assays based on DPPH and ABTS were also used to assess the antioxidant activity of the extract compared to vitamin C and vitamin E (positive controls. Results Spirulina extract did not cause cytotoxic effect on 3T3 cells within the range of concentrations tested (0 - 250 μg/mL. The extract reduced significantly (p Conclusions The results showed that aqueous extract of Spirulina has a protective effect against apoptotic cell death due to free radicals. The potential application of incorporating Spirulina into food products and beverages to enhance their antioxidant capacity is worth exploring.

  5. Neural stem cells achieve and maintain pluripotency without feeder cells.

    Directory of Open Access Journals (Sweden)

    Hyun Woo Choi

    Full Text Available BACKGROUND: Differentiated cells can be reprogrammed into pluripotency by transduction of four defined transcription factors. Induced pluripotent stem cells (iPS cells are expected to be useful for regenerative medicine as well as basic research. Recently, the report showed that mouse embryonic fibroblasts (MEF cells are not essential for reprogramming. However, in using fibroblasts as donor cells for reprogramming, individual fibroblasts that had failed to reprogram could function as feeder cells. METHODOLOGY/PRINCIPAL FINDING: Here, we show that adult mouse neural stem cells (NSCs, which are not functional feeder cells, can be reprogrammed into iPS cells using defined four factors (Oct4, Sox2, Klf4, and c-Myc under feeder-free conditions. The iPS cells, generated from NSCs expressing the Oct4-GFP reporter gene, could proliferate for more than two months (passage 20. Generated and maintained without feeder cells, these iPS cells expressed pluripotency markers (Oct4 and Nanog, the promoter regions of Oct4 and Nanog were hypomethylated, could differentiated into to all three germ layers in vitro, and formed a germline chimera. These data indicate that NSCs can achieve and maintain pluripotency under feeder-free conditions. CONCLUSION/SIGNIFICANCE: This study suggested that factors secreted by feeder cells are not essential in the initial/early stages of reprogramming and for pluripotency maintenance. This technology might be useful for a human system, as a feeder-free reprogramming system may help generate iPS cells of a clinical grade for tissue or organ regeneration.

  6. Catalytical Properties of Free and Immobilized Aspergillus niger Tannase

    Directory of Open Access Journals (Sweden)

    Abril Flores-Maltos

    2011-01-01

    Full Text Available A fungal tannase was produced, recovered, and immobilized by entrapment in calcium alginate beads. Catalytical properties of the immobilized enzyme were compared with those of the free one. Tannase was produced intracellularly by the xerophilic fungus Aspergillus niger GH1 in a submerged fermentation system. Enzyme was recovered by cell disruption and the crude extract was partially purified. The catalytical properties of free and immobilized tannase were evaluated using tannic acid and methyl gallate as substrates. KM and Vmax values for free enzyme were very similar for both substrates. But, after immobilization, KM and Vmax values increased drastically using tannic acid as substrate. These results indicated that immobilized tannase is a better biocatalyst than free enzyme for applications on liquid systems with high tannin content, such as bioremediation of tannery or olive-mill wastewater.

  7. Catalytical Properties of Free and Immobilized Aspergillus niger Tannase.

    Science.gov (United States)

    Flores-Maltos, Abril; Rodríguez-Durán, Luis V; Renovato, Jacqueline; Contreras, Juan C; Rodríguez, Raúl; Aguilar, Cristóbal N

    2011-01-01

    A fungal tannase was produced, recovered, and immobilized by entrapment in calcium alginate beads. Catalytical properties of the immobilized enzyme were compared with those of the free one. Tannase was produced intracellularly by the xerophilic fungus Aspergillus niger GH1 in a submerged fermentation system. Enzyme was recovered by cell disruption and the crude extract was partially purified. The catalytical properties of free and immobilized tannase were evaluated using tannic acid and methyl gallate as substrates. K(M) and V(max) values for free enzyme were very similar for both substrates. But, after immobilization, K(M) and V(max) values increased drastically using tannic acid as substrate. These results indicated that immobilized tannase is a better biocatalyst than free enzyme for applications on liquid systems with high tannin content, such as bioremediation of tannery or olive-mill wastewater.

  8. Human Breast Adipose-Derived Stem Cells Transfected with the Stromal Cell-Derived Factor-1 Receptor CXCR4 Exhibit Enhanced Viability in Human Autologous Free Fat Grafts

    Directory of Open Access Journals (Sweden)

    Fang-tian Xu

    2014-11-01

    Full Text Available Background: The main complication of autologous free fat tissue transplantation is fat resorption and calcification due to the ischemic necrosis of fat. The promotion of transplant neovascularization soon after autologous free fat grafts may reduce these outcomes. In adulthood, stromal cell-derived factor-1 (SDF-1 and its membrane receptor C-X-C chemokine receptor type 4 (CXCR4 are involved in the homing and migration of multiple stem cell types, neovascularization, and cell proliferation. We hypothesized that CXCR4 may improve the long-term survival of free fat tissue transplants by recruiting endothelial progenitor cells (EPCs and may therefore improve graft revascularization. In this study, we aimed to determine the effect of human breast adipose-derived stem cells (HBASCs transfected with the CXCR4 gene on the survival rate of human autologous free fat transplants in nude mice. Methods: Human breast adipose-derived stem cells (HBASCs were expanded ex vivo for 3 passages, labeled with green fluorescent protein (GFP and transfected with CXCR4 or left untransfected. Autologous fat tissues were mixed with the GFP-labeled, CXCR4-transfected HBASCs (group A, GFP-labeled HBASCs (group B, the known vascularization-promoting agent VEGF (group C, or medium (group D and then injected subcutaneously into 32 nude mice at 4 spots in a random fashion. Six months later, the transplanted tissue volume and histology were evaluated, and neo-vascularization was quantified by counting the capillaries. CXCR4 and SDF-1α mRNA expression in the transplants was determined using real-time quantitative PCR analysis (qPCR. Results: The data revealed that the control (group D transplant volume survival was 28.3 ± 4.5%. Mixing CXCR4-transfected (group A and untransfected (group B HBASCs significantly increased transplant volume survival (79.5 ± 8.3% and 67.2 ± 5.9%, respectively, whereas VEGF-transfected HBASCs (group C were less effective (41.2 ± 5.1%. Histological

  9. Asynchronous Free-Space Optical CDMA Communications System for Last-mile Access Network

    DEFF Research Database (Denmark)

    Jurado-Navas, Antonio; Raddo, Thiago R.; Sanches, Anderson L.

    2016-01-01

    We propose a new hybrid asynchronous OCDMA-FSO communications system for access network solutions. New ABER expressions are derived under gamma-gamma scintillation channels, where all users can surprisingly achieve error-free transmissions when FEC is employed.......We propose a new hybrid asynchronous OCDMA-FSO communications system for access network solutions. New ABER expressions are derived under gamma-gamma scintillation channels, where all users can surprisingly achieve error-free transmissions when FEC is employed....

  10. Feasibility study of stain-free classification of cell apoptosis based on diffraction imaging flow cytometry and supervised machine learning techniques.

    Science.gov (United States)

    Feng, Jingwen; Feng, Tong; Yang, Chengwen; Wang, Wei; Sa, Yu; Feng, Yuanming

    2018-06-01

    This study was to explore the feasibility of prediction and classification of cells in different stages of apoptosis with a stain-free method based on diffraction images and supervised machine learning. Apoptosis was induced in human chronic myelogenous leukemia K562 cells by cis-platinum (DDP). A newly developed technique of polarization diffraction imaging flow cytometry (p-DIFC) was performed to acquire diffraction images of the cells in three different statuses (viable, early apoptotic and late apoptotic/necrotic) after cell separation through fluorescence activated cell sorting with Annexin V-PE and SYTOX® Green double staining. The texture features of the diffraction images were extracted with in-house software based on the Gray-level co-occurrence matrix algorithm to generate datasets for cell classification with supervised machine learning method. Therefore, this new method has been verified in hydrogen peroxide induced apoptosis model of HL-60. Results show that accuracy of higher than 90% was achieved respectively in independent test datasets from each cell type based on logistic regression with ridge estimators, which indicated that p-DIFC system has a great potential in predicting and classifying cells in different stages of apoptosis.

  11. An autonomous fuel-cell system. Optimisation approach; Ein autonomes Brennstoffzellensystem. Optimierungsansaetze

    Energy Technology Data Exchange (ETDEWEB)

    Heideck, Guenter

    2006-07-20

    The reduction of the consumption of fossil energy sources and CO2 Emissions has become a worldwide issue. One promising way to accomplish this is by using hydrogen, which is predominantly produced by renewable energy sources, as energy storage. Fuel cell technology will play a key role in the field of converting chemically stored energy into electrical energy. This work contributes ways to make this conversion more effective. Because autonomous fuel cell systems have the largest application potential, this work concentrates on their optimization. To clarify the interrelationship of the parameters and the influencing physical factors as well as the potentials of optimization, models in the form of mathematical equation systems are given in the main part of this work. The focus is on the energy balance of the subsystems. The theoretical analyses showed that with improved efficiency of single subsystems, the system efficiency does not necessarily improve too, because the subsystems are not free of interactions. Instead, the system efficiency is improved through adapting single subsystems to match the needs of a given application. (orig.)

  12. Treatment of initial parenchymal central nervous system involvement in systemic aggressive B-cell lymphoma.

    Science.gov (United States)

    Nijland, Marcel; Jansen, Anne; Doorduijn, Jeanette K; Enting, Roelien H; Bromberg, Jacoline E C; Kluin-Nelemans, Hanneke C

    2017-09-01

    Central nervous system (CNS) involvement in systemic B-cell non-Hodgkin lymphoma (B-NHL) at diagnosis (sysCNS) is rare. We investigated the outcome of 21 patients with sysCNS, most commonly diffuse large B-cell lymphoma, treated with high dose methotrexate (HD-MTX) and R-CHOP. The median number of cycles of HD-MTX and R-CHOP was 4 (range 1-8) and 6 (range 0-8), respectively. Consolidative whole brain radiotherapy (WBRT) was given to 33% (7/21) patients. With a median follow-up of 44 months the 3-year progression free survival (PFS) and overall survival (OS) were 45% (95%CI 34-56%) and 49% (95%CI 38-60%), respectively. Over 90% of patients had an unfavorable international prognostic index score, reflected by treatment-related mortality of 19% (4/21) and relapse-related mortality of 28% (6/21). The outcome of these patients was, however, unexpectedly good when compared to secondary CNS relapses. Prospective studies are needed to define the optimal treatment for patients with sysCNS, but its rarity might be challenging.

  13. Label-free assessment of adipose-derived stem cell differentiation using coherent anti-Stokes Raman scattering and multiphoton microscopy

    Science.gov (United States)

    Mouras, Rabah; Bagnaninchi, Pierre O.; Downes, Andrew R.; Elfick, Alistair P. D.

    2012-11-01

    Adult stem cells (SCs) hold great potential as likely candidates for disease therapy but also as sources of differentiated human cells in vitro models of disease. In both cases, the label-free assessment of SC differentiation state is highly desirable, either as a quality-control technology ensuring cells to be used clinically are of the desired lineage or to facilitate in vitro time-course studies of cell differentiation. We investigate the potential of nonlinear optical microscopy as a minimally invasive technology to monitor the differentiation of adipose-derived stem cells (ADSCs) into adipocytes and osteoblasts. The induction of ADSCs toward these two different cell lineages was monitored simultaneously using coherent anti-Stokes Raman scattering, two photon excitation fluorescence (TPEF), and second harmonic generation at different time points. Changes in the cell's morphology, together with the appearance of biochemical markers of cell maturity were observed, such as lipid droplet accumulation for adipo-induced cells and the formation of extra-cellular matrix for osteo-induced cells. In addition, TPEF of flavoproteins was identified as a proxy for changes in cell metabolism that occurred throughout ADSC differentiation toward both osteoblasts and adipocytes. These results indicate that multimodal microscopy has significant potential as an enabling technology for the label-free investigation of SC differentiation.

  14. Adaptive Integral Sliding Mode Stabilization of Nonholonomic Drift-Free Systems

    Directory of Open Access Journals (Sweden)

    Waseem Abbasi

    2016-01-01

    Full Text Available This article presents adaptive integral sliding mode control algorithm for the stabilization of nonholonomic drift-free systems. First the system is transformed, by using input transform, into a special structure containing a nominal part and some unknown terms which are computed adaptively. The transformed system is then stabilized using adaptive integral sliding mode control. The stabilizing controller for the transformed system is constructed that consists of the nominal control plus a compensator control. The compensator control and the adaptive laws are derived on the basis of Lyapunov stability theory. The proposed control algorithm is applied to three different nonholonomic drift-free systems: the unicycle model, the front wheel car model, and the mobile robot with trailer model. The controllability Lie algebra of the unicycle model contains Lie brackets of depth one, the model of a front wheel car contains Lie brackets of depths one and two, and the model of a mobile robot with trailer contains Lie brackets of depths one, two, and three. The effectiveness of the proposed control algorithm is verified through numerical simulations.

  15. A sub-Kelvin cryogen-free EPR system.

    Science.gov (United States)

    Melhuish, Simon J; Stott, Chloe; Ariciu, Ana-Maria; Martinis, Lorenzo; McCulloch, Mark; Piccirillo, Lucio; Collison, David; Tuna, Floriana; Winpenny, Richard

    2017-09-01

    We present an EPR instrument built for operation at Q band below 1K. Our cryogen-free Dewar integrates with a commercial electro-magnet and bridge. A description of the cryogenic and RF systems is given, along with the adaptations to the standard EPR experiment for operation at sub-Kelvin temperatures. As a first experiment, the EPR spectra of powdered Cr 12 O 9 (OH) 3 [Formula: see text] were measured. The sub-Kelvin EPR spectra agree well with predictions, and the performance of the sub-Kelvin system at 5K is compared to that of a commercial spectrometer. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  16. DNA fragmentation: manifestation of target cell destruction mediated by cytotoxic T-cell lines, lymphotoxin-secreting helper T-cell clones, and cell-free lymphotoxin-containing supernatant

    International Nuclear Information System (INIS)

    Schmid, D.S.; Tite, J.P.; Ruddle, N.H.

    1986-01-01

    A Lyt-2 + , trinitrophenyl-specific, lymphotoxin-secreting, cytotoxic T-cell line, PCl 55, mediates the digestion of target cell DNA into discretely sized fragments. This phenomenon manifests itself within 30 min after effector cell encounter as measured by the release of 3 H counts from target cells prelabeled with [ 3 H]deoxythymidine and occurs even at very low effector to target cell ratios (0.25:1). A Lyt-1 + , ovalbumin-specific, lymphotoxin-secreting T-helper cell clone, 5.9.24, is also able to mediate fragmentation of target cell DNA over a time course essentially indistinguishable from the cytotoxic T lymphocyte-mediated hit. Cell-free lymphotoxin-containing supernatants also cause release of DNA from targets, although they require a longer time course, on the order of 24 hr. In contrast, lysis of cells by antibody plus complement or Triton X-100 does not result in DNA release even after extended periods of incubation (24 hr). All three treatments that result in the release of DNA from cells cause fragmentation of that DNA into discretely sized pieces that are multiples of 200 base pairs. The results thus suggest that cytotoxic T cells, lymphotoxin-secreting helper clones with cytolytic activity, and lymphotoxin all effect target cell destruction by means of a similar mechanism and that observed differences in time course and the absence of target cell specificity in killing mediated by lymphotoxin may simply reflect differences in the mode of toxin delivery

  17. The effect of system boundaries on the mean free path for confined gases

    Directory of Open Access Journals (Sweden)

    Sooraj K. Prabha

    2013-10-01

    Full Text Available The mean free path of rarefied gases is accurately determined using Molecular Dynamics simulations. The simulations are carried out on isothermal argon gas (Lennard-Jones fluid over a range of rarefaction levels under various confinements (unbounded gas, parallel reflective wall and explicit solid platinum wall bounded gas in a nanoscale domain. The system is also analyzed independently in constitutive sub-systems to calculate the corresponding local mean free paths. Our studies which predominate in the transition regime substantiate the boundary limiting effect on mean free paths owing to the sharp diminution in molecular free paths near the planar boundaries. These studies provide insight to the transport phenomena of rarefied gases through nanochannels which have established their potential in microscale and nanoscale heat transfer applications.

  18. Evaluation of the performance of Hy-Line Brown laying hens fed soybean or soybean-free diets using cage or free-range rearing systems.

    Science.gov (United States)

    Al-Ajeeli, M N; Leyva-Jimenez, H; Abdaljaleel, R A; Jameel, Y; Hashim, M M; Archer, G; Bailey, C A

    2018-03-01

    This study evaluated egg production and quality variables of caged and free-range Hy-Line Brown laying hens fed soybean meal (SBM) and soybean-meal-free (SBMF) diets. Hens were randomly assigned to the same 2 dietary treatments within 3 location blocks. SBM and SBMF diets with equivalent calculated nutrient content were prepared based on Hy-Line Brown rearing guidelines. The SBMF diets utilized cottonseed meal, corn distillers dried grains with solubles, corn gluten meal, and wheat middlings in place of dehulled soybean meal. The experiment was conducted between August 2015 and January of 2016 within the TAMU Poultry Research Center and data analyzed over 6 consecutive 28-day periods. Data were analyzed as a split-plot with rearing systems designated whole plots and diets designated as subplots. Hens reared in the free-range rearing system peaked a couple of wk later than those hens within the more conventional indoor caged system, and cumulative production data were considerably more variable for hens raised in the free-range environment. Cumulative egg production, feed per dozen eggs and feed conversion ratio (g feed/g egg) were 92 ± 1.23 and 86 ± 1.84%, 1.45 ± 0.02 and 1.89 ± 0.05 kg, and 2.14 ± 0.04 and 2.77 ± 0.08 (P free-range rearing systems. Cumulative egg weight, feed per dozen eggs, and feed conversion ratio were 59.9 ± 0.59 and 56.5 ± 0.60 g, 1.57 ± 0.04 and 1.77 ± 0.05 kg, and 2.24 ± 0.06 and 2.67 ± 0.08 kg (P  0.05). With respect to egg quality, there were no differences in cumulative albumen height, Haugh unit, or breaking strength, but there was a significant rearing system by diet interaction for shell thickness, with the free-range hens averaging 40.77 ± 0.19 and 39.86 ± 0.31 μm (P free-range production is more variable than traditional closed-house cage systems based on standard errors, and SBMF diets containing cottonseed meal can be used in both caged and free-range production systems without

  19. Differentiation of monkey embryonic stem cells to hepatocytes by feeder-free dispersion culture and expression analyses of cytochrome p450 enzymes responsible for drug metabolism.

    Science.gov (United States)

    Maruyama, Junya; Matsunaga, Tamihide; Yamaori, Satoshi; Sakamoto, Sakae; Kamada, Noboru; Nakamura, Katsunori; Kikuchi, Shinji; Ohmori, Shigeru

    2013-01-01

    We reported previously that monkey embryonic stem cells (ESCs) were differentiated into hepatocytes by formation of embryoid bodies (EBs). However, this EB formation method is not always efficient for assays using a large number of samples simultaneously. A dispersion culture system, one of the differentiation methods without EB formation, is able to more efficiently provide a large number of feeder-free undifferentiated cells. A previous study demonstrated the effectiveness of the Rho-associated kinase inhibitor Y-27632 for feeder-free dispersion culture and induction of differentiation of monkey ESCs into neural cells. In the present study, the induction of differentiation of cynomolgus monkey ESCs (cmESCs) into hepatocytes was performed by the dispersion culture method, and the expression and drug inducibility of cytochrome P450 (CYP) enzymes in these hepatocytes were examined. The cmESCs were successfully differentiated into hepatocytes under feeder-free dispersion culture conditions supplemented with Y-27632. The hepatocytes differentiated from cmESCs expressed the mRNAs for three hepatocyte marker genes (α-fetoprotein, albumin, CYP7A1) and several CYP enzymes, as measured by real-time polymerase chain reaction. In particular, the basal expression of cmCYP3A4 (3A8) in these hepatocytes was detected at mRNA and enzyme activity (testosterone 6β-hydroxylation) levels. Furthermore, the expression and activity of cmCYP3A4 (3A8) were significantly upregulated by rifampicin. These results indicated the effectiveness of Y-27632 supplementation for feeder-free dispersed culture and induction of differentiation into hepatocytes, and the expression of functional CYP enzyme(s) in cmESC-derived hepatic cells.

  20. A Human Amnion-Derived Extracellular Matrix-Coated Cell-Free Scaffold for Cartilage Repair: In Vitro and In Vivo Studies.

    Science.gov (United States)

    Nogami, Makiko; Kimura, Tomoatsu; Seki, Shoji; Matsui, Yoshito; Yoshida, Toshiko; Koike-Soko, Chika; Okabe, Motonori; Motomura, Hiraku; Gejo, Ryuichi; Nikaido, Toshio

    2016-04-01

    Extracellular matrix (ECM) derived from human amniotic mesenchymal cells (HAMs) has various biological activities. In this study, we developed a novel HAM-derived ECM-coated polylactic-co-glycolic acid (ECM-PLGA) scaffold, examined its property on mesenchymal cells, and investigated its potential as a cell-free scaffold for cartilage repair. ECM-PLGA scaffolds were developed by inoculating HAM on a PLGA. After decellularization by irradiation, accumulated ECM was examined. Exogenous cell growth and differentiation of rat mesenchymal stem cells (MSCs) on the ECM-PLGA were analyzed in vitro by cell attachment/proliferation assay and reverse transcription-polymerase chain reaction. The cell-free ECM-PLGA scaffolds were implanted into osteochondral defects in the trochlear groove of rat knees. After 4, 12, or 24 weeks, the animals were sacrificed and the harvested tissues were examined histologically. The ECM-PLGA contained ECM that mimicked natural amniotic stroma that contains type I collagen, fibronectin, hyaluronic acid, and chondroitin sulfates. The ECM-PLGA showed excellent properties of cell attachment and proliferation. MSCs inoculated on the ECM-PLGA scaffold showed accelerated type II collagen mRNA expression after 3 weeks in culture. The ECM-PLGA implanted into an osteochondral defect in rat knees induced gradual tissue regeneration and resulted in hyaline cartilage repair, which was better than that in the empty control group. These in vitro and in vivo experiments show that the cell-free scaffold composed of HAM-derived ECM and PLGA provides a favorable growth environment for MSCs and facilitates the cartilage repair process. The ECM-PLGA may become a "ready-made" biomaterial for cartilage repair therapy.

  1. Help system for control of JAERI FEL (Free Electron laser)

    International Nuclear Information System (INIS)

    Sugimoto, Masayoshi

    1993-01-01

    The control system of JAERI FEL (Free Electron Laser) has a help system to provide the information necessary to operate the machine and to develop the new user interface. As the control software is constructed on the MS-Windows 3.x, the hyper-text feature of the Windows help system can be accessed. It consists of three major parts: (1) on-line help, (2) full document, and (3) tutorial system. (author)

  2. Water-Free Proton-Conducting Membranes for Fuel Cells

    Science.gov (United States)

    Narayanan, Sekharipuram; Yen, Shiao-Pin

    2007-01-01

    Poly-4-vinylpyridinebisulfate (P4VPBS) is a polymeric salt that has shown promise as a water-free proton-conducting material (solid electrolyte) suitable for use in membrane/electrode assemblies in fuel cells. Heretofore, proton-conducting membranes in fuel cells have been made from perfluorinated ionomers that cannot conduct protons in the absence of water and, consequently, cannot function at temperatures >100 C. In addition, the stability of perfluorinated ionomers at temperatures >100 C is questionable. However, the performances of fuel cells of the power systems of which they are parts could be improved if operating temperatures could be raised above 140 C. What is needed to make this possible is a solid-electrolyte material, such as P4VPBS, that can be cast into membranes and that both retains proton conductivity and remains stable in the desired higher operating temperature range. A family of solid-electrolyte materials different from P4VPBS was described in Anhydrous Proton-Conducting Membranes for Fuel Cells (NPO-30493), NASA Tech Briefs, Vol. 29, No. 8 (August 2005), page 48. Those materials notably include polymeric quaternized amine salts. If molecules of such a polymeric salt could be endowed with flexible chain structures, it would be possible to overcome the deficiencies of simple organic amine salts that must melt before being able to conduct protons. However, no polymeric quaternized amine salts have yet shown to be useful in this respect. The present solid electrolyte is made by quaternizing the linear polymer poly- 4-vinylpyridine (P4VP) to obtain P4VPBS. It is important to start with P4VP having a molecular weight of 160,000 daltons because P4VPBS made from lower-molecular-weight P4VP yields brittle membranes. In an experimental synthesis, P4VP was dissolved in methanol and then reacted with an excess of sulfuric acid to precipitate P4VPBS. The precipitate was recovered, washed several times with methanol to remove traces of acid, and dried to a

  3. N-terminally truncated GADD34 proteins are convenient translation enhancers in a human cell-derived in vitro protein synthesis system.

    Science.gov (United States)

    Mikami, Satoshi; Kobayashi, Tominari; Machida, Kodai; Masutani, Mamiko; Yokoyama, Shigeyuki; Imataka, Hiroaki

    2010-07-01

    Human cell-derived in vitro protein synthesis systems are useful for the production of recombinant proteins. Productivity can be increased by supplementation with GADD34, a protein that is difficult to express in and purify from E. coli. Deletion of the N-terminal 120 or 240 amino acids of GADD34 improves recovery of this protein from E. coli without compromising its ability to boost protein synthesis in an in vitro protein synthesis system. The use of N-terminally truncated GADD34 proteins in place of full-length GADD34 should improve the utility of human cell-based cell-free protein synthesis systems.

  4. Free Radicals and Antioxidants in Cardiovascular Health and Disease

    African Journals Online (AJOL)

    Free radicals can be overproduced or the natural antioxidant system defenses weakened, first resulting in oxidative stress, and then leading to oxidative injury ... Keywords: Oxidative stress, cardiovascular disease, atherosclerosis, inflammation, cell signaling and transduction mechanisms, antioxidants, dietary phenolics.

  5. Effect of cold working on biocompatibility of Ni-free high nitrogen austenitic stainless steels using Dalton's Lymphoma cell line

    International Nuclear Information System (INIS)

    Talha, Mohd; Kumar, Sanjay; Behera, C.K.; Sinha, O.P.

    2014-01-01

    The aims of the present work are to explore the effect of cold working on in-vitro biocompatibility of indigenized low cost Ni-free nitrogen containing austenitic stainless steels (HNSs) and to compare it with conventionally used biomedical grade, i.e. AISI 316L and 316LVM, using Dalton's Lymphoma (DL) cell line. The MTT assay [3-(4,5-dimethythiazol 2-yl)-2,5-diphenyltetrazolium bromide] was performed on DL cell line for cytotoxicity evaluation and cell adhesion test. As a result, it was observed that the HNS had higher cell proliferation and cell growth and it increases by increasing nitrogen content and degree of cold working. The surface wettability of the alloys was also investigated by water contact angle measurements. The value of contact angles was found to decrease with increase in nitrogen content and degree of cold working. This indicates that the hydrophilic character increases with increasing nitrogen content and degree of cold working which further attributed to enhance the surface free energy (SFE) which would be conducive to cell adhesion which in turn increases the cell proliferation. - Graphical abstract: Effect of cold working on in-vitro biocompatibility of indigenized Ni-free nitrogen bearing austenitic stainless steels was explored using Dalton's Lymphoma cell line. Cell proliferation and cell adhesion increase by increasing the degree of cold working and nitrogen content in steel indicating that indigenized material is more biocompatible and no negative effect of cold working on these steels. - Highlights: • Effect of cold working on biocompatibility of Ni-free austenitic stainless steels • Cell proliferation and adhesion increase with nitrogen and degree of cold working. • Contact angle values decrease with nitrogen and degree of cold working

  6. Reliability issues of free-space communications systems and networks

    Science.gov (United States)

    Willebrand, Heinz A.

    2003-04-01

    Free space optics (FSO) is a high-speed point-to-point connectivity solution traditionally used in the enterprise campus networking market for building-to-building LAN connectivity. However, more recently some wire line and wireless carriers started to deploy FSO systems in their networks. The requirements on FSO system reliability, meaing both system availability and component reliability, are far more stringent in the carrier market when compared to the requirements in the enterprise market segment. This paper tries to outline some of the aspects that are important to ensure carrier class system reliability.

  7. Well-Controlled Cell-Trapping Systems for Investigating Heterogeneous Cell-Cell Interactions.

    Science.gov (United States)

    Kamiya, Koki; Abe, Yuta; Inoue, Kosuke; Osaki, Toshihisa; Kawano, Ryuji; Miki, Norihisa; Takeuchi, Shoji

    2018-03-01

    Microfluidic systems have been developed for patterning single cells to study cell-cell interactions. However, patterning multiple types of cells to understand heterogeneous cell-cell interactions remains difficult. Here, it is aimed to develop a cell-trapping device to assemble multiple types of cells in the well-controlled order and morphology. This device mainly comprises a parylene sheet for assembling cells and a microcomb for controlling the cell-trapping area. The cell-trapping area is controlled by moving the parylene sheet on an SU-8 microcomb using tweezers. Gentle downward flow is used as a driving force for the cell-trapping. The assembly of cells on a parylene sheet with round and line-shaped apertures is demonstrated. The cell-cell contacts of the trapped cells are then investigated by direct cell-cell transfer of calcein via connexin nanopores. Finally, using the device with a system for controlling the cell-trapping area, three different types of cells in the well-controlled order are assembled. The correct cell order rate obtained using the device is 27.9%, which is higher than that obtained without the sliding parylene system (0.74%). Furthermore, the occurrence of cell-cell contact between the three cell types assembled is verified. This cell-patterning device will be a useful tool for investigating heterogeneous cell-cell interactions. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Thermodynamic free energy methods to investigate shape transitions in bilayer membranes.

    Science.gov (United States)

    Ramakrishnan, N; Tourdot, Richard W; Radhakrishnan, Ravi

    2016-06-01

    The conformational free energy landscape of a system is a fundamental thermodynamic quantity of importance particularly in the study of soft matter and biological systems, in which the entropic contributions play a dominant role. While computational methods to delineate the free energy landscape are routinely used to analyze the relative stability of conformational states, to determine phase boundaries, and to compute ligand-receptor binding energies its use in problems involving the cell membrane is limited. Here, we present an overview of four different free energy methods to study morphological transitions in bilayer membranes, induced either by the action of curvature remodeling proteins or due to the application of external forces. Using a triangulated surface as a model for the cell membrane and using the framework of dynamical triangulation Monte Carlo, we have focused on the methods of Widom insertion, thermodynamic integration, Bennett acceptance scheme, and umbrella sampling and weighted histogram analysis. We have demonstrated how these methods can be employed in a variety of problems involving the cell membrane. Specifically, we have shown that the chemical potential, computed using Widom insertion, and the relative free energies, computed using thermodynamic integration and Bennett acceptance method, are excellent measures to study the transition from curvature sensing to curvature inducing behavior of membrane associated proteins. The umbrella sampling and WHAM analysis has been used to study the thermodynamics of tether formation in cell membranes and the quantitative predictions of the computational model are in excellent agreement with experimental measurements. Furthermore, we also present a method based on WHAM and thermodynamic integration to handle problems related to end-point-catastrophe that are common in most free energy methods.

  9. Roll-coating fabrication of ITO-free flexible solar cells based on a non-fullerene small molecule acceptor

    DEFF Research Database (Denmark)

    Liu, Wenqing; Shi, Hangqi; Andersen, Thomas Rieks

    2015-01-01

    We report organic solar cells (OSCs) with non-fullerene small molecule acceptors (SMAs) prepared in large area via a roll coating process. We employ all solution-processed indium tin oxide (ITO)-free flexible substrates for inverted solar cells with a new SMA of F(DPP)(2)B-2. By utilizing poly(3......-hexylthiophene) as donor blended with F(DPP)(2)B-2 as acceptor, ITO-free large-area flexible SMA based OSCs were produced under ambient conditions with the use of slot-die coating and flexographic printing methods on a lab-scale compact roll-coater that is readily transferrable to roll-to-roll processing...

  10. Nanosensors for label-free measurement of sodium ion fluxes of neuronal cells

    Energy Technology Data Exchange (ETDEWEB)

    Gebinoga, Michael, E-mail: michael.gebinoga@tu-ilmenau.de [ZIK MacroNano Microfluidics and Biosensors, Technical University Ilmenau, P.O. Box 100565, D-98684 Ilmenau (Germany); Silveira, Liele; Cimalla, Irina [ZIK MacroNano Microfluidics and Biosensors, Technical University Ilmenau, P.O. Box 100565, D-98684 Ilmenau (Germany); Dumitrescu, Andreea [University of Pennsylvania - School of Engineering and Applied Science, Philadelphia, PA 19104-6391 (United States); Kittler, Mario; Luebbers, Benedikt; Becker, Annette [ZIK MacroNano Microfluidics and Biosensors, Technical University Ilmenau, P.O. Box 100565, D-98684 Ilmenau (Germany); Lebedev, Vadim [Fraunhofer Institute for Solid State Physics, Tullastr. 7, D-79108 Freiburg (Germany); Schober, Andreas [ZIK MacroNano Microfluidics and Biosensors, Technical University Ilmenau, P.O. Box 100565, D-98684 Ilmenau (Germany)

    2010-05-25

    Novel nanosensors based on aluminium gallium nitrides (AlGaN/GaN) high electron mobility transistors have been of high interest during the last years, especially for their electrical characteristics as open gate field effect transistors. These nanosensors provide a valuable tool for high content screening in drug discovery, cell monitoring and liquid analyses focusing on applications of electrochemical detection technology. Our own measurements with these sensors confirm their pH sensitivity and in addition the possibility of detection of other ions in aqueous media. These measurements deal with the reactions of NG 108-15 (mouse neuroblastoma x rat glioma hybrid) neuronal cells in response to different acetylcholinesterase inhibitors. Our experimental approach shows some advantages. The first advantage is the label-free measurement of ion fluxes, and another advantage is the possibility non-destructively to estimate cell signals.

  11. Nanosensors for label-free measurement of sodium ion fluxes of neuronal cells

    International Nuclear Information System (INIS)

    Gebinoga, Michael; Silveira, Liele; Cimalla, Irina; Dumitrescu, Andreea; Kittler, Mario; Luebbers, Benedikt; Becker, Annette; Lebedev, Vadim; Schober, Andreas

    2010-01-01

    Novel nanosensors based on aluminium gallium nitrides (AlGaN/GaN) high electron mobility transistors have been of high interest during the last years, especially for their electrical characteristics as open gate field effect transistors. These nanosensors provide a valuable tool for high content screening in drug discovery, cell monitoring and liquid analyses focusing on applications of electrochemical detection technology. Our own measurements with these sensors confirm their pH sensitivity and in addition the possibility of detection of other ions in aqueous media. These measurements deal with the reactions of NG 108-15 (mouse neuroblastoma x rat glioma hybrid) neuronal cells in response to different acetylcholinesterase inhibitors. Our experimental approach shows some advantages. The first advantage is the label-free measurement of ion fluxes, and another advantage is the possibility non-destructively to estimate cell signals.

  12. Research on Control Strategy of Free-Piston Stirling Power Generating System

    Directory of Open Access Journals (Sweden)

    Jigui Zheng

    2017-10-01

    Full Text Available As a clean and fuel adaptive alternative power plant, the Stirling power generating system has drawn attention of experts and scholars in the energy field. In practical application, the instability of free-piston Stirling power generating system caused by abrupt load change is an inevitable problem. Thus, methods to improve the output frequency response and stability of the free-piston Stirling power generating system are necessary. The model of free-piston Stirling power generating system is built by isothermal analysis firstly, and the initial control strategy based on given voltage system is put forward. To further improve the performance of power system, a current feedback decoupling control strategy is proposed, and the mathematical model is established. The influence of full decoupled quadrature-direct (d-q axis currents is analyzed with respect to the output voltage adjusting time and fluctuation amplitude under the variations of piston displacement and output load. The simulation results show that the system performance is significantly improved, but the dynamic regulation lags caused by the decoupled current control still exist. To solve this problem and improve the performance of decoupled-state feedback current control that relies on parameter accuracy, internal model control based on sliding mode (IMC-SM current decoupling control strategy is proposed, the system model is established, and then the performance of voltage ripple in generating mode is improved. Finally, the test bench is built, and the steady state and transient voltage control performances are tested. The feasibility and priority of the control strategy is verified by experiment and simulation results.

  13. Washing of Cloth Contaminated with Radionuclides Using a Detergent-free Laundry System

    Energy Technology Data Exchange (ETDEWEB)

    Yim, Sung Paal

    2005-07-01

    In this study, a new laundry system to wash clothes without using detergent (detergent-free) was applied to wash clothes contaminated with radionuclides at the RWTF of KAERI. If the clothes contaminated with radionuclides and soil decontaminated and cleaned by washing without using detergent, the problem caused by the detergent could be solved naturally. The experiment was performed in two stages. In the first stage, washability of the processed water from the detergent=free laundry system was investigated with regard to its decontamination efficiency for the radionuclides and the detergency for the soil by using the test cloth specimens. In the second stage, real working clothes contaminated with radionuclides from the RWTF were washed by using a laundry machine equipped with a detergent-free system. Decontamination and detergency of the clothes were estimated after washing and the wastewater was also analyzed for its properties.

  14. Methane-free biogas for direct feeding of solid oxide fuel cells

    Science.gov (United States)

    Leone, P.; Lanzini, A.; Santarelli, M.; Calì, M.; Sagnelli, F.; Boulanger, A.; Scaletta, A.; Zitella, P.

    This paper deals with the experimental analysis of the performance and degradation issues of a Ni-based anode-supported solid oxide fuel cell fed by a methane-free biogas from dark-anaerobic digestion of wastes by pastry and fruit shops. The biogas is produced by means of an innovative process where the biomass is fermented with a pre-treated bacteria inoculum (Clostridia) able to completely inhibit the methanization step during the fermentation process and to produce a H 2/CO 2 mixture instead of conventional CH 4/CO 2 anaerobic digested gas (bio-methane). The proposed biogas production route leads to a biogas composition which avoids the need of introducing a reformer agent into or before the SOFC anode in order to reformate it. In order to analyse the complete behaviour of a SOFC with the bio-hydrogen fuel, an experimental session with several H 2/CO 2 synthetic mixtures was performed on an anode-supported solid oxide fuel cell with a Ni-based anode. It was found that side reactions occur with such mixtures in the typical thermodynamic conditions of SOFCs (650-800 °C), which have an effect especially at high currents, due to the shift to a mixture consisting of hydrogen, carbon monoxide, carbon dioxide and water. However, cells operated with acceptable performance and carbon deposits (typical of a traditional hydrocarbon-containing biogas) were avoided after 50 h of cell operation even at 650 °C. Experiments were also performed with traditional bio-methane from anaerobic digestion with 60/40 vol% of composition. It was found that the cell performance dropped after few hours of operation due to the formation of carbon deposits. A short-term test with the real as-produced biogas was also successfully performed. The cell showed an acceptable power output (at 800 °C, 0.35 W cm -2 with biogas, versus 0.55 W cm -2 with H 2) although a huge quantity of sulphur was present in the feeding fuel (hydrogen sulphide at 103 ppm and mercaptans up to 10 ppm). Therefore, it

  15. Methane-free biogas for direct feeding of solid oxide fuel cells

    Energy Technology Data Exchange (ETDEWEB)

    Leone, P.; Lanzini, A.; Santarelli, M.; Cali, M. [Dipartimento di Energetica, Politecnico di Torino, Corso Duca degli Abruzzi 24, 10129 Turin (Italy); Sagnelli, F.; Boulanger, A.; Scaletta, A.; Zitella, P. [BioEnergy Lab, Environment Park S.p.A., Via Livorno 60, 10144 Turin (Italy)

    2010-01-01

    This paper deals with the experimental analysis of the performance and degradation issues of a Ni-based anode-supported solid oxide fuel cell fed by a methane-free biogas from dark-anaerobic digestion of wastes by pastry and fruit shops. The biogas is produced by means of an innovative process where the biomass is fermented with a pre-treated bacteria inoculum (Clostridia) able to completely inhibit the methanization step during the fermentation process and to produce a H{sub 2}/CO{sub 2} mixture instead of conventional CH{sub 4}/CO{sub 2} anaerobic digested gas (bio-methane). The proposed biogas production route leads to a biogas composition which avoids the need of introducing a reformer agent into or before the SOFC anode in order to reformate it. In order to analyse the complete behaviour of a SOFC with the bio-hydrogen fuel, an experimental session with several H{sub 2}/CO{sub 2} synthetic mixtures was performed on an anode-supported solid oxide fuel cell with a Ni-based anode. It was found that side reactions occur with such mixtures in the typical thermodynamic conditions of SOFCs (650-800 C), which have an effect especially at high currents, due to the shift to a mixture consisting of hydrogen, carbon monoxide, carbon dioxide and water. However, cells operated with acceptable performance and carbon deposits (typical of a traditional hydrocarbon-containing biogas) were avoided after 50 h of cell operation even at 650 C. Experiments were also performed with traditional bio-methane from anaerobic digestion with 60/40 vol% of composition. It was found that the cell performance dropped after few hours of operation due to the formation of carbon deposits. A short-term test with the real as-produced biogas was also successfully performed. The cell showed an acceptable power output (at 800 C, 0.35 W cm{sup -2} with biogas, versus 0.55 W cm{sup -2} with H{sub 2}) although a huge quantity of sulphur was present in the feeding fuel (hydrogen sulphide at 103 ppm and

  16. A Pd/C-CeO2 Anode Catalyst for High-Performance Platinum-Free Anion Exchange Membrane Fuel Cells.

    Science.gov (United States)

    Miller, Hamish A; Lavacchi, Alessandro; Vizza, Francesco; Marelli, Marcello; Di Benedetto, Francesco; D'Acapito, Francesco; Paska, Yair; Page, Miles; Dekel, Dario R

    2016-05-10

    One of the biggest obstacles to the dissemination of fuel cells is their cost, a large part of which is due to platinum (Pt) electrocatalysts. Complete removal of Pt is a difficult if not impossible task for proton exchange membrane fuel cells (PEM-FCs). The anion exchange membrane fuel cell (AEM-FC) has long been proposed as a solution as non-Pt metals may be employed. Despite this, few examples of Pt-free AEM-FCs have been demonstrated with modest power output. The main obstacle preventing the realization of a high power density Pt-free AEM-FC is sluggish hydrogen oxidation (HOR) kinetics of the anode catalyst. Here we describe a Pt-free AEM-FC that employs a mixed carbon-CeO2 supported palladium (Pd) anode catalyst that exhibits enhanced kinetics for the HOR. AEM-FC tests run on dry H2 and pure air show peak power densities of more than 500 mW cm(-2) . © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Indium Tin Oxide-Free Polymer Solar Cells: Toward Commercial Reality

    DEFF Research Database (Denmark)

    Angmo, Dechan; Espinosa Martinez, Nieves; Krebs, Frederik C

    2014-01-01

    Polymer solar cell (PSC) is the latest of all photovoltaic technologies which currently lies at the brink of commercialization. The impetus for its rapid progress in the last decade has come from low-cost high throughput production possibility which in turn relies on the use of low-cost materials...... and vacuum-free manufacture. Indium tin oxide (ITO), the commonly used transparent conductor, imposes the majority of the cost of production of PSCs, limits flexibility, and is feared to create bottleneck in the dawning industry due to indium scarcity and the resulting large price fluctuations. As such...

  18. Short-term, serum-free, static culture of cord blood-derived CD34+ cells: effects of FLT3-L and MIP-1alpha on in vitro expansion of hematopoietic progenitor cells.

    Science.gov (United States)

    Capmany, G; Querol, S; Cancelas, J A; García, J

    1999-08-01

    The use of ex vivo expanded cells has been suggested as a possible means to accelerate the speed of engraftment in cord blood (CB) transplantation. The aim of this study was to fix the optimal condition for the generation of committed progenitors without affecting the stem cell compartment. Analysis of the effects of FLT3-L and MIP-1alpha when combined with SCF, IL-3 and IL-6, in short-term (6 days), serum-free expansion cultures of CB-selected CD34+ cells. An important expansion was obtained that ranged between 8-15 times for CFU-GM, 21-51 times for the BFU-E/CFU-Mix population and 11 to 30 times for CD34+ cells assessed by flow cytometry. From the combinations tested, those in which FLT3-L was present had a significant increase in the expansion of committed progenitors, while the presence of MIP-1alpha had a detrimental effect on the generation of more differentiated cells. However, stem cell candidates assessed by week 5 CAFC assay could be maintained in culture when both MIP-1a and FLT3-L were present (up to 91% recovery). This culture system was also able to expand megakaryocytic precursors as determined by the co-expression of CD34 and CD61 antigens (45-70 times), in spite of the use of cytokines non-specific for the megakaryocytic lineage. The results obtained point to the combination of SCF, IL-3, IL-6, FLT3-L and MIP-1alpha as the best suited for a pre-clinical short-term serum-free static ex vivo expansion protocol of CB CD34+ cells, since it can generate large numbers of committed progenitor cells as well as maintaining week 5 CAFC.

  19. High Efficiency Gene Correction in Hematopoietic Cells by Donor-Template-Free CRISPR/Cas9 Genome Editing

    Directory of Open Access Journals (Sweden)

    Duran Sürün

    2018-03-01

    Full Text Available The CRISPR/Cas9 prokaryotic adaptive immune system and its swift repurposing for genome editing enables modification of any prespecified genomic sequence with unprecedented accuracy and efficiency, including targeted gene repair. We used the CRISPR/Cas9 system for targeted repair of patient-specific point mutations in the Cytochrome b-245 heavy chain gene (CYBB, whose inactivation causes chronic granulomatous disease (XCGD—a life-threatening immunodeficiency disorder characterized by the inability of neutrophils and macrophages to produce microbicidal reactive oxygen species (ROS. We show that frameshift mutations can be effectively repaired in hematopoietic cells by non-integrating lentiviral vectors carrying RNA-guided Cas9 endonucleases (RGNs. Because about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that up to a quarter of all patients suffering from monogenic blood disorders could benefit from gene therapy employing personalized, donor template-free RGNs.

  20. Catalytical Properties of Free and Immobilized Aspergillus niger Tannase

    OpenAIRE

    Abril Flores-Maltos; Luis V. Rodríguez-Durán; Jacqueline Renovato; Juan C. Contreras; Raúl Rodríguez; Cristóbal N. Aguilar

    2011-01-01

    A fungal tannase was produced, recovered, and immobilized by entrapment in calcium alginate beads. Catalytical properties of the immobilized enzyme were compared with those of the free one. Tannase was produced intracellularly by the xerophilic fungus Aspergillus niger GH1 in a submerged fermentation system. Enzyme was recovered by cell disruption and the crude extract was partially purified. The catalytical properties of free and immobilized tannase were evaluated using tannic acid and methy...

  1. Fundamental Study on the Impact of Gluten-Free Starches on the Quality of Gluten-Free Model Breads

    Directory of Open Access Journals (Sweden)

    Stefan W. Horstmann

    2016-04-01

    Full Text Available Starch is widely used as an ingredient and significantly contributes to texture, appearance, and overall acceptability of cereal based foods, playing an important role due to its ability to form a matrix, entrapping air bubbles. A detailed characterisation of five gluten-free starches (corn, wheat, rice, tapioca, potato was performed in this study. In addition, the influence of these starches, with different compositional and morphological properties, was evaluated on a simple gluten-free model bread system. The morphological characterisation, evaluated using scanning electron microscopy, revealed some similarities among the starches, which could be linked to the baking performance of the breads. Moreover, the lipid content, though representing one of the minor components in starch, was found to have an influence on pasting, bread making, and staling. Quality differences in cereal root and tuber starch based breads were observed. However, under the baking conditions used, gluten-free rendered wheat starch performed best, followed by potato starch, in terms of loaf volume and cell structure. Tapioca starch and rice starch based breads were not further analysed, due to an inferior baking performance. This is the first study to evaluate gluten-free starch on a simple model bread system.

  2. Fabrication of corneal epithelial cell sheets maintaining colony-forming cells without feeder cells by oxygen-controlled method.

    Science.gov (United States)

    Nakajima, Ryota; Takeda, Shizu

    2014-01-01

    The use of murine 3T3 feeder cells needs to be avoided when fabricating corneal epithelial cell sheets for use in treating ocular surface diseases. However, the expression level of the epithelial stem/progenitor cell marker, p63, is down-regulated in feeder-free culture systems. In this study, in order to fabricate corneal epithelial cell sheets that maintain colony-forming cells without using any feeder cells, we investigated the use of an oxygen-controlled method that was developed previously to fabricate cell sheets efficiently. Rabbit limbal epithelial cells were cultured under hypoxia (1-10% O2) and under normoxia during stratification after reaching confluence. Multilayered corneal epithelial cell sheets were fabricated using an oxygen-controlled method, and immunofluorescence analysis showed that cytokeratin 3 and p63 was expressed in appropriate localization in the cell sheets. The colony-forming efficiency of the cell sheets fabricated by the oxygen-controlled method without feeder cells was significantly higher than that of cell sheets fabricated under 20% O2 without feeder cells. These results indicate that the oxygen-controlled method has the potential to achieve a feeder-free culture system for fabricating corneal epithelial cell sheets for corneal regeneration. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. A compact 3 T all HTS cryogen-free MRI system

    Science.gov (United States)

    Parkinson, B. J.; Bouloukakis, K.; Slade, R. A.

    2017-12-01

    We have designed and built a passively shielded, cryogen-free 3 T 160 mm bore bismuth strontium calcium copper oxide HTS magnet with shielded gradient coils suitable for use in small animal imaging applications. The magnet is cooled to approximately 16 K using a two-stage cryocooler and is operated at 200 A. The magnet has been passively shimmed so as to achieve ±10 parts per million (ppm) homogeneity over a 60 mm diameter imaging volume. We have demonstrated that B 0 temporal stability is fit-for-purpose despite the magnet operating in the driven mode. The system has produced good quality spin-echo and gradient echo images. This compact HTS-MRI system is emerging as a true alternative to conventional low temperature superconductor based cryogen-free MRI systems, with much more efficient cryogenics since it operates entirely from a single phase alternating current electrical supply.

  4. Consumer acceptance of eggs from Hy-Line Brown layers fed soybean or soybean-free diets using cage or free-range rearing systems.

    Science.gov (United States)

    Al-Ajeeli, M N; Miller, R K; Leyva, H; Hashim, M M; Abdaljaleel, R A; Jameel, Y; Bailey, C A

    2018-05-01

    Consumers have begun to awaken to the food on their plates with respect to human health and the environment, as well as animal welfare. They have become more demanding about what they buy or prefer in their food, such as soy-free, gluten-free, or organic products. The objective of this study was to evaluate consumer acceptance of eggs from hens fed soybean meal or soybean-free diets utilizing cottonseed meal and distillers' dried grains, using cage or free-range rearing systems. All eggs were stored at the sensory lab at Texas A&M University (TAMU) for a d prior to each test at 4°C. A panel of consumers (n = 60) made up of TAMU students, faculty, and staff, ages 18 to 50, were recruited to evaluate consumer acceptance based on 2 tests using scrambled and hard cooked eggs. Samples were placed in separate weigh boats labeled with 3-digit codes to avoid visual bias. Sensory ballots were based on overall like or dislike of flavor, texture, odor, and color using the 9-point hedonic scales. For scrambled eggs, flavor did not differ (P > 0.05), but texture liking was higher (P = 0.064) for scrambled eggs from the soybean-free diet (7.08) vs. scrambled eggs from the soybean meal diet (6.65). With respect to the hard cooked eggs, the consumer panel preferred the flavor of the eggs from the caged rearing system (7.11) vs. eggs from the free-range system (6.60; P = 0.014). Consumers liked the texture (P = 0.018) for eggs collected from hens fed soybean meal (6.91) vs. eggs from hens fed the soybean-free diet (6.30).

  5. Potential for free radical-induced lipid peroxidation as a cause of endothelial cell injury in Rocky Mountain spotted fever.

    Science.gov (United States)

    Silverman, D J; Santucci, L A

    1988-01-01

    Cells infected by Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, display unusual intracellular morphological changes characterized by dilatation of the membranes of the endoplasmic reticulum and outer nuclear envelope. These changes are consistent with those that might be expected to occur following peroxidation of membrane lipids initiated by oxygen radical species, such as the hydroxyl radical or a variety of organic radicals. Using a fluorescent probe, we have found significantly increased levels of peroxides in human endothelial cells infected by R. rickettsii. Studies with desferrioxamine, an iron chelator effective in preventing formation of the hydroxyl radical from hydrogen peroxide and the superoxide free radical, reduced peroxide levels in infected cells to those found in uninfected cells. This observation suggests that the increased peroxides in infected cells may be lipid peroxides, degradation products of free radical attack on polyenoic fatty acids. The potential for lipid peroxidation as an important mechanism in endothelial cell injury caused by R. rickettsii is discussed. Images PMID:3141280

  6. MR imaging of sickle cell patients: Comparison during pain-free and crisis states

    International Nuclear Information System (INIS)

    Brogdon, B.G.; Williams, J.P.; Mankad, V.N.; Harpen, M.D.; Moore, R.B.

    1986-01-01

    The MR imaging appearance of long bones and femoral heads of patients with sickle cell disease during a pain-free steady state and during a crisis-pain state was compared with the MR imaging appearance of matched healthy control subjects. A distinctive signal change in the narrow spaces of the long bones of patients with sickle cell disease was seen at all times. Distinct signal changes during pain crises were found in the marrow of a significant number of patients. Changes associated with aseptic necrosis, when present, did not differ from changes seen in aseptic necrosis of other causes

  7. Prospective guidance in a free-swimming cell

    NARCIS (Netherlands)

    Delafield-Butt, Jonathan T.; Pepping, Gert-Jan; McCaig, Colin D.; Lee, David N.

    A systems theory of movement control in animals is presented in this article and applied to explaining the controlled behaviour of the single-celled Paramecium caudatum in an electric field. The theory-General Tau Theory-is founded on three basic principles: (i) all purposive movement entails

  8. Label-free nonlinear optical microscopy detects early markers for osteogenic differentiation of human stem cells

    Science.gov (United States)

    Hofemeier, Arne D.; Hachmeister, Henning; Pilger, Christian; Schürmann, Matthias; Greiner, Johannes F. W.; Nolte, Lena; Sudhoff, Holger; Kaltschmidt, Christian; Huser, Thomas; Kaltschmidt, Barbara

    2016-05-01

    Tissue engineering by stem cell differentiation is a novel treatment option for bone regeneration. Most approaches for the detection of osteogenic differentiation are invasive or destructive and not compatible with live cell analysis. Here, non-destructive and label-free approaches of Raman spectroscopy, coherent anti-Stokes Raman scattering (CARS) and second harmonic generation (SHG) microscopy were used to detect and image osteogenic differentiation of human neural crest-derived inferior turbinate stem cells (ITSCs). Combined CARS and SHG microscopy was able to detect markers of osteogenesis within 14 days after osteogenic induction. This process increased during continued differentiation. Furthermore, Raman spectroscopy showed significant increases of the PO43- symmetric stretch vibrations at 959 cm-1 assigned to calcium hydroxyapatite between days 14 and 21. Additionally, CARS microscopy was able to image calcium hydroxyapatite deposits within 14 days following osteogenic induction, which was confirmed by Alizarin Red-Staining and RT- PCR. Taken together, the multimodal label-free analysis methods Raman spectroscopy, CARS and SHG microscopy can monitor osteogenic differentiation of adult human stem cells into osteoblasts with high sensitivity and spatial resolution in three dimensions. Our findings suggest a great potential of these optical detection methods for clinical applications including in vivo observation of bone tissue-implant-interfaces or disease diagnosis.

  9. Rapid and label-free separation of Burkitt's lymphoma cells from red blood cells by optically-induced electrokinetics.

    Directory of Open Access Journals (Sweden)

    Wenfeng Liang

    Full Text Available Early stage detection of lymphoma cells is invaluable for providing reliable prognosis to patients. However, the purity of lymphoma cells in extracted samples from human patients' marrow is typically low. To address this issue, we report here our work on using optically-induced dielectrophoresis (ODEP force to rapidly purify Raji cells' (a type of Burkitt's lymphoma cell sample from red blood cells (RBCs with a label-free process. This method utilizes dynamically moving virtual electrodes to induce negative ODEP force of varying magnitudes on the Raji cells and RBCs in an optically-induced electrokinetics (OEK chip. Polarization models for the two types of cells that reflect their discriminate electrical properties were established. Then, the cells' differential velocities caused by a specific ODEP force field were obtained by a finite element simulation model, thereby established the theoretical basis that the two types of cells could be separated using an ODEP force field. To ensure that the ODEP force dominated the separation process, a comparison of the ODEP force with other significant electrokinetics forces was conducted using numerical results. Furthermore, the performance of the ODEP-based approach for separating Raji cells from RBCs was experimentally investigated. The results showed that these two types of cells, with different concentration ratios, could be separated rapidly using externally-applied electrical field at a driven frequency of 50 kHz at 20 Vpp. In addition, we have found that in order to facilitate ODEP-based cell separation, Raji cells' adhesion to the OEK chip's substrate should be minimized. This paper also presents our experimental results of finding the appropriate bovine serum albumin concentration in an isotonic solution to reduce cell adhesion, while maintaining suitable medium conductivity for electrokinetics-based cell separation. In short, we have demonstrated that OEK technology could be a promising tool for

  10. Immobilization of anaerobic thermophilic bacteria for the production of cell-free thermostable. alpha. -amylases and pullulanases

    Energy Technology Data Exchange (ETDEWEB)

    Klingeberg, M [Goettingen Univ. (Germany, F.R.). Inst. fuer Mikrobiologie; Vorlop, K D [Technische Univ. Braunschweig (Germany, F.R.). Inst. fuer Technische Chemie; Antranikian, G [Technische Univ. Hamburg-Harburg, Hamburg (Germany, F. R.). Arbeitsbereich Biotechnologie 1

    1990-08-01

    For the production of cell-free thermostable {alpha}-amylases and pullulanases various anaerobic thermophilic bacteria that belong to the genera Clostridium and Thermoanaerobacter were immobilized in calcium alginate gel beads. The entrapment of bacteria was performed in full was well as in hollow spheres. An optimal limited medium, which avoided bacterial outgrowth, was developed for the cultivation of immobilized organisms at 60deg C using 0.4% starch as substrate. Compared to non-immobilized cells these techniques allowed a significant increase (up to 5.6-fold) in the specific activities of the extracellular enzymes formed. An increase in the productivity of extracellular enzymes was observed after immobilization of bacteria in full spheres. In the case of C. thermosaccharolyticum, for instance, the productivity was raised from 90 units (U)/10{sup 12} cells up to 700 U/10{sup 12} cells. Electrophoretic analysis of the secreted proteins showed that in all cases most of the amylolytic enzymes formed were released into the culture medium. Proteins that had a molecular mass of less than 450 000 daltons could easily diffuse through the gel matrix. Cultivation of immobilized bacteria in semi-continuous and fed-batch cultures was also accompanied by an elevation in the concentration of cell-free enzymes. (orig.).

  11. Gluten-free diet increases beta-cell volume and improves glucose tolerance in an animal model of type 2 diabetes

    DEFF Research Database (Denmark)

    Haupt-Jørgensen, Martin; Buschard, Karsten; Hansen, Axel Kornerup

    2016-01-01

    Background Gluten-free (GF) diet alleviates type 1 diabetes in animal models and possibly in humans. We recently showed that fatty acid-induced insulin secretion is enhanced by enzymatically digested gluten (gliadin) stimulation in INS-1E insulinoma cells. We therefore hypothesized that GF diet...... would induce beta-cell rest and ameliorate type 2 diabetes. Methods C57BL/6JBomTac (B6) mice were fed a high-fat (HF), gluten-free high-fat (GF–HF), standard (STD) or gluten-free (GF) diet for 42 weeks. Results Short-term (6–24 weeks) GF–HF versus HF feeding impaired glucose tolerance and increased...... capacity controls pancreas volume. Thus, long-term GF diets may be beneficial for obese type 2 diabetes patients and trials should be performed....

  12. Fetal bovine serum and human constitutive androstane receptor: Evidence for activation of the SV23 splice variant by artemisinin, artemether, and arteether in a serum-free cell culture system

    Energy Technology Data Exchange (ETDEWEB)

    Lau, Aik Jiang; Chang, Thomas K.H., E-mail: thomas.chang@ubc.ca

    2014-06-01

    The naturally occurring SV23 splice variant of human constitutive androstane receptor (hCAR-SV23) is activated by di-(2-ethylhexyl)phthalate (DEHP), which is detected as a contaminant in fetal bovine serum (FBS). In our initial experiment, we compared the effect of dialyzed FBS, charcoal-stripped, dextran-treated FBS (CS-FBS), and regular FBS on the basal activity and ligand-activation of hCAR-SV23 in a cell-based reporter gene assay. In transfected HepG2 cells cultured in medium supplemented with 10% FBS, basal hCAR-SV23 activity varied with the type of FBS (regular > dialyzed > CS). DEHP increased hCAR-SV23 activity when 10% CS-FBS, but not regular FBS or dialyzed FBS, was used. With increasing concentrations (1–10%) of regular FBS or CS-FBS, hCAR-SV23 basal activity increased, whereas in DEHP-treated cells, hCAR-SV23 activity remained similar (regular FBS) or slightly increased (CS-FBS). Subsequent experiments identified a serum-free culture condition to detect DEHP activation of hCAR-SV23. Under this condition, artemisinin, artemether, and arteether increased hCAR-SV23 activity, whereas they decreased it in cells cultured in medium supplemented with 10% regular FBS. By comparison, FBS increased the basal activity of the wild-type isoform of hCAR (hCAR-WT), whereas it did not affect the basal activity of the SV24 splice variant (hCAR-SV24) or ligand activation of hCAR-SV24 and hCAR-WT by 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO). The use of serum-free culture condition was suitable for detecting CITCO activation of hCAR-WT and hCAR-SV24. In conclusion, FBS leads to erroneous classification of pharmacological ligands of hCAR-SV23 in cell-based assays, but investigations on functional ligands of hCAR isoforms can be conducted in serum-free culture condition. - Highlights: • FBS leads to erroneous pharmacological classification of hCAR-SV23 ligands. • Artemisinin, artemether, and arteether activate h

  13. Gibbs free energy of formation of lanthanum rhodate by quadrupole mass spectrometer

    International Nuclear Information System (INIS)

    Prasad, R.; Banerjee, Aparna; Venugopal, V.

    2003-01-01

    The ternary oxide in the system La-Rh-O is of considerable importance because of its application in catalysis. Phase equilibria in the pseudo-binary system La 2 O 3 -Rh 2 O 3 has been investigated by Shevyakov et. al. Gibbs free energy of LaRhO 3 (s) was determined by Jacob et. al. using a solid state Galvanic cell in the temperature range 890 to 1310 K. No other thermodynamic data were available in the literature. Hence it was decided to determine Gibbs free energy of formation of LaRhO 3 (s) by an independent technique, viz. quadrupole mass spectrometer (QMS) coupled with a Knudsen effusion cell and the results are presented

  14. Diurnal Variations of Human Circulating Cell-Free Micro-RNA.

    Directory of Open Access Journals (Sweden)

    Niels H H Heegaard

    Full Text Available A 24-hour light and dark cycle-dependent rhythmicity pervades physiological processes in virtually all living organisms including humans. These regular oscillations are caused by external cues to endogenous, independent biological time-keeping systems (clocks. The rhythm is reflected by gene expression that varies in a circadian and specific fashion in different organs and tissues and is regulated largely by dynamic epigenetic and post-transcriptional mechanisms. This leads to well-documented oscillations of specific electrolytes, hormones, metabolites, and plasma proteins in blood samples. An emerging, important class of gene regulators is short single-stranded RNA (micro-RNA, miRNA that interferes post-transcriptionally with gene expression and thus may play a role in the circadian variation of gene expression. MiRNAs are promising biomarkers by virtue of their disease-specific tissue expression and because of their presence as stable entities in the circulation. However, no studies have addressed the putative circadian rhythmicity of circulating, cell-free miRNAs. This question is important both for using miRNAs as biological markers and for clues to miRNA function in the regulation of circadian gene expression. Here, we investigate 92 miRNAs in plasma samples from 24 young male, healthy volunteers repeatedly sampled 9 times during a 24-hour stay in a regulated environment. We demonstrate that a third (26/79 of the measurable plasma miRNAs (using RT-qPCR on a microfluidic system exhibit a rhythmic behavior and are distributed in two main phase patterns. Some of these miRNAs weakly target known clock genes and many have strong targets in intracellular MAPK signaling pathways. These novel findings highlight the importance of considering bio-oscillations in miRNA biomarker studies and suggest the further study of a set of specific circulating miRNAs in the regulation and functioning of biological clocks.

  15. Direct evaluation of free energy for large system through structure integration approach.

    Science.gov (United States)

    Takeuchi, Kazuhito; Tanaka, Ryohei; Yuge, Koretaka

    2015-09-30

    We propose a new approach, 'structure integration', enabling direct evaluation of configurational free energy for large systems. The present approach is based on the statistical information of lattice. Through first-principles-based simulation, we find that the present method evaluates configurational free energy accurately in disorder states above critical temperature.

  16. tRNA-mediated labelling of proteins with biotin. A nonradioactive method for the detection of cell-free translation products.

    Science.gov (United States)

    Kurzchalia, T V; Wiedmann, M; Breter, H; Zimmermann, W; Bauschke, E; Rapoport, T A

    1988-03-15

    We have developed a new method for the rapid and sensitive detection of cell-free translation products. Biotinylated lysine is incorporated into newly synthesized proteins by means of lysyl-tRNA that is modified in the epsilon-position. After electrophoresis in a dodecyl sulfate gel and blotting onto nitrocellulose, the translation products can be identified by probing with streptavidin and biotinylated alkaline phosphatase, followed by incubation with a chromogenic enzyme substrate. The non-radioactive labelling by biotin approaches in its sensitivity that obtained by radioactive amino acids. The products are absolutely stable and can be rapidly identified. The new method has been tested with different mRNAs in the cell-free translation systems of wheat germ and reticulocytes. Neither the interaction of secretory proteins with the signal recognition particle nor the in vitro translocation across the endoplasmic reticulum membrane or core glycosylation of nascent polypeptides are prevented by the incorporation of biotinylated lysine residues. The results indicate that both the ribosome and the endoplasmic reticulum membrane permit the passage of polypeptides carrying bulky groups attached to the amino acids (by atomic models it was estimated that the size of the side chain of lysine changes from approximately equal to 0.8 nm to approximately equal to 2 nm after modification.

  17. Development of a Microfluidic-Based Optical Sensing Device for Label-Free Detection of Circulating Tumor Cells (CTCs Through Their Lactic Acid Metabolism

    Directory of Open Access Journals (Sweden)

    Tzu-Keng Chiu

    2015-03-01

    Full Text Available This study reports a microfluidic-based optical sensing device for label-free detection of circulating tumor cells (CTCs, a rare cell species in blood circulation. Based on the metabolic features of cancer cells, live CTCs can be quantified indirectly through their lactic acid production. Compared with the conventional schemes for CTC detection, this label-free approach could prevent the biological bias due to the heterogeneity of the surface antigens on cancer cells. In this study, a microfluidic device was proposed to generate uniform water-in-oil cell-encapsulating micro-droplets, followed by the fluorescence-based optical detection of lactic acid produced within the micro-droplets. To test its feasibility to quantify cancer cells, experiments were carried out. Results showed that the detection signals were proportional to the number of cancer cells within the micro-droplets, whereas such signals were insensitive to the existence and number of leukocytes within. To further demonstrate its feasibility for cancer cell detection, the cancer cells with known cell number in a cell suspension was detected based on the method. Results revealed that there was no significant difference between the detected number and the real number of cancer cells. As a whole, the proposed method opens up a new route to detect live CTCs in a label-free manner.

  18. THE THIOREDOXIN SYSTEM IN REGULATING MCF-7 CELL PROLIFERATION UNDER REDOX STATUS MODULATION

    Directory of Open Access Journals (Sweden)

    E. A. Stepovaya

    2016-01-01

    Full Text Available Introduction. Despite the available data on tumor cell functioning under the conditions of free radical-mediated oxidation, the mechanisms of redox regulation, cell proliferation management and apoptosis avoidance remain understudied.The objective of the study was to identify the role of the thioredoxin system in regulating MCF-7 breast cancer cell proliferation under redox status modulation with 1.4-dithioerythritol.Material and methods. The studies were conducted on the MCF-7 breast cancer cell line, grown in adherent cell culture. Cell redox status was modulated with5 mM N-ethylmaleimide – an SH group and peptide inhibitor and5 mM 1.4-dithioerythritol – a thiol group protector. The cell cycle was evaluated by flow cytometry, the same technique was used to measure the reactive oxygen species concentration. The levels of reduced and oxidized glutathione and the activity of thioredoxin reductase were identified by spectrophotometry. The intracellular concentrations of thioredoxin, cyclin E and cyclin-dependent kinase 2 were determined by Western blot analysis.Results and discussion. The essential role of the thioredoxin system in regulating MCF-7 breast cancer cell proliferation was exhibited. S-phase arrest under the effect of N-ethylmaleimide and G0/G1-phase arrest under the effect of 1.4-dithioerythritol are associated with the changes in the activity of redox-sensitive protein complexes (cyclins and cyclin-dependent kinases that regulate cell proliferation.Conclusion. Redoxdependent modulation of proliferation regulating intracellular protein activity occurs due to the thioredoxin system. This is a promising research area for seeking molecular targets of breast cell malignization. 

  19. Chlorine-Incorporation-Induced Formation of the Layered Phase for Antimony-Based Lead-Free Perovskite Solar Cells.

    Science.gov (United States)

    Jiang, Fangyuan; Yang, Dongwen; Jiang, Youyu; Liu, Tiefeng; Zhao, Xingang; Ming, Yue; Luo, Bangwu; Qin, Fei; Fan, Jiacheng; Han, Hongwei; Zhang, Lijun; Zhou, Yinhua

    2018-01-24

    The environmental toxicity of Pb in organic-inorganic hybrid perovskite solar cells remains an issue, which has triggered intense research on seeking alternative Pb-free perovskites for solar applications. Halide perovskites based on group-VA cations of Bi 3+ and Sb 3+ with the same lone-pair ns 2 state as Pb 2+ are promising candidates. Herein, through a joint experimental and theoretical study, we demonstrate that Cl-incorporated methylammonium Sb halide perovskites (CH 3 NH 3 ) 3 Sb 2 Cl X I 9-X show promise as efficient solar absorbers for Pb-free perovskite solar cells. Inclusion of methylammonium chloride into the precursor solutions suppresses the formation of the undesired zero-dimensional dimer phase and leads to the successful synthesis of high-quality perovskite films composed of the two-dimensional layered phase favored for photovoltaics. Solar cells based on the as-obtained (CH 3 NH 3 ) 3 Sb 2 Cl X I 9-X films reach a record-high power conversion efficiency over 2%. This finding offers a new perspective for the development of nontoxic and low-cost Sb-based perovskite solar cells.

  20. Protein-free transfection of CHO host cells with an IgG-fusion protein: selection and characterization of stable high producers and comparison to conventionally transfected clones.

    Science.gov (United States)

    Lattenmayer, Christine; Loeschel, Martina; Schriebl, Kornelia; Steinfellner, Willibald; Sterovsky, Thomas; Trummer, Evelyn; Vorauer-Uhl, Karola; Müller, Dethardt; Katinger, Hermann; Kunert, Renate

    2007-04-15

    In order to improve the current techniques of cell cultivation in the absence of serum, we have developed a protein-free transfection protocol for CHO cells, based on the Nucleofector technology. After starting with a heterogeneous pool of primary transfectants which express the fusion protein EpoFc, we isolated single clones and compared them with parallel clones generated by lipofection in serum-dependent cultivation. Our intensive characterization program was based on determination of specific productivity (q(p)) and analysis of genetic parameters. In two nucleofection experiments, transfection with 5 microg of DNA resulted in best productivities of the primary cell pools. After subcloning, the q(p) could be raised up to 27 pg x cells(-1) x day(-1). While the serum-dependent transfectants exhibited specific productivities up to 57 pg x cells(-1) x day(-1) in serum-dependent cultivation, a significant decrease that resulted in the range of q(p) of the protein-free transfectants was observed after switching to protein-free conditions. Investigation of genetic parameters revealed higher mRNA levels and gene copy numbers (GCN) for the protein-free adapted serum-dependent transfectants. Therefore, we assume that problems during protein-free adaptation (PFA) lead to a less efficient translation machinery after serum deprivation. We describe the generation of stable-producing recombinant CHO clones by protein-free transfection of a protein-free adapted host cell line, which reduces the risk of adverse clonal changes after PFA. The main advantage of this approach is the earlier predictability of clone behavior, which makes the generation of production clones by protein-free transfection, a viable and highly efficient strategy for recombinant cell line development. (c) 2006 Wiley Periodicals, Inc.

  1. Round-Robin Studies on Roll-Processed ITO-free Organic Tandem Solar Cells Combined with Inter-Laboratory Stability Studies

    DEFF Research Database (Denmark)

    Livi, Francesco; Søndergaard, Roar R.; Andersen, Thomas Rieks

    2015-01-01

    Roll-processed, indium tin oxide (ITO)-free, flexible, organic tandem solar cells and modules have been realized and used in round-robin studies as well as in parallel inter-laboratory stability studies. The tandem cells/modules show no significant difference in comparison to their single...

  2. Tungsten-188/carrier-free rhenium-188 perrhenic acid generator system

    International Nuclear Information System (INIS)

    Knapp, F.F. Jr.; Lisic, E.C.; Mirzadeh, S.; Callahan, A.P.

    1993-01-01

    A generator system for providing a carrier-free radioisotope in the form of an acid comprises a chromatography column in tandem fluid connection with an ion exchange column, the chromatography column containing a charge of a radioactive parent isotope. The chromatography column, charged with a parent isotope, is eluted with an alkali metal salt solution to generate the radioisotope in the form of an intermediate solution, which is passed through the ion-exchange column to convert the radioisotope to a carrier-free acid form

  3. Cytoprotective Mechanisms Mediated by Polyphenols from Chilean Native Berries against Free Radical-Induced Damage on AGS Cells.

    Science.gov (United States)

    Ávila, Felipe; Theoduloz, Cristina; López-Alarcón, Camilo; Dorta, Eva; Schmeda-Hirschmann, Guillermo

    2017-01-01

    The prevalence of cytoprotective mechanisms induced by polyphenols such as activation of intracellular antioxidant responses (ICM) and direct free radical scavenging was investigated in native Chilean species of strawberries, raspberries, and currants. Human gastric epithelial cells were co- and preincubated with polyphenolic-enriched extracts (PEEs) from Chilean raspberries ( Rubus geoides ), strawberries ( Fragaria chiloensis ssp. chiloensis f . chiloensis ), and currants ( Ribes magellanicum ) and challenged with peroxyl and hydroxyl radicals. Cellular protection was determined in terms of cell viability, glyoxalase I and glutathione s-transferases activities, and carboxymethyl lysine (CML) and malondialdehyde levels. Our results indicate that cytoprotection induced by ICM was the prevalent mechanism for Rubus geoides and F. chiloensis . This agreed with increased levels of glyoxalase I and glutathione S-transferase activities in cells preincubated with PEEs. ORAC index indicated that F. chiloensis was the most efficient peroxyl radical scavenger. Moreover, ICM mediated by F. chiloensis was effective in protecting cells from CML accumulation in contrast to the protective effects induced by free radical scavenging. Our results indicate that although both polyphenol-mediated mechanisms can exert protective effects, ICM was the most prevalent in AGS cells. These results suggest a potential use of these native berries as functional food.

  4. Pyrrhocoricin, a proline-rich antimicrobial peptide derived from insect, inhibits the translation process in the cell-free Escherichia coli protein synthesis system.

    Science.gov (United States)

    Taniguchi, Masayuki; Ochiai, Akihito; Kondo, Hiroshi; Fukuda, Shun; Ishiyama, Yohei; Saitoh, Eiichi; Kato, Tetsuo; Tanaka, Takaaki

    2016-05-01

    Previous studies have shown that pyrrhocoricin, a proline-rich antimicrobial peptide (PrAMP), killed sensitive species in a dose-dependent manner by specifically binding to DnaK. Here, on the basis of the finding that DnaK-deficient Escherichia coli strains are susceptible to PrAMPs, we used pyrrhocoricin to investigate internal targets other than DnaK. Using conventional antibiotics (bleomycin, streptomycin, and fosfomycin) that have known modes of action, first, we validated the availability of an assay using a cell-free rapid translation system (RTS), which is an in vitro protein synthesis system based on E. coli lysate, for evaluating inhibition of protein synthesis. We found that, similarly to bleomycin and streptomycin, pyrrhocoricin inhibited GFP synthesis in RTS in a concentration-dependent manner. In addition, blockage of transcription and translation steps in RTS was individually estimated using RT-PCR after gene expression to determine mRNA products and using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the amounts of GFP expressed from purified mRNA, respectively. The results demonstrated that this inhibition of GFP synthesis by pyrrhocoricin did not occur at the transcription step but rather at the translation step, in a manner similar to that of GFP synthesis by streptomycin, an inhibitor of the translation step by causing misreading of tRNA. These results suggest that RTS is a powerful assay system for determining if antimicrobial peptides inhibit protein synthesis and its transcription and/or translation steps. This is the first study to have shown that pyrrhocoricin inhibited protein synthesis by specifically repressing the translation step. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  5. Antibiotics-free stable polyhydroxyalkanoate (PHA) production from carbon dioxide by recombinant cyanobacteria.

    Science.gov (United States)

    Akiyama, Hideo; Okuhata, Hiroshi; Onizuka, Takuo; Kanai, Shozo; Hirano, Masahiko; Tanaka, Satoshi; Sasaki, Ken; Miyasaka, Hitoshi

    2011-12-01

    A practical antibiotics-free plasmid expression system in cyanobacteria was developed by using the complementation of cyanobacterial recA null mutation with the EscherichiacolirecA gene on the plasmid. This system was applied to the production of polyhydroxyalkanoate (PHA), a biodegradable plastic, and the transgenic cyanobacteria stably maintained the pha genes for PHA production in the antibiotics-free medium, and accumulated up to 52% cell dry weight of PHA. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. Determination of free cisplatin in medium by differential pulse polarography after ultrasound and cisplatin treatment of a cancer cell culture

    International Nuclear Information System (INIS)

    Bernard, Vladan; Skorpikova, Jirina; Mornstein, Vojtech; Fojt, Lukas

    2011-01-01

    The in vitro study was carried out for detection of the cisplatin in free form and in culture medium, depending on various conditions of sonodynamic human ovarian cancer cells A2780 treatment by differential pulse polarography (DPP). For sonodynamic treatment, we used cisplatin alone and combined cisplatin/ultrasound treatments. The ultrasound exposure intensity of 1.0 and 2.0 Wcm 2 in far field for incubation periods 1, 24 and 48 h was used. The parameters of DPP measurements were - 1 s drop time, 5 mV.s -1 voltage scan rate, 50 mV modulation amplitude and negative scanning direction; platinum wire served as counter electrode and Ag|AgCl|3 M KCI as reference electrode. The results showed the dependence of free platinum quantities in culture medium on incubation time and treatment protocol. We found difference in concentration of free cisplatin between conventional application of cisplatin and sonodynamic treatment. The sonodynamic combined treatment of cisplatin and ultrasound field showed a higher cisplatin content in the culture medium than cisplatin treatment alone; a difference of 20% was observed for incubation time 48 h. The results also showed the influence of a time sequence of ultrasound and cytostatics in the sonodynamic treatment. The highest amount of free cisplatin in the solution was found for primary application of cisplatin and the subsequent ultrasound exposure. The quantity of free cisplatin increased with time, namely for time intervals 1-24 h. There was no difference between the DPP signal of cisplatin in reaction mixture containing cells in small quantities and micro-filtered mixture without cells. Thus, the DPP method is suitable for the detection and quantification of free cisplatin in the culture medium of cell suspension. Ultrasound field can be important factor during cytostatic therapy. (author)

  7. Free radical scavenging activities measured by electron spin resonance spectroscopy and B16 cell antiproliferative behaviors of seven plants.

    Science.gov (United States)

    Calliste, C A; Trouillas, P; Allais, D P; Simon, A; Duroux, J L

    2001-07-01

    In an effort to discover new antioxidant natural compounds, seven plants that grow in France (most of them in the Limousin countryside) were screened. Among these plants, was the extensively studied Vitis vinifera as reference. For each plant, sequential percolation was realized with five solvents of increasing polarities (hexane, chloroform, ethyl acetate, methanol, and water). Free radical scavenging activities were examined in different systems using electron spin resonance (ESR) spectroscopy. These assays were based on the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH), the hydroxyl radicals generated by a Fenton reaction, and the superoxide radicals generated by the X/XO system. Antiproliferative behavior was studied on B16 melanoma cells. ESR results showed that three plants (Castanea sativa, Filipendula ulmaria, and Betula pendula) possessed, for the most polar fractions (presence of phenolic compounds), high antioxidant activities in comparison with the Vitis vinifera reference. Gentiana lutea was the only one that presented a hydroxyl scavenging activity for the ethyl acetate and chloroform fractions. The antiproliferative test results showed that the same three plants are the most effective, but for the apolar fractions (chloroform and hexane).

  8. Coatings for Fuel Cell Propulsion Compressor Bearings, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — Fuel cell air handling systems require clean and contaminant-free inlet air, which dictates that oil-free, motorized, compressor/expander systems should be used....

  9. Label-free imaging of mammalian cell nucleoli by Raman microspectroscopy.

    Science.gov (United States)

    Schulze, H Georg; Konorov, Stanislav O; Piret, James M; Blades, Michael W; Turner, Robin F B

    2013-06-21

    The nucleolus is a prominent subnuclear structure whose major function is the transcription and assembly of ribosome subunits. The size of the nucleolus varies with the cell cycle, proliferation rate and stress. Changes in nucleolar size, number, chemical composition, and shape can be used to characterize malignant cells. We used spontaneous Raman microscopy as a label-free technique to examine nucleolar spatial and chemical features. Raman images of the 1003 cm(-1) phenylalanine band revealed large, well-defined subnuclear protein structures in MFC-7 breast cancer cells. The 783 cm(-1) images showed that nucleic acids were similarly distributed, but varied more in intensity, forming observable high-intensity regions. High subnuclear RNA concentrations were observed within some of these regions as shown by 809 cm(-1) Raman band images. Principal component analyses of sub-images and library spectra validated the subnuclear presence of RNA. They also revealed that an actin-like protein covaried with DNA within the nucleolus, a combination that accounted for 64% or more of the spectral variance. Embryonic stem cells are another rapidly proliferating cell type, but their nucleoli were not as large or well defined. Estimating the size of the larger MCF-7 nucleolus was used to show the utility of Raman microscopy for morphometric analyses. It was concluded that imaging based on Raman microscopy provides a promising new method for the study of nucleolar function and organization, in the evaluation of drug and experimental effects on the nucleolus, and in clinical diagnostics and prognostics.

  10. Artificial intelligence in label-free microscopy biological cell classification by time stretch

    CERN Document Server

    Mahjoubfar, Ata; Jalali, Bahram

    2017-01-01

    This book introduces time-stretch quantitative phase imaging (TS-QPI), a high-throughput label-free imaging flow cytometer developed for big data acquisition and analysis in phenotypic screening. TS-QPI is able to capture quantitative optical phase and intensity images simultaneously, enabling high-content cell analysis, cancer diagnostics, personalized genomics, and drug development. The authors also demonstrate a complete machine learning pipeline that performs optical phase measurement, image processing, feature extraction, and classification, enabling high-throughput quantitative imaging that achieves record high accuracy in label -free cellular phenotypic screening and opens up a new path to data-driven diagnosis. • Demonstrates how machine learning is used in high-speed microscopy imaging to facilitate medical diagnosis; • Provides a systematic and comprehensive illustration of time stretch technology; • Enables multidisciplinary application, including industrial, biomedical, and artificial intell...

  11. Protective effect of aqueous extract from Spirulina platensis against cell death induced by free radicals.

    Science.gov (United States)

    Chu, Wan-Loy; Lim, Yen-Wei; Radhakrishnan, Ammu Kutty; Lim, Phaik-Eem

    2010-09-21

    Spirulina is a commercial alga well known to contain various antioxidants, especially phycocyanin. Apart from being sold as a nutraceutical, Spirulina is incorporated as a functional ingredient in food products and beverages. Most of the previous reports on antioxidant activity of Spirulina were based on chemical rather than cell-based assays. The primary objective of this study was to assess the antioxidant activity of aqueous extract from Spirulina based on its protective effect against cell death induced by free radicals. The antioxidant activity of the cold water extract from food-grade Spirulina platensis was assessed using both chemical and cell-based assays. In the cell-based assay, mouse fibroblast cells (3T3) cells were incubated for 1 h in medium containing aqueous extract of Spirulina or vitamin C (positive control) at 25, 125 and 250 μg/mL before the addition of 50 μM 1,1-diphenyl-2-picrylhydrazyl (DPPH) or 3-ethylbenzothiazoline-6-sulfonic acid (ABTS). The cells were incubated for another 24 h before being assessed for cell death due to apoptosis using the Cell Death Detection ELISA Kit. Spectrophotometric assays based on DPPH and ABTS were also used to assess the antioxidant activity of the extract compared to vitamin C and vitamin E (positive controls). Spirulina extract did not cause cytotoxic effect on 3T3 cells within the range of concentrations tested (0 - 250 μg/mL). The extract reduced significantly (p assay, the radical scavenging activity of the extract was higher than phycocyanin and was at least 50% of vitamin C and vitamin E. Based on the ABTS assay, the antioxidant activity of the extract at 50 μmug/mL was as good as vitamin C and vitamin E. The results showed that aqueous extract of Spirulina has a protective effect against apoptotic cell death due to free radicals. The potential application of incorporating Spirulina into food products and beverages to enhance their antioxidant capacity is worth exploring.

  12. Cell-free assay measuring repair DNA synthesis in human fibroblasts

    International Nuclear Information System (INIS)

    Ciarrocchi, G.; Linn, S.

    1978-01-01

    Osmotic disruption of confluent cultured human fibroblasts that have been irradiated or exposed to chemical carcinogens allows the specific measurement of repair DNA synthesis using dTTP as a precursor. Fibroblasts similarly prepared from various xeroderma pigmentosum cell lines show the deficiencies of uv-induced DNA synthesis predicted from in vivo studies, while giving normal responses to methylmethanesulfonate. A pyrimidine-dimer-specific enzyme, T4 endonuclease V, stimulated the rate of uv-induced repair synthesis with normal and xeroderma pigmentosum cell lines. This system should prove useful for identifying agents that induce DNA repair, and cells that respond abnormally to such induction. It should also be applicable to an in vitro complementation assay with repair-defective cells and proteins obtained from repair-proficient cells. Finally, by using actively growing fibroblasts and thymidine in the system, DNA replication can be measured and studied in vitro

  13. In Vivo Evaluation of Biocompatibility and Chondrogenic Potential of a Cell-Free Collagen-Based Scaffold

    Directory of Open Access Journals (Sweden)

    Giovanna Calabrese

    2017-11-01

    Full Text Available Injured articular cartilage has a limited innate regenerative capacity, due to the avascular nature and low cellularity of the tissue itself. Although several approaches have been proposed to repair the joint cartilage, none of them has proven to be effective. The absence of suitable therapeutic options has encouraged tissue-engineering approaches combining specific cell types and biomaterials. In the present work, we have evaluated the potential of a cell-free Collagen I-based scaffold to promote the augmentation of cartilage-like phenotype after subcutaneous implantation in the mouse. Forty female mice were grafted subcutaneously with scaffolds, while four additional mice without scaffold were used as negative controls. The effects of scaffold were evaluated at 1, 2, 4, 8, or 16 weeks after implantation. Immunohistochemical analysis shows the expression of typical cartilage markers, including type-II Collagen, Aggrecan, Matrilin-1 and Sox 9. These data are also confirmed by qRT-PCR that further show that both COL2A1 and COL1A1 increase over time, but the first one increases more rapidly, thus suggesting a typical cartilage-like address. Histological analysis shows the presence of some pericellular lacunae, after 8 and 16 weeks. Results suggest that this scaffold (i is biocompatible in vivo, (ii is able to recruit host cells (iii induce chondrogenic differentiation of host cells. Such evidences suggest that this cell-free scaffold is promising and represents a potential approach for cartilage regeneration.

  14. Label free quantitative proteomics analysis on the cisplatin resistance in ovarian cancer cells.

    Science.gov (United States)

    Wang, F; Zhu, Y; Fang, S; Li, S; Liu, S

    2017-05-20

    Quantitative proteomics has been made great progress in recent years. Label free quantitative proteomics analysis based on the mass spectrometry is widely used. Using this technique, we determined the differentially expressed proteins in the cisplatin-sensitive ovarian cancer cells COC1 and cisplatin-resistant cells COC1/DDP before and after the application of cisplatin. Using the GO analysis, we classified those proteins into different subgroups bases on their cellular component, biological process, and molecular function. We also used KEGG pathway analysis to determine the key signal pathways that those proteins were involved in. There are 710 differential proteins between COC1 and COC1/DDP cells, 783 between COC1 and COC1/DDP cells treated with cisplatin, 917 between the COC1/DDP cells and COC1/DDP cells treated with LaCl3, 775 between COC1/DDP cells treated with cisplatin and COC1/DDP cells treated with cisplatin and LaCl3. Among the same 411 differentially expressed proteins in cisplatin-sensitive COC1 cells and cisplain-resistant COC1/DDP cells before and after cisplatin treatment, 14% of them were localized on the cell membrane. According to the KEGG results, differentially expressed proteins were classified into 21 groups. The most abundant proteins were involved in spliceosome. This study lays a foundation for deciphering the mechanism for drug resistance in ovarian tumor.

  15. The Effect of Dopant-Free Hole-Transport Polymers on Charge Generation and Recombination in Cesium-Bismuth-Iodide Solar Cells.

    Science.gov (United States)

    Zhu, Huimin; Johansson, Malin B; Johansson, Erik M J

    2018-03-22

    The photovoltaic characteristics of CsBi 3 I 10 -based solar cells with three dopant-free hole-conducting polymers are investigated. The effect on charge generation and charge recombination in the solar cells using the different polymers is studied and the results indicate that the choice of polymer strongly affects the device properties. Interestingly, for the solar cell with poly[[2,3-bis(3-octyloxyphenyl)-5,8-quinoxalinediyl]-2,5-thiophenediyl] (TQ1), the photon-to-current conversion spectrum is highly improved in the red wavelength region, suggesting that the polymer also contributes to the photocurrent generation in this case. This report provides a new direction for further optimization of Bi-halide solar cells by using dopant-free hole-transporting polymers and shows that the energy levels and the interaction between the Bi-halide and the conducting polymers are very important for solar cell performance. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Cell-free DNA in healthy individuals, noncancerous disease and strong prognostic value in colorectal cancer

    DEFF Research Database (Denmark)

    Spindler, Karen-Lise Garm; Appelt, Ane L; Pallisgaard, Niels

    2014-01-01

    The purpose was to investigate total cell-free DNA (cfDNA) in colorectal cancer (CRC) patients during treatment with second-line chemotherapy and in healthy controls and patients with different comorbidities. Patient treated with second-line irinotecan for metastatic CRC (n = 100), a cohort...

  17. [Comparison of pedicle and free tissue transfers in the German DRG system].

    Science.gov (United States)

    Lotter, O; Stahl, S; Hohenstein, C; Schaller, H-E; Jaminet, P

    2011-12-01

    Diagnosis-Related Groups (DRGs) are a patient classification system grouping related types of patients to the resources they have consumed. In this analysis, we compared pedicle and free flaps in plastic and reconstructive surgery in the actual German DRG system. After grouping common flaps while systematically modifying the diagnosis, the operative procedure(s), and the receptor site, reimbursement and thresholds of length of stay were identified. The mean value of the average length of stay was higher in free flaps as compared to pedicle flaps (15 vs. 9 days) and the mean reimbursement in free flaps was almost twice as high as in pedicle flaps (8 936 € vs. 4 582 €). Regarding the diagnosis, third-grade open fractures in pedicle flaps and full-thickness burns in free flaps are in the vanguard of reimbursement. Higher DRG conformity is generally found with free flaps. Different possibilities in coding the procedures and the strong dependence on the underlying diagnosis lead to variations of remuneration and length of stay, which are not explainable and sometimes even seem paradoxical. Furthermore, mixed calculation creates DRGs that lose the ability to describe the real effort. © Georg Thieme Verlag KG Stuttgart · New York.

  18. An Extraordinary Accumulation of (-)-Pinoresinol in Cell-Free Extracts of Forsythia intermedia: Evidence for Enantiospecific Reduction of (+)-Pinoresinol

    Science.gov (United States)

    Katayama, Takeshi; Davin, Laurence B.; Lewis, Norman G.

    1992-01-01

    Stereoselective and enantiospecific transformation mechanisms in lignan biogenesis are only now yielding to scientific inquiry: it has been shown that soluble cell-free preparations from Forsythia intermedia catalysis the formation of the enantiomerically pure lignan, (-)-secoisolariciresinol, when incubated with coniferyl alcohol in the presence of NAD(P)H and H2O2. Surprisingly, (-)-pinoresinol also accumulates in this soluble cell-free assay mixture in greater than 96% enantiomeric excess, even though it is not the naturally occurring antipode present in Forsythia sp. But these soluble cell-free preparations do not engender stereoselective coupling; instead, racemic pinoresinols are first formed, catalysed by an H2O2-dependent peroxidase reaction. An enantiospecific NAD(P)H reductase then converts (+)- pinoresinol, and not the (-)-antipode, into (-)-secoisolariciresinol. Stereoselective syntheis of(+)-pinoresinol from E-coniferyl alcohol is, however, catalysed by an insoluble enzyme preparation in F. suspensa, obtained following removal of readily soluble and ionically bound enzymes; no exogenously supplied cofactors were required other than oxygen, although the reaction was stimulated by NAD-malate addition. Thus, the overall biochemical pathway to enantiomerically pure (-)-secoisolariciresinol has been delineated.

  19. Systems biomechanics of the cell

    CERN Document Server

    Maly, Ivan V

    2013-01-01

    Systems Biomechanics of the Cell attempts to outline systems biomechanics of the cell as an emergent and promising discipline. The new field owes conceptually to cell mechanics, organism-level systems biomechanics, and biology of biochemical systems. Its distinct methodology is to elucidate the structure and behavior of the cell by analyzing the unintuitive collective effects of elementary physical forces that interact within the heritable cellular framework. The problematics amenable to this approach includes the variety of cellular activities that involve the form and movement of the cell body and boundary (nucleus, centrosome, microtubules, cortex, and membrane). Among the elementary system effects in the biomechanics of the cell, instability of symmetry, emergent irreversibility, and multiperiodic dissipative motion can be noted. Research results from recent journal articles are placed in this unifying framework. It is suggested that the emergent discipline has the potential to expand the spectrum of ques...

  20. Free radical scavenging injectable hydrogels for regenerative therapy

    Energy Technology Data Exchange (ETDEWEB)

    Komeri, Remya [Sree Chitra Tirunal Institute for Medical Sciences and Technology, Polymer Science Division, BMT Wing, Thiruvananthapuram 695 012, Kerala State (India); Thankam, Finosh Gnanaprakasam [Dept. of Biomedical Sciences, Creighton University, 2500 California Plaza, Omaha NE68178 (United States); Muthu, Jayabalan, E-mail: mjayabalan52@gmail.com [Sree Chitra Tirunal Institute for Medical Sciences and Technology, Polymer Science Division, BMT Wing, Thiruvananthapuram 695 012, Kerala State (India)

    2017-02-01

    Pathological free radicals generated from inflamed and infarcted cardiac tissues interferes natural tissue repair mechanisms. Hypoxic microenvironment at the injured zone of non-regenerating cardiac tissues hinders the therapeutic attempts including cell therapy. Here we report an injectable, cytocompatible, free radical scavenging synthetic hydrogel formulation for regenerative therapy. New hydrogel (PEAX-P) is prepared with D-xylitol-co-fumarate-co-poly ethylene adipate-co-PEG comaromer (PEAX) and PEGDiacrylate. PEAX-P hydrogel swells 4.9 times the initial weight and retains 100.07 kPa Young modulus at equilibrium swelling, which is suitable for cardiac applications. PEAX-P hydrogel retains elastic nature even at 60% compressive strain, which is favorable to fit with the dynamic and elastic natural tissue counterparts. PEAX-P hydrogel scavenges 51% DPPH radical, 40% hydroxyl radicals 41% nitrate radicals with 31% reducing power. The presence of hydrogel protects 62% cardiomyoblast cells treated with stress inducing media at LD 50 concentration. The free hydroxyl groups in sugar alcohols of the comacromer influence the free radical scavenging. Comparatively, PEAX-P hydrogel based on xylitol evinces slightly lower scavenging characteristics than with previously reported PEAM-P hydrogel containing mannitol having more hydroxyl groups. The possible free radical scavenging mechanism of the present hydrogel relies on the free π electrons associated with uncrosslinked fumarate bonds, hydrogen atoms associated with sugar alcohols/PEG and radical dilution by free water in the matrix. Briefly, the present PEAX-P hydrogel is a potential injectable system for combined antioxidant and regenerative therapy. - Graphical abstract: Injectable hydrogel with inherent free radical scavenging property for regenerative tissue engineering application. - Highlights: • Novel injectable hydrogel (PEAX-P) is prepared using D-xylitol-co-fumarate-co-poly ethylene adipate-co-PEG comaromer