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Sample records for cell free system

  1. 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

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

    African Journals Online (AJOL)

    Circulating cell free DNA as a predictor of systemic lupus erythematosus severity and monitoring of therapy. Olfat M. Hendy, Tawfik Abdel Motalib, Mona A. El Shafie, Fatma A. Khalaf, Sobhy E. Kotb, Aziza Khalil, Salwa R. Ali ...

  3. 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...

  4. Cloning and expression of the HIV protein in Escherichia coli cell-free system.

    Science.gov (United States)

    Chen, Haiqin; Xu, Zhinan; Yin, Xiufei; Cen, Peilin

    2007-11-01

    Sixteen kinds of human immunodeficiency virus (HIV) target genes were cloned by polymerase chain reaction (PCR) amplification, and specific plasmids were constructed as the templates for the expression of these genes in the cell-free system. Similarly, the linear PCR templates of these genes for cell-free protein expression were also constructed by using two PCR amplification process. These different templates can be employed to biosynthesize HIV proteins in the cell-free system simultaneously and can be adapted for some high-throughput processes. HIV protease (P10) was performed as a target protein, and two different templates (plasmid and PCR product) were prepared and used for P10 expression in the Escherichia coli cell-free system. The target protein P10 was detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels either by using a plasmid template or by a PCR template. These results are promising and helpful to develop a high throughput process for drug discovery.

  5. IRES-mediated translation of membrane proteins and glycoproteins in eukaryotic cell-free systems.

    Directory of Open Access Journals (Sweden)

    Andreas K Brödel

    Full Text Available Internal ribosome entry site (IRES elements found in the 5' untranslated region of mRNAs enable translation initiation in a cap-independent manner, thereby representing an alternative to cap-dependent translation in cell-free protein expression systems. However, IRES function is largely species-dependent so their utility in cell-free systems from different species is rather limited. A promising approach to overcome these limitations would be the use of IRESs that are able to recruit components of the translation initiation apparatus from diverse origins. Here, we present a solution to this technical problem and describe the ability of a number of viral IRESs to direct efficient protein expression in different eukaryotic cell-free expression systems. The IRES from the intergenic region (IGR of the Cricket paralysis virus (CrPV genome was shown to function efficiently in four different cell-free systems based on lysates derived from cultured Sf21, CHO and K562 cells as well as wheat germ. Our results suggest that the CrPV IGR IRES-based expression vector is universally applicable for a broad range of eukaryotic cell lysates. Sf21, CHO and K562 cell-free expression systems are particularly promising platforms for the production of glycoproteins and membrane proteins since they contain endogenous microsomes that facilitate the incorporation of membrane-spanning proteins and the formation of post-translational modifications. We demonstrate the use of the CrPV IGR IRES-based expression vector for the enhanced synthesis of various target proteins including the glycoprotein erythropoietin and the membrane proteins heparin-binding EGF-like growth factor receptor as well as epidermal growth factor receptor in the above mentioned eukaryotic cell-free systems. CrPV IGR IRES-mediated translation will facilitate the development of novel eukaryotic cell-free expression platforms as well as the high-yield synthesis of desired proteins in already established

  6. IRES-Mediated Translation of Membrane Proteins and Glycoproteins in Eukaryotic Cell-Free Systems

    Science.gov (United States)

    Brödel, Andreas K.; Sonnabend, Andrei; Roberts, Lisa O.; Stech, Marlitt; Wüstenhagen, Doreen A.; Kubick, Stefan

    2013-01-01

    Internal ribosome entry site (IRES) elements found in the 5′ untranslated region of mRNAs enable translation initiation in a cap-independent manner, thereby representing an alternative to cap-dependent translation in cell-free protein expression systems. However, IRES function is largely species-dependent so their utility in cell-free systems from different species is rather limited. A promising approach to overcome these limitations would be the use of IRESs that are able to recruit components of the translation initiation apparatus from diverse origins. Here, we present a solution to this technical problem and describe the ability of a number of viral IRESs to direct efficient protein expression in different eukaryotic cell-free expression systems. The IRES from the intergenic region (IGR) of the Cricket paralysis virus (CrPV) genome was shown to function efficiently in four different cell-free systems based on lysates derived from cultured Sf21, CHO and K562 cells as well as wheat germ. Our results suggest that the CrPV IGR IRES-based expression vector is universally applicable for a broad range of eukaryotic cell lysates. Sf21, CHO and K562 cell-free expression systems are particularly promising platforms for the production of glycoproteins and membrane proteins since they contain endogenous microsomes that facilitate the incorporation of membrane-spanning proteins and the formation of post-translational modifications. We demonstrate the use of the CrPV IGR IRES-based expression vector for the enhanced synthesis of various target proteins including the glycoprotein erythropoietin and the membrane proteins heparin-binding EGF-like growth factor receptor as well as epidermal growth factor receptor in the above mentioned eukaryotic cell-free systems. CrPV IGR IRES-mediated translation will facilitate the development of novel eukaryotic cell-free expression platforms as well as the high-yield synthesis of desired proteins in already established systems. PMID

  7. IRES-mediated translation of membrane proteins and glycoproteins in eukaryotic cell-free systems.

    Science.gov (United States)

    Brödel, Andreas K; Sonnabend, Andrei; Roberts, Lisa O; Stech, Marlitt; Wüstenhagen, Doreen A; Kubick, Stefan

    2013-01-01

    Internal ribosome entry site (IRES) elements found in the 5' untranslated region of mRNAs enable translation initiation in a cap-independent manner, thereby representing an alternative to cap-dependent translation in cell-free protein expression systems. However, IRES function is largely species-dependent so their utility in cell-free systems from different species is rather limited. A promising approach to overcome these limitations would be the use of IRESs that are able to recruit components of the translation initiation apparatus from diverse origins. Here, we present a solution to this technical problem and describe the ability of a number of viral IRESs to direct efficient protein expression in different eukaryotic cell-free expression systems. The IRES from the intergenic region (IGR) of the Cricket paralysis virus (CrPV) genome was shown to function efficiently in four different cell-free systems based on lysates derived from cultured Sf21, CHO and K562 cells as well as wheat germ. Our results suggest that the CrPV IGR IRES-based expression vector is universally applicable for a broad range of eukaryotic cell lysates. Sf21, CHO and K562 cell-free expression systems are particularly promising platforms for the production of glycoproteins and membrane proteins since they contain endogenous microsomes that facilitate the incorporation of membrane-spanning proteins and the formation of post-translational modifications. We demonstrate the use of the CrPV IGR IRES-based expression vector for the enhanced synthesis of various target proteins including the glycoprotein erythropoietin and the membrane proteins heparin-binding EGF-like growth factor receptor as well as epidermal growth factor receptor in the above mentioned eukaryotic cell-free systems. CrPV IGR IRES-mediated translation will facilitate the development of novel eukaryotic cell-free expression platforms as well as the high-yield synthesis of desired proteins in already established systems.

  8. Cell-Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems.

    Science.gov (United States)

    Zemella, Anne; Thoring, Lena; Hoffmeister, Christian; Kubick, Stefan

    2015-11-01

    From its start as a small-scale in vitro system to study fundamental translation processes, cell-free protein synthesis quickly rose to become a potent platform for the high-yield production of proteins. In contrast to classical in vivo protein expression, cell-free systems do not need time-consuming cloning steps, and the open nature provides easy manipulation of reaction conditions as well as high-throughput potential. Especially for the synthesis of difficult to express proteins, such as toxic and transmembrane proteins, cell-free systems are of enormous interest. The modification of the genetic code to incorporate non-canonical amino acids into the target protein in particular provides enormous potential in biotechnology and pharmaceutical research and is in the focus of many cell-free projects. Many sophisticated cell-free systems for manifold applications have been established. This review describes the recent advances in cell-free protein synthesis and details the expanding applications in this field. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  9. Cell-free protein synthesis: Pros and cons of prokaryotic and eukaryotic systems. Review

    OpenAIRE

    Zemella, Anne; Thoring, Lena; Hoffmeister, Christian; Kubick, Stefan

    2015-01-01

    From its start as a small-scale in vitro system to study fundamental translation processes, cell-free protein synthesis quickly rose to become a potent platform for the high-yield production of proteins. In contrast to classical in vivo protein expression, cell-free systems do not need time-consuming cloning steps, and the open nature provides easy manipulation of reaction conditions as well as high-throughput potential. Especially for the synthesis of difficult to express proteins, such as t...

  10. A highly controllable reconstituted cell-free system--a breakthrough in protein synthesis research.

    Science.gov (United States)

    Ohashi, Hiroyuki; Kanamori, Takashi; Shimizu, Yoshihiro; Ueda, Takuya

    2010-04-01

    The PURE system is a highly controllable cell-free protein synthesis system composed of individually prepared components that are required for protein synthesis in Escherichia coli. The PURE system contains neither nucleases nor proteases, both of which degrade DNA or mRNA templates and proteins. The protein products are easily purified using affinity chromatography to remove the tagged protein factors. The PURE system should help to create new fields in protein research.

  11. New bioproduction systems: from molecular circuits to novel reactor concepts in cell-free biotechnology.

    Science.gov (United States)

    Rupp, Steffen

    2013-01-01

    : The last decades witnessed a strong growth in several areas of biotechnology, especially in fields related to health, as well as in industrial biotechnology. Advances in molecular engineering now enable biotechnologists to design more efficient pathways in order to convert a larger spectrum of renewable resources into industrially used biofuels and chemicals as well as into new pharmaceuticals and therapeutic proteins. In addition material sciences advanced significantly making it more and more possible to integrate biology and engineering. One of the key questions currently is how to develop new ways of engineering biological systems to cope with the complexity and limitations given by the cell. The options to integrate biology with classical engineering advanced cell free technologies in the recent years significantly. Cell free protein production using cellular extracts is now a well-established universal technology for production of proteins derived from many organisms even at the milligram scale. Among other applications it has the potential to supply the demand for a multitude of enzymes and enzyme variants facilitating in vitro metabolic engineering. This review will briefly address the recent achievements and limitations of cell free conversions. Especially, the requirements for reactor systems in cell free biotechnology, a currently underdeveloped field, are reviewed and some perspectives are given on how material sciences and biotechnology might be able to advance these new developments in the future.

  12. 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.

  13. Expression of Trans-Membrane Proteins in vitro Using a Cell Free System

    Science.gov (United States)

    Weisse, Natalie; Noireaux, Vincent; Chalmeau, Jerome

    2010-10-01

    Trans-membrane proteins represent a significant portion of the proteins expressed by cells. The expression of proteins in vitro, however, remains a challenge. Numerous expression approaches have been developed with cell free expression (CFE) being one of the most promising. CFE is based on a transcription-translation system that has been extracted from E. coli bacteria. Adding the desired DNA allows expression of a selected protein, and in the presence of phospholipids the expression of trans-membrane proteins becomes possible. In order to express trans-membrane proteins in a closed native environment, the cell free system (CFS) is encapsulated with a phospholipid bilayer, creating an artificial cell. To verify protein expression, AquaporinZ (AqpZ), a well-known trans-membrane protein tagged with a green fluorescent protein (eGFP), was used so the expressed proteins could be seen under a fluorescent microscope. These artificial cells will serve as an experimental platform for testing the viability of the expressed trans-membrane proteins. Results from the manipulation of these artificial cells by attaching them to the slide surface through streptavidin-biotin bonding will be presented.

  14. 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.

  15. 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.

  16. Generation of transgene-free mouse induced pluripotent stem cells using an excisable lentiviral system.

    Science.gov (United States)

    Varga, E; Nemes, C; Davis, R P; Ujhelly, O; Klincumhom, N; Polgar, Z; Muenthaisong, S; Pirity, M K; Dinnyes, A

    2014-04-01

    One goal of research using induced pluripotent stem cell (iPSC) is to generate patient-specific cells which can be used to obtain multiple types of differentiated cells as disease models. Minimally or non-integrating methods to deliver the reprogramming genes are considered to be the best but they may be inefficient. Lentiviral delivery is currently among the most efficient methods but it integrates transgenes into the genome, which may affect the behavior of the iPSC if integration occurs into an important locus. Here we designed a polycistronic lentiviral construct containing four pluripotency genes with an EGFP selection marker. The cassette was excisable with the Cre-loxP system making possible the removal of the integrated transgenes from the genome. Mouse embryonic fibroblasts were reprogrammed using this viral system, rapidly resulting in large number of iPSC colonies. Based on the lowest EGFP expression level, one parental line was chosen for excision. Introduction of the Cre recombinase resulted in transgene-free iPSC subclones. The effect of the transgenes was assessed by comparing the parental iPSC with two of its transgene-free subclones. Both excised and non-excised iPSCs expressed standard pluripotency markers. The subclones obtained after Cre recombination were capable of differentiation in vitro, in contrast to the parental, non-excised cells and formed germ-line competent chimeras in vivo. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Coarse-Grained Dynamics of Protein Synthesis in a Cell-Free System

    Science.gov (United States)

    Karzbrun, Eyal; Shin, Jonghyeon; Bar-Ziv, Roy H.; Noireaux, Vincent

    2011-01-01

    A complete gene expression reaction is reconstituted in a cell-free system comprising the entire endogenous transcription, translation, as well as mRNA and protein degradation machinery of E. coli. In dissecting the major reaction steps, we derive a coarse-grained enzymatic description of biosynthesis and degradation, from which ten relevant rate constants and concentrations are determined. Governed by zeroth-order degradation, protein expression follows a sharp transition from undetectable levels to constant-rate accumulation, without reaching steady state.

  18. The good of two worlds: increasing complexity in cell-free systems.

    Science.gov (United States)

    Billerbeck, Sonja; Härle, Johannes; Panke, Sven

    2013-12-01

    In vitro biocatalytic systems have moved far beyond established uses in food, diagnostic, and chemical applications. As new strategies to construct and manage multiple enzymes in ever more complex systems are developed, novel applications emerge. In the field of chemistry, complex protein networks are applied to enable the production of fine chemicals, such as dihydroxyacetone phosphate, and even bulk chemicals, such as biofuels, from cheap sugars. Cell-free protein synthesis is applied to expanding protein and nucleic acid biochemistry and enabling novel assay formats, while programmable DNA-circuits can be exploited to engineer sensitive detection methods. Novel developments in chemical analytics such as real-time mass spectrometry to follow the metabolism online, directed physical assembly of network members facilitating substrate channeling, and encapsulation forming biofunctional subunits enable a better control and potential for optimization. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Scavenging of superoxide anion by phosphorylethanolamine: studies in human neutrophils and in a cell free system.

    Science.gov (United States)

    Gordon, L I; Weiss, D; Prachand, S; Weitzman, S A

    1991-01-01

    On the basis of previous observations, we attempted to characterize the effects of various products of phospholipid hydrolysis on neutrophil (PMN) respiratory burst activity. We studied the effects of phosphorylcholine (PC) and phosphorylethanoline (PE) on superoxide anion production in PMN and in cell free system. We found that PE but not PC inhibited measured superoxide anion, but that this was not due to inhibition of cellular superoxide generation but to scavenging of generated superoxide anion. Further, utilizing a system based upon the photo-oxidation of O-dianisidine sensitized by riboflavin, we were able to determine that the scavenging effect of PE was not superoxide dismutase (SOD)-like but rather a general scavenging or glutathione (GSH)-like effect. These data underscore the importance of identifying the mechanism of inhibition of superoxide generation by putative inhibitors as being due to a direct cellular effect or to a scavenging property.

  20. Site-Specific Cleavage of Ribosomal RNA in Escherichia coli-Based Cell-Free Protein Synthesis Systems.

    Directory of Open Access Journals (Sweden)

    Jurek Failmezger

    Full Text Available Cell-free protein synthesis, which mimics the biological protein production system, allows rapid expression of proteins without the need to maintain a viable cell. Nevertheless, cell-free protein expression relies on active in vivo translation machinery including ribosomes and translation factors. Here, we examined the integrity of the protein synthesis machinery, namely the functionality of ribosomes, during (i the cell-free extract preparation and (ii the performance of in vitro protein synthesis by analyzing crucial components involved in translation. Monitoring the 16S rRNA, 23S rRNA, elongation factors and ribosomal protein S1, we show that processing of a cell-free extract results in no substantial alteration of the translation machinery. Moreover, we reveal that the 16S rRNA is specifically cleaved at helix 44 during in vitro translation reactions, resulting in the removal of the anti-Shine-Dalgarno sequence. These defective ribosomes accumulate in the cell-free system. We demonstrate that the specific cleavage of the 16S rRNA is triggered by the decreased concentrations of Mg2+. In addition, we provide evidence that helix 44 of the 30S ribosomal subunit serves as a point-of-entry for ribosome degradation in Escherichia coli. Our results suggest that Mg2+ homeostasis is fundamental to preserving functional ribosomes in cell-free protein synthesis systems, which is of major importance for cell-free protein synthesis at preparative scale, in order to create highly efficient technical in vitro systems.

  1. 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

  2. A validated system for ligation-free USER™ -based assembly of expression vectors for mammalian cell engineering

    DEFF Research Database (Denmark)

    Lund, Anne Mathilde; Kildegaard, Helene Faustrup; Hansen, Bjarne Gram

    The development in the field of mammalian cell factories require fast and high-throughput methods, this means a high need for simpler and more efficient cloning techniques. For optimization of protein expression by genetic engineering and for allowing metabolic engineering in mammalian cells, a n...... versatile expression vector system was developed. This vector system applies the ligation-free uracilexcision cloning technique to construct mammalian expression vectors of multiple parts and with maximum flexibility....

  3. Expression of Green Fluorescent Protein (GFP using In Vitro translation cell free system

    Directory of Open Access Journals (Sweden)

    M Mohamadipoor

    2009-03-01

    Full Text Available ABSTRACT Background and the purpose of the study: One of the major concerns about recombinant protein production is its possible toxicity for the organism. Purification of the recombinant protein is another challenge in this respect. Recently In Vitro translation cell free system that provides a coupled transcription-translation reaction for protein synthesis to overcome the above mentioned problems has been emerged. The aim of this study was expression of GFP as a marker for gene expression and protein in In Vitro translation system. Methods: pIVEX2.3-GFP plasmid was cloned to E. coli   and the plasmid DNA extracted. In Vitro translation was performed with RTS 100 E. coli Hy kit according to manufacture's instructions. Expression of recombinant fusion protein, His- GFP, was determined by SDS-PAGE, ELISA and western blot analysis. Results: Expected size of recombinant protein was detected in SDS-PAGE and further confirmed by western blot analysis and ELISA. Major conclusion: Results showed that In Vitro translation is suitable for expression of recombinant protein and fusion of the recombinant protein with His-tag facilitates the purification.

  4. Study of messenger RNA inactivation and protein degradation in an Escherichia coli cell-free expression system

    Directory of Open Access Journals (Sweden)

    Noireaux Vincent

    2010-07-01

    Full Text Available Abstract Background A large amount of recombinant proteins can be synthesized in a few hours with Escherichia coli cell-free expression systems based on bacteriophage transcription. These cytoplasmic extracts are used in many applications that require large-scale protein production such as proteomics and high throughput techniques. In recent years, cell-free systems have also been used to engineer complex informational processes. These works, however, have been limited by the current available cell-free systems, which are not well adapted to these types of studies. In particular, no method has been proposed to increase the mRNA inactivation rate and the protein degradation rate in cell-free reactions. The construction of in vitro informational processes with interesting dynamics requires a balance between mRNA and protein synthesis (the source, and mRNA inactivation and protein degradation (the sink. Results Two quantitative studies are presented to characterize and to increase the global mRNA inactivation rate, and to accelerate the degradation of the synthesized proteins in an E. coli cell-free expression system driven by the endogenous RNA polymerase and sigma factor 70. The E. coli mRNA interferase MazF was used to increase and to adjust the mRNA inactivation rate of the Firefly luciferase (Luc and of the enhanced green fluorescent protein (eGFP. Peptide tags specific to the endogenous E. coli AAA + proteases were used to induce and to adjust the protein degradation rate of eGFP. Messenger RNA inactivation rate, protein degradation rate, maturation time of Luc and eGFP were measured. Conclusions The global mRNA turnover and the protein degradation rate can be accelerated and tuned in a biologically relevant range in a cell-free reaction with quantitative procedures easy to implement. These features broaden the capabilities of cell-free systems with a better control of gene expression. This cell-free extract could find some applications in

  5. 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

  6. Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

    Science.gov (United States)

    Shin, Jonghyeon; Caschera, Filippo; Murray, Richard M.; Noireaux, Vincent

    2013-01-01

    Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard." PMID:24084388

  7. 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.

  8. Evaluation of human MRC-5 cells as a feeder layer in a xenobiotic-free culture system for conjunctival epithelial progenitor cells.

    Science.gov (United States)

    Schrader, Stefan; Tuft, Stephen J; Beaconsfield, Michele; Borrelli, Maria; Geerling, Gerd; Daniels, Julie T

    2012-12-01

    Aim of this study was to evaluate the efficiency of human MRC-5 cells to act as a feeder layer for conjunctival epithelial cells in order to develop a complete xenobiotic-free culture system for the expansion of conjunctival epithelial progenitor cells for clinical applications. Human conjunctival epithelial cells were expanded from a bulbar biopsy, in a completely xenobiotic-free culture system using growth arrested MRC-5 cells as a feeder layer, without (MRC-5/No Serum) and in the presence of 5% (MRC-5/HS 5%) or 10% (MRC-5/HS 10%) human serum. The total cell count, the surface area as well as the total colony-forming efficiency (CFE), the percentage of aborted colonies and the expression of putative progenitor cell markers p63α, ABCG2, CK15 was compared to the gold standard culture system (GS) in which growth arrested 3T3 feeder cells and feotal calf serum were used. The epithelial cell count revealed significantly less proliferation in the MRC-5/No Serum group compared to the GS conditions. All groups showed immunoreactivity to CK19; however, more differentiated epithelial cells were observed in the MRC-5/No Serum- and MRC-5/HS 10%-group and less immunoreactivity to p63 α and ABCG2 was found in these groups compared to GS and MRC-5/HS 5% conditions. This was in accordance with CFE results, were the MRC-5/HS 5% group showed similar CFE results compared to the GS group, while in the MRC-5/No Serum- and MRC-5/HS 10%-group significantly lower CFE's were observed. Our results indicate that a completely xenobiotic-free culture system using MRC-5 cells as a feeder layer in combination with human serum can be successfully used to expand conjunctival epithelial cells with progenitor cell characteristics and might be a useful tool for the safe expansion of these cells for clinical use.

  9. The circulating cell-free microRNA profile in systemic sclerosis is distinct from both healthy controls and systemic lupus erythematosus

    DEFF Research Database (Denmark)

    Steen, Samantha O; Iversen, Line V; Carlsen, Anting Liu

    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...

  10. Defining minimum essential factors to derive highly pure human endothelial cells from iPS/ES cells in an animal substance-free system.

    Science.gov (United States)

    Wu, Yu-Ting; I-Shing Yu; Tsai, Kuen-Jer; Shih, Chien-Yu; Hwang, Shiaw-Min; Su, Ih-Jen; Chiang, Po-Min

    2015-04-13

    It is desirable to obtain unlimited supplies of endothelial cells for research and therapeutics. However, current methods of deriving endothelial cells from humans suffer from issues, such as limited supplies, contamination from animal substances, and lengthy/complicated procedures. In this article we developed a way to differentiate human iPS and ES cells to highly pure endothelial cells in 5 days. The chemically defined system is robust, easy to perform, and free of animal substances. Using the system, we verified that combined TGFβ and canonical Wnt agonists are essential and sufficient for iPS/ES cell-to-mesoderm transition. Besides, VEGF-KDR signaling alone is required for endothelial formation at high density while supplementation with FGF allows for colonial endothelial differentiation. Finally, anti-adsorptive agents could enrich the endothelial output by allowing selective attachment of the endothelial precursors. The system was validated to work on multiple iPS/ES cells lines to produce endothelial cells capable of forming capillary-like structures in vitro and integrating into host vasculature in vivo. In sum, the simple yet robust differentiation system permits the unlimited supply of human endothelial cells. The defined and animal substance-free nature of the system is compatible with clinical applications and characterization of endothelial differentiation in an unbiased manner.

  11. 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.

  12. Systems Biocatalysis: Development and engineering of cell-free "artificial metabolisms" for preparative multi-enzymatic synthesis.

    Science.gov (United States)

    Fessner, Wolf-Dieter

    2015-12-25

    Systems Biocatalysis is an emerging concept of organizing enzymes in vitro to construct complex reaction cascades for an efficient, sustainable synthesis of valuable chemical products. The strategy merges the synthetic focus of chemistry with the modular design of biological systems, which is similar to metabolic engineering of cellular production systems but can be realized at a far lower level of complexity from a true reductionist approach. Such operations are free from material erosion by competing metabolic pathways, from kinetic restrictions by physical barriers and regulating circuits, and from toxicity problems with reactive foreign substrates, which are notorious problems in whole-cell systems. A particular advantage of cell-free concepts arises from the inherent opportunity to construct novel biocatalytic reaction systems for the efficient synthesis of non-natural products ("artificial metabolisms") by using enzymes specifically chosen or engineered for non-natural substrate promiscuity. Examples illustrating the technology from our laboratory are discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Artificial OFF-Riboswitches That Downregulate Internal Ribosome Entry without Hybridization Switches in a Eukaryotic Cell-Free Translation System.

    Science.gov (United States)

    Ogawa, Atsushi; Masuoka, Hiroki; Ota, Tsubasa

    2017-09-15

    We constructed novel artificial riboswitches that function in a eukaryotic translation system (wheat germ extract), by rationally implanting an in vitro-selected aptamer into the intergenic internal ribosome entry site (IRES) of Plautia stali intestine virus. These eukaryotic OFF-riboswitches (OFF-eRSs) ligand-dose-dependently downregulate IRES-mediated translation without hybridization switches, which typical riboswitches utilize for gene regulation. The hybridization-switch-free mechanism not only allows for easy design but also requires less energy for regulation, resulting in a higher switching efficiency than hybridization-switch-based OFF-eRSs provide. In addition, even a small ligand such as theophylline can induce satisfactory repression, in contrast to other types of OFF-eRSs that modulate the 5' cap-dependent canonical translation. Because our proposed hybridization-switch-free OFF-eRSs are based on a versatile IRES that functions well in many types of eukaryotic translation systems, they would be widely usable elements for synthetic gene circuits in both cell-free and cell-based synthetic biology.

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

    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...

  15. 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...

  16. 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...

  17. 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.

  18. Development of a xeno-free non-contact co-culture system for derivation and maintenance of embryonic stem cells using a novel human endometrial cell line.

    Science.gov (United States)

    Desai, Nina; Ludgin, Jennifer; Goldberg, Jeffrey; Falcone, Tommaso

    2013-06-01

    Mouse embryonic fibroblast feeder layers (MEF) have conventionally been used to culture and maintain the pluripotency of embryonic stem cells (ESC). This study explores the potential of using a novel human endometrial cell line to develop a non-xeno, non-contact co-culture system for ESC propagation and derivation. Such xeno-free systems may prove essential for the establishment of clinical grade human ESC lines suitable for therapeutic application. A novel line of human endometrial cells were seeded in a 6-well dish. Filter inserts containing mouse ESCs were placed on these wells and passaged 2-3 times per week. Inner cell masses derived from mouse blastocysts were also cultured on transwells in the presence of the feeder layer. In both cases, staining for SSEA-1, SOX-2, OCT-4 and alkaline phosphatase were used to monitor the retention of stem cells. ESC colonies retained their stem cell morphology and attributes for over 120 days in culture and 44 passages to date. Inner cell mass derived ESC cultures were maintained in a pluripotent state for 45 days, through 6 passages with retention of all stem cell characteristics. The stem cell colonies expressed stem cell specific markers SSEA-1, Sox 2, Oct-4 and alkaline phosphatase. Upon removal of the human feeder layer, there was a distinct change in cell morphology within the colonies and evidence of ESC differentiation. Human feeder layers offer a simple path away from the use of MEF feeder cells or MEF conditioned medium for ESC culture. Furthermore, indirect co-culture using porous membranes to separate the two cell types can prevent contamination of stem cell preparations with feeder cells during passaging.

  19. 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.

  20. A miRNA-responsive cell-free translation system facilitates isolation of hepatitis C virus miRNP complexes.

    Science.gov (United States)

    Bradrick, Shelton S; Nagyal, Simardeep; Novatt, Hilary

    2013-08-01

    Micro(mi)RNAs are 21- to 23-nt RNAs that regulate multiple biological processes. In association with Argonaute (Ago) proteins and other factors that form the RNA-induced silencing complex (RISC), miRNAs typically bind mRNA 3' untranslated regions (UTRs) and repress protein production through antagonizing translation and transcript stability. For a given mRNA-miRNA interaction, cis-acting RNA elements and trans-acting RNA-binding proteins (RBPs) may influence mRNA fate. This is particularly true of the hepatitis C virus (HCV) genome which interacts with miR-122, an abundant liver miRNA. miR-122 binding to HCV RNA considerably stimulates virus replication in cultured cells and primates, but the mechanism(s) and associated host factors required for enhancement of HCV replication have not been fully elucidated. We recapitulated miR-122-HCV RNA interactions in a cell-free translation system derived from cells that express miR-122. Specifically, lysates produced from HEK-293 cells that inducibly transcribe and process pri-miR-122 were characterized alongside those from isogenic cells lacking miR-122 expression. We observed a stimulatory effect of miR-122 on HCV reporter mRNAs in a manner that depended on expression of miR-122 and intact target sites within the HCV 5' UTR. We took advantage of this system to affinity-purify miR-122-HCV RNP complexes. Similar to functional assays, we found that association of immobilized HCV internal ribosome entry site (IRES) RNA with endogenous Ago2 requires both miR-122 expression and intact miR-122 target sites in cis. This combined approach may be generalizable to affinity purification of miRNP complexes for selected target mRNAs, allowing identification of miRNP components and RBPs that may contribute to regulation.

  1. Impact of Cu(2+) ions on the structure of colistin and cell-free system nucleic acid degradation.

    Science.gov (United States)

    Stokowa-Sołtys, Kamila; Kasprowicz, Aleksandra; Wrzesiński, Jan; Ciesiołka, Jerzy; Gaggelli, Nicola; Gaggelli, Elena; Valensin, Gianni; Jeżowska-Bojczuk, Małgorzata

    2015-10-01

    Colistin and transition metal ions are commonly used as feed additives for livestock animals. This work presents the results of an analysis of combined potentiometric and spectroscopic (UV-vis, EPR, CD, NMR) data which lead to conclude that colistin is able to effectively chelate copper(II) ions. In cell-free system the oxidative activity of the complex manifests itself in the plasmid DNA destruction with simultaneous generation of reactive OH species, when accompanied by hydrogen peroxide or ascorbic acid. The degradation of RNA occurs most likely via a hydrolytic mechanism not only for complexed compound but also colistin alone. Therefore, huge amounts of the used antibiotic for nontherapeutic purposes might have a potential influence on livestock health. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Repair of X-ray-induced single-strand breaks by a cell-free system

    International Nuclear Information System (INIS)

    Seki, Shuji; Ikeda, Shogo; Tsutui, Ken; Teraoka, Hirobumi

    1990-01-01

    Repair of X-ray-induced single-strand breaks of DNA was studied in vitro using an exonuclease purified from mouse ascites sarcoma (SR-C3H/He) cells. X-ray-dose-dependent unscheduled DNA synthesis was primed by the exonuclease. Repair of X-ray-induced single-strand breaks in pUC19 plasmid DNA was demonstrated by agarose gel electrophoresis after incubating the damaged DNA with the exonuclease, DNA polymerase (Klenow fragment of DNA polymerase I or DNA polymerase β purified from SR-C3H/He cells), four deoxynucleoside triphosphates, ATP and DNA ligase (T4 DNA ligase or DNA ligase I purified from calf thymus). The present results suggested that the exonuclease is involved in the initiation of repair of X-ray-induced single-strand breaks in removing 3' ends of X-ray-damaged DNA. (author)

  3. A New Cell-Free System to Study BRCA1 Function

    Science.gov (United States)

    2016-06-01

    are BRCA1-dependent will be functionally characterized. For example, MCM2-7 with the ubiquitylation site(s) mutated to arginine will be expressed in...any) are functional for DNA replication. Once a functional MCM2-7 complex is obtained, we will mutate key lysine residues to identify those whose...resection by antagonizing 53BP1. More recently, they discovered that loss of 53BP1 does not rescue the ICL sensitivity observed in BRCA1-deficient cells

  4. A combined cell-free transcription-translation system from Saccharomyces cerevisiae for rapid and robust protein synthe.

    Science.gov (United States)

    Gan, Rui; Jewett, Michael C

    2014-05-01

    Cell-free protein synthesis (CFPS) provides a valuable platform for understanding, using, and expanding the capabilities of the translation apparatus. For example, high-throughput CFPS is helping to address the increasing discrepancy between genome sequence data and their translation products. Here, we report the development of a combined cell-free transcription-translation (Tx/Tl) system from Saccharomyces cerevisiae that is suitable for such efforts. First, we show the ability to enable translation initiation in a cap-independent manner. The performance of various genetic elements was assessed, including 5'-UTR, 3'-UTR, and length of poly(A) tail. A specific vector harboring the 5'-UTR fragment of the Ω sequence from the tobacco mosaic virus and a poly(A) tail of 50 nucleotides led to optimal performance. Second, we developed a simple, two-step polymerase chain reaction (PCR) method for high-throughput production of linear templates for yeast CFPS. This procedure allows all functional elements needed for Tx/Tl to be added to an open-reading frame directly by overlap extension PCR. Our two-step PCR method was successfully applied to three reporter proteins: luciferase, green fluorescence protein, and chloramphenicol acetyl transferase, yielding 7 to 12.5 μg mL-1 active protein after 1.5-h batch reactions. Surprisingly, the linear templates outperformed plasmid DNA by up to 60%. Hence, the presented CFPS method has the potential to rapidly prepare tens to thousands of DNA templates without time-consuming cloning work. Further, it holds promise for fast and convenient optimization of expression constructs, study of internal ribosome entry site, and production of protein libraries for genome-scale studies. See accompanying commentary by Russ and Dueber DOI: 10.1002/biot.201400071. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. 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.

  6. Engineering of ribosomal shunt-modulating eukaryotic ON riboswitches by using a cell-free translation system.

    Science.gov (United States)

    Ogawa, Atsushi

    2015-01-01

    A number of natural and artificial bacterial riboswitches have been reported thus far. However, they generally function only in bacteria, not in eukaryotes. This is because of the differences of expression mechanisms (transcription, translation, and so on) between these two main types of organisms. For example, the mechanism of translation initiation is quite different between bacteria and eukaryotes, especially in ribosome loading on mRNA. While the bacterial ribosome binds to a well-conserved, internal sequence some bases before the start codon to initiate translation, the eukaryotic one is loaded on the 5' terminus with the help of certain eukaryotic initiation factors. This means not only that bacterial riboswitches regulating translation initiation are not available in eukaryotic translation systems, but also that it is physically difficult to construct eukaryotic ON riboswitches that regulate the eukaryotic canonical translation initiation, because an aptamer cannot be inserted upstream of the ribosome loading site. However, the mechanism of noncanonical translation initiation via "ribosomal shunt" enables us to design translation initiation-modulating (specifically, ribosomal shunt-modulating) eukaryotic ON riboswitches. This chapter describes a facile method for engineering these ribosomal shunt-modulating eukaryotic ON riboswitches by using a cell-free translation system. Because these riboswitches do not require hybridization switching thanks to a unique shunting mechanism, they have the major advantages of a low energy requirement for upregulation and relatively straightforward design over common hybridization switch-based ON riboswitches. © 2015 Elsevier Inc. All rights reserved.

  7. 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

  8. 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...

  9. 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

    The incidence of cancer is increasing worldwide and metastatic disease, through the spread of circulating tumor cells (CTCs), is responsible for the majority of the cancer deaths. Accurate monitoring of CTC levels in blood provides clinical information supporting therapeutic decision making, and ...

  10. Enhanced functional expression of aquaporin Z via fusion of in situ cleavable leader peptides in Escherichia coli cell-free system.

    Science.gov (United States)

    Zhang, Xu; Lian, Jiazhang; Kai, Lei; Huang, Lei; Cen, Peilin; Xu, Zhinan

    2014-02-05

    Aquaporin Z (AqpZ) is a water channel protein from Escherichia coli and has attracted many attentions to develop the biomimetic water filtration technology. Cell-free protein synthesis (CFPS) system, one of the most complex multi-enzymatic systems, has the ability of producing the integral membrane protein in vitro. To enhance the synthesis of AqpZ in E. coli cell-free system, several natural leader peptides were respectively fused at the N-terminus and were verified to enhance the expression level significantly. Moreover, the supplementation of detergents or liposome could activate leader peptidase from the cell-free extract and provide hydrophobic environment for proper folding of AqpZ. Thus, the release of mature AqpZ via the in situ removal of leader peptide was achieved, with a specific water transport activity of (2.1 ± 0.1) × 10⁻¹⁴ cm³ s⁻¹ monomer⁻¹. Using this in situ removable leader peptide strategy, the transcription-translation, leader sequence cleavage and membrane protein folding were integrated into a simple process in the cell-free system, providing a convenient approach to enhance the expression of target proteins, especially those membrane proteins difficult to achieve. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Antioxidant effectiveness of coffee extracts and selected constituents in cell-free systems and human colon cell lines.

    Science.gov (United States)

    Bakuradze, Tamara; Lang, Roman; Hofmann, Thomas; Stiebitz, Herbert; Bytof, Gerhard; Lantz, Ingo; Baum, Matthias; Eisenbrand, Gerhard; Janzowski, Christine

    2010-12-01

    Epidemiological studies suggest that coffee can reduce the risk of degenerative diseases such as diabetes type 2, cardiovascular disease and cancer. These beneficial effects have partly been attributed to the antioxidant activity of coffee. We determined composition and antioxidant potential of differentially roasted coffee extracts and investigated the impact of selected original constituents and roast products. Parameters studied were direct antioxidant activity (trolox equivalent antioxidant capacity/oxygen radical absorbing capacity), cellular reactive oxygen species (ROS) level, DNA damage and protein expression of NAD(P)H: quinone oxidoreductase, γ-glutamylcysteine ligase and glutathione reductase in HT-29/Caco-2 cells at 24-h incubation. All extracts showed distinct direct antioxidant activity: medium roasts>light roast AB1 (caffeoylquinic acid (CQA)-rich Arabica Brazil extract); dark roast AB2 (N-methylpyridinium (NMP)-rich Arabica Brazil extract), and diminished t-butylhydroperoxide-induced ROS level in HT-29 cells (AB2>medium roasts>AB1). NAD(P)H:quinone oxidoreductase 1 expression and γ-glutamylcysteine ligase expression were distinctly induced by AB1 and 5-CQA, but not by AB2 and NMP. 5-CQA and caffeic acid exhibited highest trolox equivalent antioxidant capacity/oxygen radical absorbing capacity values (5-CQA: 1.3/3.5 mM and caffeic acid: 1.3/3.9 mM trolox); ROS level was distinctly diminished by 5-CQA (≥3 μM), catechol (30 μM) and trigonelline (≥30 μM), whereas menadione-induced DNA damage in Caco-2 cells was reduced by NMP compounds (1-30 μM). The results emphasize that both original constituents and roast products contribute to the cellular antioxidant effectiveness of coffee. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Cell-Free Synthetic Biology: Engineering Beyond the Cell.

    Science.gov (United States)

    Perez, Jessica G; Stark, Jessica C; Jewett, Michael C

    2016-12-01

    Cell-free protein synthesis (CFPS) technologies have enabled inexpensive and rapid recombinant protein expression. Numerous highly active CFPS platforms are now available and have recently been used for synthetic biology applications. In this review, we focus on the ability of CFPS to expand our understanding of biological systems and its applications in the synthetic biology field. First, we outline a variety of CFPS platforms that provide alternative and complementary methods for expressing proteins from different organisms, compared with in vivo approaches. Next, we review the types of proteins, protein complexes, and protein modifications that have been achieved using CFPS systems. Finally, we introduce recent work on genetic networks in cell-free systems and the use of cell-free systems for rapid prototyping of in vivo networks. Given the flexibility of cell-free systems, CFPS holds promise to be a powerful tool for synthetic biology as well as a protein production technology in years to come. Copyright © 2016 Cold Spring Harbor Laboratory Press; all rights reserved.

  13. A Review of Hydrogen Production by Photosynthetic Organisms Using Whole-Cell and Cell-Free Systems.

    Science.gov (United States)

    Martin, Baker A; Frymier, Paul D

    2017-10-01

    Molecular hydrogen is a promising currency in the future energy economy due to the uncertain availability of finite fossil fuel resources and environmental effects from their combustion. It also has important uses in the production of fertilizers and platform chemicals as well as in upgrading conventional fuels. Conventional methods for producing molecular hydrogen from natural gas produce carbon dioxide and use a finite resource as feedstock. However, these issues can be overcome by using light energy from the Sun combined with microorganisms and their molecular machinery capable of photosynthesis. In the presence of light, the proteins involved in photosynthesis coupled with appropriate catalysts in higher plants, algae, and cyanobacteria can produce molecular hydrogen, and optimization via genetic modifications and biomolecular engineering further improves production rates. In this review, we will discuss techniques that have been utilized to improve rates of hydrogen production in biological systems based on the protein machinery of photosynthesis coupled with appropriate catalysts. We will also suggest areas for improvement and future directions for work in the field.

  14. Integrated lipase production and in situ biodiesel synthesis in a recombinant Pichia pastoris yeast: an efficient dual biocatalytic system composed of cell free enzymes and whole cell catalysts

    Science.gov (United States)

    2014-01-01

    Background Lipase-catalyzed biotransformation of acylglycerides or fatty acids into biodiesel via immobilized enzymes or whole cell catalysts has been considered as one of the most promising methods to produce renewable and environmentally friendly alternative liquid fuels, thus being extensively studied so far. In all previously pursued approaches, however, lipase enzymes are prepared in an independent process separated from enzymatic biodiesel production, which would unavoidably increase the cost and energy consumption during industrial manufacture of this cost-sensitive energy product. Therefore, there is an urgent need to develop novel cost-effective biocatalysts and biocatalytic processes with genuine industrial feasibility. Result Inspired by the consolidated bioprocessing of lignocellulose to generate bioethanol, an integrated process with coupled lipase production and in situ biodiesel synthesis in a recombinant P. pastoris yeast was developed in this study. The novel and efficient dual biocatalytic system based on Thermomyces lanuginosus lipase took advantage of both cell free enzymes and whole cell catalysts. The extracellular and intracellular lipases of growing yeast cells were simultaneously utilized to produce biodiesel from waste cooking oils in situ and in one pot. This integrated system effectively achieved 58% and 72% biodiesel yield via concurrent esterified-transesterified methanolysis and stepwise hydrolysis-esterification at 3:1 molar ratio between methanol and waste cooking oils, respectively. Further increasing the molar ratio of methanol to waste cooking oils to 6:1 led to an 87% biodiesel yield using the stepwise strategy. Both water tolerance and methanol tolerance of this novel system were found to be significantly improved compared to previous non-integrated biodiesel production processes using separately prepared immobilized enzymes or whole cell catalysts. Conclusion We have proposed a new concept of integrated biodiesel production

  15. Derivation, Expansion, and Motor Neuron Differentiation of Human-Induced Pluripotent Stem Cells with Non-Integrating Episomal Vectors and a Defined Xenogeneic-free Culture System.

    Science.gov (United States)

    Hu, Wentao; He, Yongpei; Xiong, Yongjie; Lu, Hong; Chen, Hong; Hou, Limin; Qiu, Zhandong; Fang, Yu; Zhang, Suming

    2016-04-01

    Induced pluripotent stem cells (iPSCs) generated from patient-derived somatic cells provides the opportunity for model development in order to study patient-specific disease states with the potential for drug discovery. However, use of lentivirus and exposure of iPSCs to animal-derived products limit their therapeutic utility and affect lineage differentiation and subsequent downstream functionality of iPSC derivatives. Within the context of this study, we describe a simple and practical protocol enabling the efficient reprogramming of terminally differentiated adult fibroblasts into integration-free human iPSCs (hiPSCs) using a combination of episomal plasmids with small molecules (SMs). Using this approach, there was a 10-fold increase in reprogramming efficiency over single plasmid vector-based methods. We obtained approximately 100 iPSCs colonies from 1 × 10(5) human adult dermal fibroblasts (HADFs) and achieved approximately 0.1% reprogramming efficiencies. Concurrently, we developed a highly conducive culture system using xeno-free media and human vitronectin. The resulting hiPSCs were free of DNA integration and had completely lost episomal vectors, maintained long-term self-renewal, featured a normal karyotype, expressed pluripotent stem cell markers, and possessed the capability of differentiating into components of all three germ layers in vivo. Finally, we demonstrate that the integration-free hiPSCs could be differentiated into motor neurons under xeno-free culture conditions. This induction method will promote the derivation of patient-specific integration-free and xeno-free iPSCs and improve the strategy for motor neuron derivation. Our approach provides a useful tool for human disease models, drug screen, and clinical applications.

  16. 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

  17. 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

  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. 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.

  20. Label-Free Biosensors for Cell Biology

    Directory of Open Access Journals (Sweden)

    Ye Fang

    2011-01-01

    Full Text Available Label-free biosensors for studying cell biology have finally come of age. Recent developments have advanced the biosensors from low throughput and high maintenance research tools to high throughput and low maintenance screening platforms. In parallel, the biosensors have evolved from an analytical tool solely for molecular interaction analysis to powerful platforms for studying cell biology at the whole cell level. This paper presents historical development, detection principles, and applications in cell biology of label-free biosensors. Future perspectives are also discussed.

  1. Influence of Mabs on PrP(Sc formation using in vitro and cell-free systems.

    Directory of Open Access Journals (Sweden)

    Binggong Chang

    Full Text Available PrP(Sc is believed to serve as a template for the conversion of PrP(C to the abnormal isoform. This process requires contact between the two proteins and implies that there may be critical contact sites that are important for conversion. We hypothesized that antibodies binding to either PrP(cor PrP(Sc would hinder or prevent the formation of the PrP(C-PrP(Sc complex and thus slow down or prevent the conversion process. Two systems were used to analyze the effect of different antibodies on PrP(Sc formation: (i neuroblastoma cells persistently infected with the 22L mouse-adapted scrapie stain, and (ii protein misfolding cyclic amplification (PMCA, which uses PrP(Sc as a template or seed, and a series of incubations and sonications, to convert PrP(C to PrP(Sc. The two systems yielded similar results, in most cases, and demonstrate that PrP-specific monoclonal antibodies (Mabs vary in their ability to inhibit the PrP(C-PrP(Sc conversion process. Based on the numerous and varied Mabs analyzed, the inhibitory effect does not appear to be epitope specific, related to PrP(C conformation, or to cell membrane localization, but is influenced by the targeted PrP region (amino vs carboxy.

  2. 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

  3. 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.

  4. 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

  5. Fuel-Free Bio-photoelectrochemical Cells Based on a Water/Oxygen Circulation System with a Ni:FeOOH/BiVO4Photoanode.

    Science.gov (United States)

    Zhang, He; Yu, You; Zhang, Lingling; Dong, Shaojun

    2018-02-05

    A bio-photoelectrochemical cell (BPEC) based on a fuel-free self-circulation water-oxygen-water system was fabricated. It consists of Ni:FeOOH modified n-type bismuth vanadate (BiVO 4 ) photoanode and laccase catalyzed biocathode. In this BPEC, irradiation of the photoanode generates photocurrent for photo-oxidation of water to oxygen, which is reduced to water again at the laccase biocathode. Of note, the by-products of two electrode reactions could continue to be reacted, which means the H 2 O and O 2 molecules are retained in an infinite loop of water-oxygen-water without any sacrificial chemical components. As a result, the assembled fuel-free BPEC exhibits good performance with an open-circuit potential of 0.97 V and a maximum power density of 205 μW cm -2 at 0.44 V. This BPEC based on a self-circulation system offers a fuel-free model to enhance multiple energy conversion and application in reality. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Chromatographic analyses of Lavandula angustifolia and Rosmarinus officinalis extracts and their biological effects in mammalian cells and cell-free systems.

    Science.gov (United States)

    Horvathova, E; Kozics, K; Melusova, M; Galova, E; Sevcovicova, A; Kusznierewicz, B; Chmiel, T; Slamenova, D

    2017-01-01

    Knowledge of biological properties of natural compounds allows to understand their therapeutic value, efficacy and security. We investigated: composition of Lavandula angustifolia (LA) and Rosmarinus officinalis (RO) extracts, their antioxidant capacity, cytotoxicity and genotoxicity, their DNA-protective potential against DNA damage induced in hamster V79 cells by several genotoxins or in plasmid DNA by Fe2+ ions and activity of antioxidant enzymes in cells treated with these extracts. Higher cytotoxicity, observed at higher concentrations of extracts, was accompanied by the increased level of single-strand (ss) DNA breaks as well as formamidopyrimidine DNA glycosylase (Fpg) sensitive sites. LA and RO extracts were able to protect DNA of hamster cells as well as plasmid DNA against ss DNA breaks induced by genotoxins and Fe2+. LA extract mildly increased the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT), while RO extract decreased the activity of SOD, but increased the activity of CAT and GPx. Cell-free tests confirmed antioxidant activity of both extracts. The biological properties of LA and RO extracts showed that they could have a positive impact on human health.

  7. 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...... and the effect of the grid line resistance are explored for a series of devices. Laser-beam-induced current (LBIC) mapping is used for quality control of the devices. A theoretical modeling study is presented that enables the identification of the most rational cell dimension for the grids with different...... 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...

  8. Fuel cell system with interconnect

    Energy Technology Data Exchange (ETDEWEB)

    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.

  9. 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

  10. Deep Learning in Label-free Cell Classification

    Science.gov (United States)

    Chen, Claire Lifan; Mahjoubfar, Ata; Tai, Li-Chia; Blaby, Ian K.; Huang, Allen; Niazi, Kayvan Reza; Jalali, Bahram

    2016-03-01

    Label-free cell analysis is essential to personalized genomics, cancer diagnostics, and drug development as it avoids adverse effects of staining reagents on cellular viability and cell signaling. However, currently available label-free cell assays mostly rely only on a single feature and lack sufficient differentiation. Also, the sample size analyzed by these assays is limited due to their low throughput. Here, we integrate feature extraction and deep learning with high-throughput quantitative imaging enabled by photonic time stretch, achieving record high accuracy in label-free cell classification. Our system captures quantitative optical phase and intensity images and extracts multiple biophysical features of individual cells. These biophysical measurements form a hyperdimensional feature space in which supervised learning is performed for cell classification. We compare various learning algorithms including artificial neural network, support vector machine, logistic regression, and a novel deep learning pipeline, which adopts global optimization of receiver operating characteristics. As a validation of the enhanced sensitivity and specificity of our system, we show classification of white blood T-cells against colon cancer cells, as well as lipid accumulating algal strains for biofuel production. This system opens up a new path to data-driven phenotypic diagnosis and better understanding of the heterogeneous gene expressions in cells.

  11. 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 ...

  12. Cell-free synthetic biology forin vitroprototype 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).

  13. Effect of streptomycin on the stoichiometry of GTP hydrolysis in a poly(U)-dependent cell-free translation system.

    Science.gov (United States)

    Smailov, S K; Gavrilova, L P

    1985-11-11

    The technique of Sepharose-bound template translation has been used to estimate the stoichiometry of GTP hydrolysis during peptide elongation in the presence of streptomycin. The presence of streptomycin has been shown to have no great effect on the elongation rate and the stoichiometry of GTP hydrolysis during codon-specific peptide elongation in the poly(U)-directed translation system: the molar ratio of hydrolysed GTP to incorporated phenylalanine was about 2. At the same time streptomycin exerted a significant effect during misreading when a ribosome-bound peptide in the poly(U)-programmed system was elongated by leucine or isoleucine residues: the miselongation was stimulated and hence the ratio of hydrolysed GTP per peptide bond was strongly reduced, as compared with the excessive GTP hydrolysis which is characteristic of the misreading system in the absence of streptomycin [(1984) FEBS Lett. 178, 283-287]. The conclusion has been made that streptomycin blocks the stage of correction ('proof-reading') following GTP hydrolysis during EF-Tu-dependent aminoacyl-tRNA binding.

  14. Cell-Free Synthesis Meets Antibody Production: A Review

    Directory of Open Access Journals (Sweden)

    Marlitt Stech

    2015-01-01

    Full Text Available Engineered antibodies are key players in therapy, diagnostics and research. In addition to full size immunoglobulin gamma (IgG molecules, smaller formats of recombinant antibodies, such as single-chain variable fragments (scFv and antigen binding fragments (Fab, have emerged as promising alternatives since they possess different advantageous properties. Cell-based production technologies of antibodies and antibody fragments are well-established, allowing researchers to design and manufacture highly specific molecular recognition tools. However, as these technologies are accompanied by the drawbacks of being rather time-consuming and cost-intensive, efficient and powerful cell-free protein synthesis systems have been developed over the last decade as alternatives. So far, prokaryotic cell-free systems have been the focus of interest. Recently, eukaryotic in vitro translation systems have enriched the antibody production pipeline, as these systems are able to mimic the natural pathway of antibody synthesis in eukaryotic cells. This review aims to overview and summarize the advances made in the production of antibodies and antibody fragments in cell-free systems.

  15. 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.

  16. 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.

  17. Antigen-specific in vitro expansion of functional redirected NY-ESO-1-specific human CD8+ T-cells in a cell-free system.

    Science.gov (United States)

    Jakka, Gopinadh; Schuberth, Petra C; Thiel, Markus; Held, Gerhard; Stenner, Frank; Van Den Broek, Maries; Renner, Christoph; Mischo, Axel; Petrausch, Ulf

    2013-10-01

    Tumors can be targeted by the adoptive transfer of chimeric antigen receptor (CAR) redirected T-cells. Antigen-specific expansion protocols are needed to generate large quantities of redirected T-cells. We aimed to establish a protocol to expand functional active NY-ESO-1-specific redirected human CD8(+) T-cells. The anti-idiotypic Fab antibody A4 with specificity for HLA-A 0201/NY-ESO-1157-165 was tested by competition assays using a HLA-A 0201/NY-ESO-1157-165 tetramer. HLA-A 0201/NY-ESO-1157-165 redirected T-cells were generated, expanded and tested for CAR expression, cytokine release, in vitro cytolysis and protection against xenografted HLA-A 0201/NY-ESO-1157-165-positive multiple myeloma cells. A4 demonstrated antigen-specific binding to HLA-A 0201/NY-ESO-1157-165 redirected T-cells. Expansion with A4 resulted in 98% of HLA-A 0201/NY-ESO-1157-165 redirected T-cells. A4 induced strong proliferation, resulting in a 300-fold increase of redirected T-cells. After expansion protocols, redirected T-cells secreted Interleukin-2, (IL-2), interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) and lysed target cells in vitro and were protective in vivo. A4 expanded HLA-A 0201/NY-ESO-1157-165 redirected T-cells with preservation of antigen-specific function.

  18. 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.

  19. 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

  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

    2011-08-01

    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.

  1. Expression regulation by a methyl-CpG binding domain in an E. coli based, cell-free TX-TL system

    Science.gov (United States)

    Schenkelberger, M.; Shanak, S.; Finkler, M.; Worst, E. G.; Noireaux, V.; Helms, V.; Ott, A.

    2017-04-01

    Cytosine methylation plays an important role in the epigenetic regulation of eukaryotic gene expression. The methyl-CpG binding domain (MBD) is common to a family of eukaryotic transcriptional regulators. How MBD, a stretch of about 80 amino acids, recognizes CpGs in a methylation dependent manner, and as a function of sequence, is only partly understood. Here we show, using an Escherichia coli cell-free expression system, that MBD from the human transcriptional regulator MeCP2 performs as a specific, methylation-dependent repressor in conjunction with the BDNF (brain-derived neurotrophic factor) promoter sequence. Mutation of either base flanking the central CpG pair changes the expression level of the target gene. However, the relative degree of repression as a function of MBD concentration remains unaltered. Molecular dynamics simulations that address the DNA B fiber ratio and the handedness reveal cooperative transitions in the promoter DNA upon MBD binding that correlate well with our experimental observations. We suggest that not only steric hindrance, but also conformational changes of the BDNF promoter as a result of MBD binding are required for MBD to act as a specific inhibitory element. Our work demonstrates that the prokaryotic transcription machinery can reproduce features of epigenetic mammalian transcriptional regulatory elements.

  2. 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.

  3. Preparation of efficient excision repair competent cell-free extracts from C. reinhardtii cells.

    Directory of Open Access Journals (Sweden)

    Vishalsingh Chaudhari

    Full Text Available Chlamydomonas reinhardtii is a prospective model system for understanding molecular mechanisms associated with DNA repair in plants and algae. To explore this possibility, we have developed an in vitro repair system from C. reinhardtii cell-free extracts that can efficiently repair UVC damage (Thymine-dimers in the DNA. We observed that excision repair (ER synthesis based nucleotide incorporation, specifically in UVC damaged supercoiled (SC DNA, was followed by ligation of nicks. Photoreactivation efficiently competed out the ER in the presence of light. In addition, repair efficiency in cell-free extracts from ER deficient strains was several fold lower than that of wild-type cell extract. Interestingly, the inhibitor profile of repair DNA polymerase involved in C. reinhardtii in vitro ER system was akin to animal rather than plant DNA polymerase. The methodology to prepare repair competent cell-free extracts described in the current study can aid further molecular characterization of ER pathway in C. reinhardtii.

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

    Science.gov (United States)

    Edmonds, Jessica

    2015-01-01

    Aurora Flight Sciences, in partnership with Draper Laboratory, has developed a miniaturized system to count white blood cells in microgravity environments. The system uses MEMS technology to simultaneously count total white blood cells, the five white blood cell differential subgroups, and various lymphocyte subtypes. The OILWBCS-MEMS detection technology works by immobilizing an array of white blood cell-specific antibodies on small, gold-coated membranes. When blood flows across the membranes, specific cells' surface protein antigens bind to their corresponding antibodies. This binding can be measured and correlated to cell counts. In Phase I, the partners demonstrated surface chemistry sensitivity and specificity for total white blood cells and two lymphocyte subtypes. In Phase II, a functional prototype demonstrated end-to-end operation. This rugged, miniaturized device requires minimal blood sample preparation and will be useful for both space flight and terrestrial applications.

  5. 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)

  6. Localization of BiP to translating ribosomes increases soluble accumulation of secreted eukaryotic proteins in an E. coli cell-free system

    OpenAIRE

    Welsh, John P.; Bonomo, Jeanne; Swartz, James R.

    2011-01-01

    The endoplasmic reticulum (ER) resident Hsp70 chaperone, BiP, docks to the Sec translocon and interacts co-translationally with polypeptides entering the ER to encourage proper folding. In order to recreate this interaction in E. coli cell-free protein synthesis (CFPS) reactions, a fusion protein was formed between the ribosome binding portion of the E. coli protein Trigger Factor (TF) and BiP. The biophysical affinity to ribosomes as well as the characteristic Hsp70 ATPase activity were both...

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

    Directory of Open Access Journals (Sweden)

    Ji Hyae Lim

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

  8. 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.

  9. 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.

  10. Differential activity profiles of translation inhibitors in whole-cell and cell-free approaches.

    Science.gov (United States)

    Weidlich, M; Klammt, C; Bernhard, F; Karas, M; Stein, T

    2008-02-01

    Evaluation of the activity profiles of standard prokaryotic translation inhibitors with different physicochemical properties under whole-cell and cell-free conditions. The minimal inhibitory concentration values (cell-free/whole-cell microg ml(-1)) for three aminoglycosides (neomycin, 0.01/6.92; paromomycin, 0.7/1.96; streptomycin 1.45/1.57), three macrolides (erythromycin, 1.53/56.9; josamycin, 1.61/87.7; oleandomycin, 5.12/565.9), chloramphenicol (11.9/3.04), and two tetracyclines (tetracycline hydrochloride, not determined/0.63; minocycline hydrochloride, 2.53/1.09), towards Escherichia coli A19 cells were determined with a microtitre plate-based broth dilution method and compared with values determined in a coupled transcription/translation system based on a S30 extract of the same E. coli strain (cell-free) for the production of the green fluorescent protein. The analysed prokaryotic translation inhibitors showed substance-specific activity profiles under cell-free vs whole-cell conditions that are explainable by the physicochemical properties of the molecules. This study shows the advantages and limits of cell- free transcription/translation (CFTT) experiments for the discovery of novel antimicrobials. The main advantage is the direct access of the target structures (ribosomes) for the inhibitors, and our results provide an estimation of the concentration necessary to detect new agents. The main limitations are that the inhibitory properties of different agents in CFTT experiments do not necessarily reflect their growth inhibition activity in cell cultures.

  11. Expression screening of fusion partners from an E. coli genome for soluble expression of recombinant proteins in a cell-free protein synthesis system.

    Science.gov (United States)

    Ahn, Jin-Ho; Keum, Jung-Won; Kim, Dong-Myung

    2011-01-01

    While access to soluble recombinant proteins is essential for a number of proteome studies, preparation of purified functional proteins is often limited by the protein solubility. In this study, potent solubility-enhancing fusion partners were screened from the repertoire of endogenous E. coli proteins. Based on the presumed correlation between the intracellular abundance and folding efficiency of proteins, PCR-amplified ORFs of a series of highly abundant E. coli proteins were fused with aggregation-prone heterologous proteins and then directly expressed for quantitative estimation of the expression efficiency of soluble translation products. Through two-step screening procedures involving the expression of 552 fusion constructs targeted against a series of cytokine proteins, we were able to discover a number of endogenous E. coli proteins that dramatically enhanced the soluble expression of the target proteins. This strategy of cell-free expression screening can be extended to quantitative, global analysis of genomic resources for various purposes.

  12. Rapid cell-free forward engineering of novel genetic ring oscillators.

    Science.gov (United States)

    Niederholtmeyer, Henrike; Sun, Zachary Z; Hori, Yutaka; Yeung, Enoch; Verpoorte, Amanda; Murray, Richard M; Maerkl, Sebastian J

    2015-10-05

    While complex dynamic biological networks control gene expression in all living organisms, the forward engineering of comparable synthetic networks remains challenging. The current paradigm of characterizing synthetic networks in cells results in lengthy design-build-test cycles, minimal data collection, and poor quantitative characterization. Cell-free systems are appealing alternative environments, but it remains questionable whether biological networks behave similarly in cell-free systems and in cells. We characterized in a cell-free system the 'repressilator', a three-node synthetic oscillator. We then engineered novel three, four, and five-gene ring architectures, from characterization of circuit components to rapid analysis of complete networks. When implemented in cells, our novel 3-node networks produced population-wide oscillations and 95% of 5-node oscillator cells oscillated for up to 72 hr. Oscillation periods in cells matched the cell-free system results for all networks tested. An alternate forward engineering paradigm using cell-free systems can thus accurately capture cellular behavior.

  13. Chemical Screening Using Cell-FreeXenopusEgg Extract.

    Science.gov (United States)

    Broadus, Matthew R; Lee, Ethan

    2018-02-23

    Most drug screening methods use purified proteins, cultured cells, and/or small model organisms such as Xenopus , zebrafish, flies, or nematodes. These systems have proven successes in drug discovery, but they also have weaknesses. Although purified cellular components allow for identification of compounds with activity against specific targets, such systems lack the complex biological interactions present in cellular and organismal screens. In vivo systems overcome these weaknesses, but the lack of cellular permeability, efflux by cellular pumps, and/or toxicity can be major limitations. Xenopus laevis egg extract, a concentrated and biologically active cytosol, can potentially overcome these weaknesses. Drug interactions occur in a near-physiological milieu, thereby functioning in a "truer" endogenous manner than purified components. Also, Xenopus egg extract is a cell-free system that lacks intact plasma membranes that could restrict drug access to potential targets. Finally, Xenopus egg extract is readily manipulated at the protein level: Proteins are easily depleted or added to the system, an important feature for analyzing drug effects in disease states. Thus, Xenopus egg extract offers an attractive media for screening drugs that merges strengths of both in vitro and in vivo systems. © 2018 Cold Spring Harbor Laboratory Press.

  14. 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.

  15. Cell-free translation of murine coronavirus RNA.

    OpenAIRE

    Leibowitz, J L; Weiss, S R; Paavola, E; Bond, C W

    1982-01-01

    The coding assignments of the intracellular murine hepatitis virus-specific subgenomic RNA species and murine hepatitis virion RNA have been investigated by cell-free translation. The six murine hepatitis virus-specific subgenomic RNAs were partially purified by agarose gel electrophoresis and translated in an mRNA-dependent rabbit reticulocyte lysate, and the cell-free translation products were characterized by gel electrophoresis, immunoprecipitation, and tryptic peptide mapping. These stud...

  16. Bioinformatics analysis of circulating cell-free DNA sequencing data.

    Science.gov (United States)

    Chan, Landon L; Jiang, Peiyong

    2015-10-01

    The discovery of cell-free DNA molecules in plasma has opened up numerous opportunities in noninvasive diagnosis. Cell-free DNA molecules have become increasingly recognized as promising biomarkers for detection and management of many diseases. The advent of next generation sequencing has provided unprecedented opportunities to scrutinize the characteristics of cell-free DNA molecules in plasma in a genome-wide fashion and at single-base resolution. Consequently, clinical applications of circulating cell-free DNA analysis have not only revolutionized noninvasive prenatal diagnosis but also facilitated cancer detection and monitoring toward an era of blood-based personalized medicine. With the remarkably increasing throughput and lowering cost of next generation sequencing, bioinformatics analysis becomes increasingly demanding to understand the large amount of data generated by these sequencing platforms. In this Review, we highlight the major bioinformatics algorithms involved in the analysis of cell-free DNA sequencing data. Firstly, we briefly describe the biological properties of these molecules and provide an overview of the general bioinformatics approach for the analysis of cell-free DNA. Then, we discuss the specific upstream bioinformatics considerations concerning the analysis of sequencing data of circulating cell-free DNA, followed by further detailed elaboration on each key clinical situation in noninvasive prenatal diagnosis and cancer management where downstream bioinformatics analysis is heavily involved. We also discuss bioinformatics analysis as well as clinical applications of the newly developed massively parallel bisulfite sequencing of cell-free DNA. Finally, we offer our perspectives on the future development of bioinformatics in noninvasive diagnosis. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  17. 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.

  18. Free space optical alignment system using GPS

    Science.gov (United States)

    Saw, Wee-Leong; Refai, Hazem H.; Sluss, James J., Jr.

    2005-04-01

    This paper presents results from an ongoing effort at the University of Oklahoma to develop a real-time active alignment system for free-space optical communication system. An initial prototype of a FSO active alignment system using Global Positioning System (GPS) sensors, two gimbals, and point-to-point spread spectrum RF communication is described. The positions of both FSO transceivers are exchanged over the radio frequency (RF) communication link. A controller uses the exchanged information to calculate azimuth and elevation bearings to achieve initial alignment between the transceivers. The gimbals are used to steer the beams. The paper also presents a binary scan algorithm developed to expedite the initial alignment process. The algorithm incorporates power measurements as feed back to the original transceiver for comparison. In minimizing convergence time, simulation results confirm that the algorithm performs better than raster scan, spiral scan, and raster spiral scan algorithms, all of which are used in laser satellite communications. The results also show that the initial design is not able to achieve real-time alignment. For real-time alignment, different augmenting technologies (for example, steering mirrors) should be considered.

  19. Escherichia coli NemA is an efficient chromate reductase that can be biologically immobilized to provide a cell free system for remediation of hexavalent chromium.

    Directory of Open Access Journals (Sweden)

    Katherine J Robins

    Full Text Available Hexavalent chromium is a serious and widespread environmental pollutant. Although many bacteria have been identified that can transform highly water-soluble and toxic Cr(VI to insoluble and relatively non-toxic Cr(III, bacterial bioremediation of Cr(VI pollution is limited by a number of issues, in particular chromium toxicity to the remediating cells. To address this we sought to develop an immobilized enzymatic system for Cr(VI remediation. To identify novel Cr(VI reductase enzymes we first screened cell extracts from an Escherichia coli library of soluble oxidoreductases derived from a range of bacteria, but found that a number of these enzymes can reduce Cr(VI indirectly, via redox intermediates present in the crude extracts. Instead, activity assays for 15 candidate enzymes purified as His6-tagged proteins identified E. coli NemA as a highly efficient Cr(VI reductase (k(cat/K(M= 1.1×10(5 M(-1 s(-1 with NADH as cofactor. Fusion of nemA to the polyhydroxyalkanoate synthase gene phaC from Ralstonia eutropha enabled high-level biosynthesis of functionalized polyhydroxyalkanoate granules displaying stable and active NemA on their surface. When these granules were combined with either Bacillus subtilis glucose dehydrogenase or Candida boidinii formate dehydrogenase as a cofactor regenerating partner, high levels of chromate transformation were observed with only low initial concentrations of expensive NADH cofactor being required, the overall reaction being powered by consumption of the cheap sacrificial substrates glucose or formic acid, respectively. This system therefore offers promise as an economic solution for ex situ Cr(VI remediation.

  20. Flexible ITO-Free Polymer Solar Cells

    DEFF Research Database (Denmark)

    Angmo, Dechan; Krebs, Frederik C

    2013-01-01

    Indium tin oxide (ITO) is the material-of-choice for transparent conductors in any optoelectronic application. However, scarce resources of indium and high market demand of ITO have created large price fluctuations and future supply concerns. In polymer solar cells (PSCs), ITO is the single......-most cost driving factor due to expensive raw materials and processing. Given the limited lifetime and stability of PSCs as compared with other mature technologies such as silicon-based solar cells, the technological future of PSCs beyond that of academic interests rests in reducing cost of production...

  1. AMMONIA-FREE NOx CONTROL SYSTEM

    Energy Technology Data Exchange (ETDEWEB)

    Song Wu; Zhen Fan; Andrew H. Seltzer; Richard G. Herman

    2006-06-01

    This report describes a novel NOx control system that has the potential to drastically reduce cost, and enhance performance, operation and safety of power plant NOx control. The new system optimizes the burner and the furnace to achieve very low NOx levels and to provide an adequate amount of CO, and uses the CO for reducing NO both in-furnace and over a downstream AFSCR (ammonia-free selective catalytic reduction) reactor. The AF-SCR combines the advantages of the highly successful SCR technology for power plants and the TWC (three-way catalytic converter) widely used on automobiles. Like the SCR, it works in oxidizing environment of combustion flue gas and uses only base metal catalysts. Like the TWC, the AF-SCR removes NO and excess CO simultaneously without using any external reagent, such as ammonia. This new process has been studied in a development program jointed funded by the US Department of Energy and Foster Wheeler. The report outlines the experimental catalyst work performed on a bench-scale reactor, including test procedure, operating conditions, and results of various catalyst formulations. Several candidate catalysts, prepared with readily available transition metal oxides and common substrate materials, have shown over 80-90% removal for both NO and CO in oxidizing gas mixtures and at elevated temperatures. A detailed combustion study of a 400 MWe coal-fired boiler, applying computational fluid dynamics techniques to model boiler and burner design, has been carried out to investigate ways to optimize the combustion process for the lowest NOx formation and optimum CO/NO ratios. Results of this boiler and burner optimization work are reported. The paper further discusses catalyst scale-up considerations and the conceptual design of a 400 MWe size AF-SCR reactor, as well as economics analysis indicating large cost savings of the ammonia-free NOx control process over the current SCR technology.

  2. Variations of cell performance in ITO-free organic solar cells with increasing cell areas

    International Nuclear Information System (INIS)

    Yeo, Jun-Seok; Yun, Jin-Mun; Kim, Junkyung; Na, Seok-In; Kim, Seok-Soon; Kim, Dong-Yu

    2011-01-01

    This study examined the effects of a cell area on the cell performances in ITO-free organic solar cells (OSCs) based on poly(3-hexylthiophene) (P3HT) and 1-(3-methoxycarbonyl)-propyl-1-phenyl-(6,6)C 61 (PCBM). Highly conductive poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) films with two different sheet resistances (R sh ) were used as polymeric transparent anodes for cost-effective ITO-free OSCs. Changes in the power conversion efficiency (PCE), the fill factor (FF), the short-circuit current (J sc ), and the open-circuit voltage (V oc ) that resulted from changing the cell area or sheet resistance of transparent electrodes were systematically investigated. With increasing cell area from 4.5 to 49.5 mm 2 , the device performance of ITO-free OSCs was continuously decreased mainly due to the decrease in the FF and the series resistance (R s ). In addition, the performance of OSCs was critically dependent on R sh of the PEDOT:PSS electrode. Upon reducing R sh of the polymer anode from ∼200 to ∼90 Ω/□, the FF and PCE showed better values at an identical large cell area and exhibited a relieved cell performance degradation with increasing cell area, suggesting that the sheet resistance of transparent electrodes is a dominant factor to limit cell efficiencies in practical large-area solar cells

  3. A new chemotherapy agent-free theranostic system composed of graphene oxide nano-complex and aptamers for treatment of cancer cells.

    Science.gov (United States)

    Bahreyni, Amirhossein; Yazdian-Robati, Rezvan; Hashemitabar, Shirin; Ramezani, Mohammad; Ramezani, Pouria; Abnous, Khalil; Taghdisi, Seyed Mohammad

    2017-06-30

    The common cancer treatment strategies like chemotherapy and radiotherapy are nonspecific and can trigger severe side effects by damaging normal cells. So, targeted cancer therapies, such as apoptosis induction, have attracted great attention in recent years. In this project, two nano-complexes, MUC1 aptamer-NAS-24 aptamer-Graphene oxide (GO) and MUC1 aptamer-Cytochrome C aptamer-GO, were designed to induce cell programmed death in MDA-MB-231 and MCF-7 cells (breast cancer cell lines) and to verify the level of apoptosis in both cell lines. MUC1 aptamer was a molecular recognition probe that led the internalization of two nano-complexes into MDA-MB-231 and MCF-7 cells (MUC1 positive cells) but not into HepG2 cell (liver cancer cell line, MUC1 negative cells). The apoptosis induction relied on binding of NAS-24 aptamer to its target, vimentin, in MDA-MB-231 and MCF-7 (target cells) with different levels of vimentin content. The function of first nano-complex was confirmed by binding of FAM-labeled cytochrome C aptamer to its target (cytochrome C) which was released from mitochondria, based on the function of the first nano-complex. Fluorometric analysis and gel retardation assay proved the formation of nano-complexes. The results of flow cytometry and fluorescence microscopy indicated efficient apoptosis induction just in target cells (MDA-MB-231 and MCF-7 cells) but not in non-target cells (HepG2 cell). The results of MTT assay also confirmed cell death process. Overall, our results proved excellent targeted apoptosis in breast cancer cells by designed nano-complexes which can be applied as an efficient cancer therapy method. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. 21 CFR 862.1695 - Free thyroxine test system.

    Science.gov (United States)

    2010-04-01

    ...) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862... to measure free (not protein bound) thyroxine (thyroid hormone) in serum or plasma. Levels of free...

  5. 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.

  6. Pollution-free energy supply system. Mukogai energy kyokyu system

    Energy Technology Data Exchange (ETDEWEB)

    Inoue, H.; Tsukamoto, M.; Hayashibara, M. (Hitachi Ltd., Tokyo (Japan))

    1991-08-19

    This invention aims to solve the problems of stable supply of natural energy and the construction cost, enhancing the power output per unit area, producing a hydrogen gas which is free from pollution, thus stably supplying power and hydrogen fuel. In this invention, integration is made of the systems such as a light/heat collection system wherein the solar light is collected and converted into heat, a thermal power generating system which converts the heat into electricity, a wind power generating system wherein a wind receiving blade is rotated by the wind power and the rotation force is converted into electricity and a wave power generating system which converts the tidal force into electricity and a current power generating system. By this arrangement, the take out of electric energy per unit area was increased. In addition, hydrogen is produced by an electrolysis of water by utilizing this electric power. 3 figs.

  7. 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

  8. 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.

  9. The Shipboard Employment of a Free Electron Laser Weapon System

    Science.gov (United States)

    2003-12-01

    EMPLOYMENT OF A FREE ELECTRON LASER WEAPON SYSTEM by Gregory G. Allgaier December 2003 Thesis Advisor: William Colson Second Reader...Free Electron Laser Weapon System 6. AUTHOR(S) Allgaier, Gregory G. 5. FUNDING NUMBERS 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) Naval...release; distribution is unlimited. THE SHIPBOARD EMPLOYMENT OF A FREE ELECTRON LASER WEAPON SYSTEM Gregory G. Allgaier Lieutenant, United

  10. Facts on the fragmentation of antigens in presenting cells, on the association of antigen fragments with MHC molecules in cell-free systems, and speculation on the cell biology of antigen processing

    DEFF Research Database (Denmark)

    Werdelin, O; Mouritsen, S; Petersen, B L

    1988-01-01

    The processing of a protein antigen is a multi-step event taking place in antigen-presenting cells. Processing is a prerequisite for the recognition of most antigens by T lymphocytes. The antigen is ingested by endocytosis, transported to an acid cellular compartment and subjected to proteolytic...... fragmentation. Some of the antigen fragments bind to MHC class II molecules and are transported to the surface of the antigen-presenting cell where the actual presentation to T lymphocytes occurs. The nature of the processed antigen, how and where it is derived and subsequently becomes associated with MHC...

  11. Current collecting grids for ITO-free solar cells

    NARCIS (Netherlands)

    Galagan, Y.O.; Zimmermann, B.; Coenen, E.W.C.; Jorgensen, M.; Tanenbaum, D.M.; Krebs, F.C.; Gorter, H.; Sabik, S.; Slooff, L.H.; Veenstra, S.C.; Kroon, J.M.; Andriessen, H.A.J.M.

    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. Modular PEM Fuel Cell SCADA & Simulator System

    Directory of Open Access Journals (Sweden)

    Francisca Segura

    2015-09-01

    Full Text Available The paper presents a Supervision, Control, Data Acquisition and Simulation (SCADA & Simulator system that allows for real-time training in the actual operation of a modular PEM fuel cell system. This SCADA & Simulator system consists of a free software tool that operates in real time and simulates real situations like failures and breakdowns in the system. This developed SCADA & Simulator system allows us to properly operate a fuel cell and helps us to understand how fuel cells operate and what devices are needed to configure and run the fuel cells, from the individual stack up to the whole fuel cell system. The SCADA & Simulator system governs a modular system integrated by three PEM fuel cells achieving power rates higher than tens of kilowatts.

  13. Free Radical Timer of Aging: from Chemistry of Free Radicals to Systems Theory of Reliability.

    Science.gov (United States)

    Koltover, Vitaly K

    2017-01-01

    There are two generally known concepts in biology of aging. Accordingly to the first one, there is a program of aging. The alternative concept advocates that aging proceeds stochastically. In this area of research, free radical-theory of aging, which was put forward by Denham Harman in fifties of XXth century, has determined the most heuristic line. The goal of this review is to demonstrate how the aging program and the aging stochastics are united on the basis of the systems theory of reliability. On this basis, universal features of aging, such as the exponential growth of mortality rate with time and correlation of longevity with the species-specific resting metabolism, are naturally explained. The stochastic malfunctions of the mitochondrial electron transport nanoreactors, which produce the oxygen anion-radicals (O2•-) as by-products of respiration, seem to be of first importance. As a reducing agent, O2•- affects the ratio of NAD(P)H/NAD(P)+ and, by changing the activity of sirtuins, slows down renewal of biomolecular constructs. As a consequence, the oxidative-stress products and other metabolic slag accumulate with the resulting impetus to autophagic or apoptotic cell death accompanied with age-associated clinical disorders. Based on this reliability-theory approach, one can estimate that the longevity of human brain could reach 250 years should the antioxidant defense against the free-radical failures be perfect. Thus, the free-radical redox timer serves as effective stochastic mechanism of realization of the programmed deficiency in reliability of biomolecular constructs.

  14. Solar cell concentrating system

    International Nuclear Information System (INIS)

    Garg, H.P.; Sharma, V.K.; Agarwal, R.K.

    1986-11-01

    This study reviews fabrication techniques and testing facilities for different solar cells under concentration which have been developed and tested. It is also aimed to examine solar energy concentrators which are prospective candidates for photovoltaic concentrator systems. This may provide an impetus to the scientists working in the area of solar cell technology

  15. 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.

  16. Characterization of biomaterial-free cell sheets cultured from human oral mucosal epithelial cells.

    Science.gov (United States)

    Hyun, Dong Won; Kim, Yun Hee; Koh, Ah Young; Lee, Hyun Ju; Wee, Won Ryang; Jeon, Saewha; Kim, Mee Kum

    2017-03-01

    The purpose of this study was to report the characteristics of biomaterial-free sheets cultured from human oral mucosal epithelial cells without fibrin support, in vitro and after transplantation to limbal-deficient models. Human oral mucosal epithelial cells and limbal epithelial cells were cultured for 2 weeks, and the colony-forming efficiency (CFE) rates were compared. Markers of stem cells (p63), cell proliferation (Ki-67) and epithelial differentiation (cytokeratin; K1, K3, K4, K13) were observed in colonies and in biomaterial-free sheets. Biomaterial-free sheets which had been detached with 1% dispase or biomaterial-free sheets generated by fibrin support were transplanted to 12 limbal-deficient rabbit models. In vitro cell viability, in vivo stability and cytokeratin characteristics of biomaterial-free sheets were compared with those of sheets formed by fibrin-coated culture 1 week after transplantation. Mean CFE rate was significantly higher in human oral mucosal epithelial cells (44.8%) than in human limbal epithelial cells(17.7%). K3 and K4 were well expressed in both colonies and sheets. Biomaterial-free sheets had two to six layers of stratified cells and showed an average of 79.8% viable cells in the sheets after detachment. Cytokeratin expressions of biomaterial-free sheets were comparable to those of sheets cultured by fibrin support, in limbal-deficient models. Both p63 and Ki-67 were well expressed in colonies, isolated sheets and sheets transplanted to limbal-deficient models. Our results suggest that biomaterial-free sheets cultured from human oral mucosal epithelial cells without fibrin support can be an alternative option for cell therapy in use for the treatment of limbal-deficient diseases. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

  17. Communication System and Avionics for Deployable Small Free Flying Payloads

    Data.gov (United States)

    National Aeronautics and Space Administration — This IRAD will develop a core part of the communication system for the small free flying payloads.  In particular, this portion of the system will consist of the...

  18. 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)

  19. 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

  20. Systems and Methods for Derivative-Free Adaptive Control

    Science.gov (United States)

    Yucelen, Tansel (Inventor); Kim, Kilsoo (Inventor); Calise, Anthony J. (Inventor)

    2015-01-01

    An adaptive control system is disclosed. The control system can control uncertain dynamic systems. The control system can employ one or more derivative-free adaptive control architectures. The control system can further employ one or more derivative-free weight update laws. The derivative-free weight update laws can comprise a time-varying estimate of an ideal vector of weights. The control system of the present invention can therefore quickly stabilize systems that undergo sudden changes in dynamics, caused by, for example, sudden changes in weight. Embodiments of the present invention can also provide a less complex control system than existing adaptive control systems. The control system can control aircraft and other dynamic systems, such as, for example, those with non-minimum phase dynamics.

  1. Defective thymine dimer excision by cell-free extracts of xeroderma pigmentosum cells

    International Nuclear Information System (INIS)

    Mortelmans, K.; Friedberg, E.C.; Slor, H.; Thomas, G.; Cleaver, J.E.

    1976-01-01

    Crude extracts of normal human diploid fibroblasts and of human peripheral blood lymphocytes excise thymine dimers from purified ultraviolet-irradiated DNA, or from the DNA presumably present as chromatin in unfractionated cell-free preparations of cells that had been labeled with [ 3 H]thymidine. Extracts of xeroderma pigmentosum cells from complementation groups A, C, and D also excise thymine dimers from purified DNA, but extracts of group A cells do not excise dimers from the DNA of radioactively labeled unfractionated cell-free preparations

  2. Cell-Free DNA in Metastatic Colorectal Cancer

    DEFF Research Database (Denmark)

    Spindler, Karen-Lise G; Boysen, Anders K; Pallisgård, Niels

    2017-01-01

    -analysis of the prognostic value of total cfDNA in patients with metastatic colorectal cancer (mCRC) treated with chemotherapy. In addition, we report on the overall performance of cfDNA as source for KRAS mutation detection. MATERIALS AND METHODS: A systematic literature search of PubMed and Embase was performed by two...... 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....

  3. 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...... over the active area initially progressing to a larger graduation in areas with different performance. Over time coating defects also become much more apparent in the LBIC images....

  4. Ribosome-mediated synthesis of natural product-like peptides via cell-free translation.

    Science.gov (United States)

    Maini, Rumit; Umemoto, Shiori; Suga, Hiroaki

    2016-10-01

    Peptide natural products (PNPs) represent a unique class of compounds known for their fascinating structural motifs with important biological activities. Lately, PNPs have garnered a lot of interest for their application in drug discovery. Nevertheless, lack of diversity oriented synthetic/biosynthetic platforms to generate large natural product-like libraries has limited their development as peptide therapeutics. The promiscuity of cell-free translation has allowed for the synthesis of artificial PNPs having complex structural features. Modified cell-free translation systems coupled with the display technologies have generated diverse natural product-like peptide libraries and led to the discovery of several biologically active molecules. Such technologies have drastically decreased the time to obtain peptide drug leads and therefore, revolutionized the field of peptide drug discovery. In this account, we review recent developments in the synthesis of natural product-like peptides via cell-free translation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Neurogenic Effects of Cell-Free Extracts of Adipose Stem Cells.

    Directory of Open Access Journals (Sweden)

    Jae-Jun Ban

    Full Text Available Stem-cell-based therapies are regarded as promising treatments for neurological disorders, and adipose-derived stem cells (ASCs are a feasible source of clinical application of stem cell. Recent studies have shown that stem cells have a therapeutic potential for use in the treatment of various illnesses through paracrine action. To examine the effects of cell components of ASCs on neural stem cells (NSCs, we treated cell-free extracts of ASCs (CFE-ASCs containing various components with brain-derived NSCs. To elucidate the effects of CFE-ASCs in NSC proliferation, we treated mouse subventricular zone-derived cultured NSCs with various doses of CFE-ASCs. As a result, CFE-ASCs were found to induce the proliferation of NSCs under conditions of growth factor deprivation in a dose-dependent manner (p<0.01. CFE-ASCs increase the expression of neuron and astrocyte differentiation markers including Tuj-1 (p<0.05 and glial fibrillary acidic protein (p<0.01 without altering the cell's fate in differentiating NSCs. In addition, treatment with CFE-ASCs induces an increase in neurite numbers (p<0.01 and lengths of NSCs (p<0.05. Furthermore, CFE-ASCs rescue the hydrogen peroxide-induced reduction of NSCs' viability (p<0.05 and neurite branching (p<0.01. Findings from our study indicate that CFE-ASCs support the survival, proliferation and differentiation of NSCs accompanied with neurite outgrowth, suggesting that CFE-ASCs can modulate neurogenesis in the central nervous system.

  6. 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

  7. Cell-free (RNA and cell-associated (DNA HIV-1 and postnatal transmission through breastfeeding.

    Directory of Open Access Journals (Sweden)

    James Ndirangu

    Full Text Available Transmission through breastfeeding remains important for mother-to-child transmission (MTCT in resource-limited settings. We quantify the relationship between cell-free (RNA and cell-associated (DNA shedding of HIV-1 virus in breastmilk and the risk of postnatal HIV-1 transmission in the first 6 months postpartum.Thirty-six HIV-positive mothers who transmitted HIV-1 by breastfeeding were matched to 36 non-transmitting HIV-1 infected mothers in a case-control study nested in a cohort of HIV-infected women. RNA and DNA were quantified in the same breastmilk sample taken at 6 weeks and 6 months. Cox regression analysis assessed the association between cell-free and cell-associated virus levels and risk of postnatal HIV-1 transmission.There were higher median levels of cell-free than cell-associated HIV-1 virus (per ml in breastmilk at 6 weeks and 6 months. Multivariably, adjusting for antenatal CD4 count and maternal plasma viral load, at 6 weeks, each 10-fold increase in cell-free or cell-associated levels (per ml was significantly associated with HIV-1 transmission but stronger for cell-associated than cell-free levels [2.47 (95% CI 1.33-4.59 vs. aHR 1.52 (95% CI, 1.17-1.96, respectively]. At 6 months, cell-free and cell-associated levels (per ml in breastmilk remained significantly associated with HIV-1 transmission but was stronger for cell-free than cell-associated levels [aHR 2.53 (95% CI 1.64-3.92 vs. 1.73 (95% CI 0.94-3.19, respectively].The findings suggest that cell-associated virus level (per ml is more important for early postpartum HIV-1 transmission (at 6 weeks than cell-free virus. As cell-associated virus levels have been consistently detected in breastmilk despite antiretroviral therapy, this highlights a potential challenge for resource-limited settings to achieve the UNAIDS goal for 2015 of eliminating vertical transmission. More studies would further knowledge on mechanisms of HIV-1 transmission and help develop more effective

  8. 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

  9. Fuel cell systems

    International Nuclear Information System (INIS)

    Kotevski, Darko

    2003-01-01

    Fuel cell systems are an entirely different approach to the production of electricity than traditional technologies. They are similar to the batteries in that both produce direct current through electrochemical process. There are six types of fuel cells each with a different type of electrolyte, but they all share certain important characteristics: high electrical efficiency, low environmental impact and fuel flexibility. Fuel cells serve a variety of applications: stationary power plants, transport vehicles and portable power. That is why world wide efforts are addressed to improvement of this technology. (Original)

  10. 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) were...... selected as model drugs. Self-diffusion nuclear magnetic resonance studies were performed and confirmed the entrapment of drugs in micelles in Brij 35 and sodium taurodeoxycholate (TDC)/phosphatidylcholine (PC) micellar solutions. The FDS measurements indicated that with a constant level of drug...

  11. 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.

  12. 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

  13. Hydrodynamic and label-free sorting of circulating tumor cells from whole blood

    Science.gov (United States)

    Geislinger, Thomas M.; Stamp, Melanie E. M.; Wixforth, Achim; Franke, Thomas

    2015-11-01

    We demonstrate continuous, passive, and label-free sorting of different in vitro cancer cell lines (MV3, MCF7, and HEPG2) as model systems for circulating tumor cells (CTCs) from undiluted whole blood employing the non-inertial lift effect as driving force. This purely viscous, repulsive cell-wall interaction is sensitive to cell size and deformability differences and yields highly efficient cell separation and high enrichment factors. We show that the performance of the device is robust over a large range of blood cell concentrations and flow rates as well as for the different cell lines. The collected samples usually contain more than 90% of the initially injected CTCs and exhibit average enrichment factors of more than 20 for sorting from whole blood samples.

  14. Ammonia-Free NOx Control System

    Energy Technology Data Exchange (ETDEWEB)

    Song Wu; Zhen Fan; Andrew H. Seltzer; Richard G. Herman

    2005-03-31

    Research is being conducted under United States Department of Energy (DOE) Contract DE-FC26-03NT41865 to develop a new technology to achieve very low levels of NOx emissions from pulverized coal fired boiler systems by employing a novel system level integration between the PC combustion process and the catalytic NOx reduction with CO present in the combustion flue gas. The combustor design and operating conditions will be optimized to achieve atypical flue gas conditions. This approach will not only suppress NOx generation during combustion but also further reduce NOx over a downstream catalytic reactor that does not require addition of an external reductant, such as ammonia.

  15. Ammonia-Free NOx Control System

    Energy Technology Data Exchange (ETDEWEB)

    Song Wu; Zhen Fan; Andrew H. Seltzer

    2005-06-30

    Research is being conducted under United States Department of Energy (DOE) Contract DEFC26-03NT41865 to develop a new technology to achieve very low levels of NOx emissions from pulverized coal fired boiler systems by employing a novel system level integration between the PC combustion process and the catalytic NOx reduction with CO present in the combustion flue gas. The combustor design and operating conditions will be optimized to achieve atypical flue gas conditions. This approach will not only suppress NOx generation during combustion but also further reduce NOx over a downstream catalytic reactor that does not require addition of an external reductant, such as ammonia.

  16. Free positioning for inductive wireless power system

    NARCIS (Netherlands)

    Waffenschmidt, E.

    2012-01-01

    In inductive wireless power transmission system a lateral displacement of the receiver coil to the transmitter coil leads to a change ofthe coupling factor and thus an unwanted variation of the power transfer. Here, an algorithm to determine the turn distribution to achieve homogeneous coupling

  17. Systemic translocation of Salmonella enterica serovar Dublin in cattle occurs predominantly via efferent lymphatics in a cell-free niche and requires type III secretion system 1 (T3SS-1) but not T3SS-2.

    Science.gov (United States)

    Pullinger, Gillian D; Paulin, Susan M; Charleston, Bryan; Watson, Patricia R; Bowen, Alison J; Dziva, Francis; Morgan, Eirwen; Villarreal-Ramos, Bernardo; Wallis, Timothy S; Stevens, Mark P

    2007-11-01

    Salmonella enterica is an important diarrheal pathogen, and infections may involve severe systemic sequelae depending on serovar- and host-specific factors. The molecular mechanisms underlying translocation of host-restricted and -specific serovars of S. enterica from the intestines to distal organs are ill defined. By surgical cannulation of lymph and blood vessels draining the distal ileum in cattle, S. enterica serovar Dublin was observed to translocate predominantly via mesenteric lymph nodes to efferent lymphatics in a manner that correlates with systemic virulence, since the fowl typhoid-associated serovar Gallinarum translocated at a significantly lower level. While both S. enterica serovars Dublin and Gallinarum were intracellular while in the intestinal mucosa and associated with major histocompatibility complex class II-positive cells, the bacteria were predominantly extracellular within efferent lymph. Screening of a library of signature-tagged serovar Dublin mutants following oral inoculation of calves defined the role of 36 virulence-associated loci in enteric and systemic phases of infection. The number and proportion of tagged clones reaching the liver and spleen early after oral infection were identical to the values in efferent lymph, implying that this may be a relevant mode of dissemination. Coinfection studies confirmed that lymphatic translocation requires the function of type III secretion system 1 (T3SS-1) but, remarkably, not T3SS-2. This is the first description of the mode and genetics of systemic translocation of serovar Dublin in its natural host.

  18. 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

  19. Free-virus and cell-to-cell transmission in models of equine infectious anemia virus infection.

    Science.gov (United States)

    Allen, Linda J S; Schwartz, Elissa J

    2015-12-01

    Equine infectious anemia virus (EIAV) is a lentivirus in the retrovirus family that infects horses and ponies. Two strains, referred to as the sensitive strain and the resistant strain, have been isolated from an experimentally-infected pony. The sensitive strain is vulnerable to neutralization by antibodies whereas the resistant strain is neutralization-insensitive. The sensitive strain mutates to the resistant strain. EIAV may infect healthy target cells via free virus or alternatively, directly from an infected target cell through cell-to-cell transfer. The proportion of transmission from free-virus or from cell-to-cell transmission is unknown. A system of ordinary differential equations (ODEs) is formulated for the virus-cell dynamics of EIAV. In addition, a Markov chain model and a branching process approximation near the infection-free equilibrium (IFE) are formulated. The basic reproduction number R0 is defined as the maximum of two reproduction numbers, R0s and R0r, one for the sensitive strain and one for the resistant strain. The IFE is shown to be globally asymptotically stable for the ODE model in a special case when the basic reproduction number is less than one. In addition, two endemic equilibria exist, a coexistence equilibrium and a resistant strain equilibrium. It is shown that if R0>1, the infection persists with at least one of the two strains. However, for small infectious doses, the sensitive strain and the resistant strain may not persist in the Markov chain model. Parameter values applicable to EIAV are used to illustrate the dynamics of the ODE and the Markov chain models. The examples highlight the importance of the proportion of cell-to-cell versus free-virus transmission that either leads to infection clearance or to infection persistence with either coexistence of both strains or to dominance by the resistant strain. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Ammonia-Free NOx Control System

    Energy Technology Data Exchange (ETDEWEB)

    Song Wu; Zhen Fan; Andrew H. Seltzer; Richard G. Herman

    2004-09-30

    Research is being conducted under United States Department of Energy (DOE) Contract DEFC26-03NT41865 to develop a new technology to achieve very low levels of NOx emissions from pulverized coal fired boiler systems by employing a novel system level integration between the PC combustion process and the catalytic NOx reduction with CO present in the combustion flue gas. The combustor design and operating conditions will be optimized to achieve atypical flue gas conditions. This approach will not only suppress NOx generation during combustion but also further reduce NOx over a downstream catalytic reactor that does not require addition of an external reductant, such as ammonia. This report describes the work performed during the July 1 to September 30, 2004 time period.

  1. Ammonia-Free NOx Control System

    Energy Technology Data Exchange (ETDEWEB)

    Song Wu; Zhen Fan; Andrew H. Seltzer

    2005-09-30

    Research is being conducted under United States Department of Energy (DOE) Contract DEFC26-03NT41865 to develop a new technology to achieve very low levels of NOx emissions from pulverized coal fired boiler systems by employing a novel system level integration between the PC combustion process and the catalytic NOx reduction with CO present in the combustion flue gas. The combustor design and operating conditions will be optimized to achieve atypical flue gas conditions. This approach will not only suppress NOx generation during combustion but also further reduce NOx over a downstream catalytic reactor that does not require addition of an external reductant, such as ammonia. This report describes the work performed during the July 1 to September 30, 2005 time period.

  2. Ammonia-Free NOx Control System

    Energy Technology Data Exchange (ETDEWEB)

    Song Wu; Zhen Fan; Richard G. Herman

    2004-12-31

    Research is being conducted under United States Department of Energy (DOE) Contract DEFC26-03NT41865 to develop a new technology to achieve very low levels of NOx emissions from pulverized coal fired boiler systems by employing a novel system level integration between the PC combustion process and the catalytic NOx reduction with CO present in the combustion flue gas. The combustor design and operating conditions will be optimized to achieve atypical flue gas conditions. This approach will not only suppress NOx generation during combustion but also further reduce NOx over a downstream catalytic reactor that does not require addition of an external reductant, such as ammonia. This report describes the work performed during the October 1 to December 30, 2004 time period.

  3. Free-Form Kinetic Reciprocal System

    DEFF Research Database (Denmark)

    Parigi, Dario; Sassone, Mario

    2011-01-01

    Kinetic Reciprocal System (KRS) are innovative moveable structures based on the principle of reciprocity [1] with internal pin-slot constraints [2]. The analysis of KRS kinematic and static determinacy is developed through the construction of kinematic matrices, accordingly with [3...... of the required kinetic behaviour and its validity has been tested on physical models. A set of case studies and possible applications are described, focusing on the morphologic variety of results and on the efficiency of the kinetic performance....

  4. Cell-Free DNA Screening for Aneuploidy and Microdeletion Syndromes.

    Science.gov (United States)

    Shaffer, Brian L; Norton, Mary E

    2018-03-01

    Cell-free DNA (cfDNA) screening for the common aneuploidies is an accurate noninvasive screen for the common autosomal and sex chromosome aneuploidies. However, cfDNA screening should not be considered a diagnostic test, and the positive predictive value should be used in counseling women with a positive test regarding the option for diagnostic testing. Compared with traditional screening, cfDNA may not detect as many chromosomal abnormalities of importance. Furthermore, due to the low prevalence of recurrent copy number variants, the clinical utility in screening for microdeletions and duplications is uncertain and is not recommended for the general obstetric population. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Efficient method to create integration-free, virus-free, Myc and Lin28-free human induced pluripotent stem cells from adherent cells

    Science.gov (United States)

    Kamath, Anant; Ternes, Sara; McGowan, Stephen; English, Anthony; Mallampalli, Rama; Moy, Alan B

    2017-01-01

    Aim: Nonviral induced pluripotent stem cell (IPSC) reprogramming is not efficient without the oncogenes, Myc and Lin28. We describe a robust Myc and Lin28-free IPSC reprogramming approach using reprogramming molecules. Methods: IPSC colony formation was compared in the presence and absence of Myc and Lin28 by the mixture of reprogramming molecules and episomal vectors. Results: While more colonies were observed in cultures transfected with the aforementioned oncogenes, the Myc and Lin28-free method achieved the same reprogramming efficiency as reports that used these oncogenes. Further, all colonies were fully reprogrammed based on expression of SSEA4, even in the absence of Myc and Lin28. Conclusion: This approach satisfies an important regulatory pathway for developing IPSC cell therapies with lower clinical risk. PMID:28884008

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  7. 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.

  8. Free Radicals Mediate Systemic Acquired Resistance

    Directory of Open Access Journals (Sweden)

    Caixia Wang

    2014-04-01

    Full Text Available Systemic acquired resistance (SAR is a form of resistance that protects plants against a broad spectrum of secondary infections. However, exploiting SAR for the protection of agriculturally important plants warrants a thorough investigation of the mutual interrelationships among the various signals that mediate SAR. Here, we show that nitric oxide (NO and reactive oxygen species (ROS serve as inducers of SAR in a concentration-dependent manner. Thus, genetic mutations that either inhibit NO/ROS production or increase NO accumulation (e.g., a mutation in S-nitrosoglutathione reductase [GSNOR] abrogate SAR. Different ROS function additively to generate the fatty-acid-derived azelaic acid (AzA, which in turn induces production of the SAR inducer glycerol-3-phosphate (G3P. Notably, this NO/ROS→AzA→G3P-induced signaling functions in parallel with salicylic acid-derived signaling. We propose that the parallel operation of NO/ROS and SA pathways facilitates coordinated regulation in order to ensure optimal induction of SAR.

  9. Label-free haemogram using wavelength modulated Raman spectroscopy for identifying immune-cell subset

    Science.gov (United States)

    Ashok, Praveen C.; Praveen, Bavishna B.; Campbell, Elaine C.; Dholakia, Kishan; Powis, Simon J.

    2014-03-01

    Leucocytes in the blood of mammals form a powerful protective system against a wide range of dangerous pathogens. There are several types of immune cells that has specific role in the whole immune system. The number and type of immune cells alter in the disease state and identifying the type of immune cell provides information about a person's state of health. There are several immune cell subsets that are essentially morphologically identical and require external labeling to enable discrimination. Here we demonstrate the feasibility of using Wavelength Modulated Raman Spectroscopy (WMRS) with suitable machine learning algorithms as a label-free method to distinguish between different closely lying immune cell subset. Principal Component Analysis (PCA) was performed on WMRS data from single cells, obtained using confocal Raman microscopy for feature reduction, followed by Support Vector Machine (SVM) for binary discrimination of various cell subset, which yielded an accuracy >85%. The method was successful in discriminating between untouched and unfixed purified populations of CD4+CD3+ and CD8+CD3+ T lymphocyte subsets, and CD56+CD3- natural killer cells with a high degree of specificity. It was also proved sensitive enough to identify unique Raman signatures that allow clear discrimination between dendritic cell subsets, comprising CD303+CD45+ plasmacytoid and CD1c+CD141+ myeloid dendritic cells. The results of this study clearly show that WMRS is highly sensitive and can distinguish between cell types that are morphologically identical.

  10. 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.

  11. 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.

  12. 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.

  13. 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)

  14. 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.

  15. Imaging free radicals in organelles, cells, tissue, and in vivo with immuno-spin trapping.

    Science.gov (United States)

    Mason, Ronald Paul

    2016-08-01

    The accurate and sensitive detection of biological free radicals in a reliable manner is required to define the mechanistic roles of such species in biochemistry, medicine and toxicology. Most of the techniques currently available are either not appropriate to detect free radicals in cells and tissues due to sensitivity limitations (electron spin resonance, ESR) or subject to artifacts that make the validity of the results questionable (fluorescent probe-based analysis). The development of the immuno-spin trapping technique overcomes all these difficulties. This technique is based on the reaction of amino acid- and DNA base-derived radicals with the spin trap 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) to form protein- and DNA-DMPO nitroxide radical adducts, respectively. These adducts have limited stability and decay to produce the very stable macromolecule-DMPO-nitrone product. This stable product can be detected by mass spectrometry, NMR or immunochemistry by the use of anti-DMPO nitrone antibodies. The formation of macromolecule-DMPO-nitrone adducts is based on the selective reaction of free radical addition to the spin trap and is thus not subject to artifacts frequently encountered with other methods for free radical detection. The selectivity of spin trapping for free radicals in biological systems has been proven by ESR. Immuno-spin trapping is proving to be a potent, sensitive (a million times higher sensitivity than ESR), and easy (not quantum mechanical) method to detect low levels of macromolecule-derived radicals produced in vitro and in vivo. Anti-DMPO antibodies have been used to determine the distribution of free radicals in cells and tissues and even in living animals. In summary, the invention of the immuno-spin trapping technique has had a major impact on the ability to accurately and sensitively detect biological free radicals and, subsequently, on our understanding of the role of free radicals in biochemistry, medicine and toxicology. Published by

  16. Classification of blood cells and tumor cells using label-free ultrasound and photoacoustics.

    Science.gov (United States)

    Strohm, Eric M; Kolios, Michael C

    2015-08-01

    A label-free method that can identify cells in a blood sample using high frequency photoacoustic and ultrasound signals is demonstrated. When the wavelength of the ultrasound or photoacoustic wave is similar to the size of a single cell (frequencies of 100-500 MHz), unique periodic features occur within the ultrasound and photoacoustic power spectrum that depend on the cell size, structure, and morphology. These spectral features can be used to identify different cell types present in blood, such as red blood cells (RBCs), white blood cells (WBCs), and circulating tumor cells. Circulating melanoma cells are ideal for photoacoustic detection due to their endogenous optical absorption properties. Using a 532 nm pulsed laser and a 375 MHz transducer, the ultrasound and photoacoustic signals from RBCs, WBCs, and melanoma cells were individually measured in an acoustic microscope to examine how the signals change between cell types. A photoacoustic and ultrasound signal was detected from RBCs and melanoma cells; only an ultrasound signal was detected from WBCs. The different cell types were distinctly separated using the ultrasound and photoacoustic signal amplitude and power spectral periodicity. The size of each cell was also estimated from the spectral periodicity. For the first time, sound waves generated using pulse-echo ultrasound and photoacoustics have been used to identify and size single cells, with applications toward counting and identifying cells, including circulating melanoma cells. © 2015 International Society for Advancement of Cytometry.

  17. Fuel cell system with interconnect

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhien; Goettler, Richard

    2016-12-20

    The present invention includes an integrated planar, series connected fuel cell system having electrochemical cells electrically connected via interconnects, wherein the anodes of the electrochemical cells are protected against Ni loss and migration via an engineered porous anode barrier layer.

  18. 'Sit and tilt' preparation for subscapular system free flaps.

    Science.gov (United States)

    Mark, J; Patwa, H; Costello, M S; Patil, Y

    2018-02-01

    The ablation of advanced head and neck cancer often results in large three-dimensional defects that require free tissue transfer to optimally address functional and cosmetic issues. The subscapular system is a highly versatile donor site for flaps used for head and neck reconstruction. Traditional methods of harvesting subscapular flaps require repositioning and re-preparing, which significantly increases the operative time and prevents simultaneous harvesting of the flap. This paper presents our experience of a single-stage 'sit and tilt' technique, which provides a convenient method for harvesting subscapular system free flaps without significant repositioning. This technique was used for a variety of head and neck defects, and body habitus did not seem to affect free tissue harvesting. It is hoped that utilisation of this preparation and harvesting technique will make head and neck surgeons more willing to take advantage of the subscapular system.

  19. 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.

  20. 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.

  1. Numerical Optimization of Lead Free Perovskite Solar Cell

    Science.gov (United States)

    Hossain, Mohammad; Daif, Ounsi; Amin, Nowshad; Alharbi, Fahhad; Tabet, Nouar

    Alternative perovskite solar cells are emerging rapidly to compete with other conventional photovoltaic materials. However, toxic lead content of organic-inorganic perovskite is an issue to be addressed briefly. In this work, we propose to use organo-tin halide perovskite absorber layer to replace toxic Pb-based light harvester. We perform a numerical optimization of the layer thicknesses both from the optical and electrical points of view, using SCAPS (Solar Cell Capacitance Simulator) and analytical calculations. We study two types of electron conducting materials (n-ZnO and n-TiO2). Both ZnO/CH3NH3SnI3/Spiro-OMETAD and TiO2/CH3NH3SnI3/Spiro-OMETAD structures show the conversion efficiencies above 15% of (respectively 15.44% and 15.64%). Accordingly, we present a fully optimized device architecture. The analyzed results indicate the feasible fabrication of high efficiency Pb-free perovskite solar cells.

  2. 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

  3. The shipboard employment of a free electron laser weapon system

    OpenAIRE

    Allgaier, Gregory G.

    2003-01-01

    Approved for Public Release; Distribution is Unlimited A megawatt (MW) class Free Electron Laser (FEL) shows promise as a new weapon for antiship cruise missile defense. An FEL weapon system delivers energy at the speed of light at controllable energy levels, giving the war fighter new engagement options. Considerations for this weapon system include employment, design, and stability. In order to reach a MW class laser, system parameters must be optimized and the high power optical beam mu...

  4. 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.

  5. Whole-cell biosensor for label-free detection of GPCR-mediated drug responses in personal cell lines.

    Science.gov (United States)

    Hillger, Julia M; Schoop, Jeffison; Boomsma, Dorret I; Slagboom, P Eline; IJzerman, Adriaan P; Heitman, Laura H

    2015-12-15

    Deciphering how genetic variation in drug targets such as G protein-coupled receptors (GPCRs) affects drug response is essential for precision medicine. GPCR signaling is traditionally investigated in artificial cell lines which do not provide sufficient physiological context. Patient-derived cell lines such as lymphoblastoid cell lines (LCLs) could represent the ideal cellular model system. Here we describe a novel label-free, whole-cell biosensor method for characterizing GPCR-mediated drug responses in LCLs. Generally, such biosensor technology is deemed only compatible with adherent cell lines. We optimized and applied the methodology to study cellular adhesion properties as well as GPCR drug responses in LCLs, which are suspension cells. Coating the detector surface with the extracellular matrix protein fibronectin resulted in cell adherence and allowed detection of cellular responses. A prototypical GPCR present on these cells, i.e. the cannabinoid receptor 2 (CB2), was selected for pharmacological characterization. Receptor activation with the agonist JWH133, blockade by antagonist AM630 as well as downstream signaling inhibition by PTX could be monitored sensitively and receptor-specifically. Potencies and effects were comparable between LCLs of two genetically unrelated individuals, providing the proof-of-principle that this biosensor technology can be applied to LCLs, despite their suspension cell nature, in order to serve as an in vitro model system for the evaluation of individual genetic influences on GPCR-mediated drug responses. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Long-term serial cultivation of mouse induced pluripotent stem cells in serum-free and feeder-free defined medium.

    Science.gov (United States)

    Yamasaki, Sachiko; Nabeshima, Kou; Sotomaru, Yusuke; Taguchi, Yuki; Mukasa, Hanae; Furue, Miho K; Sato, J Denry; Okamoto, Tetsuji

    2013-01-01

    Mouse embryonic stem (mES) cells and mouse induced pluripotent stem (miPS) cells are commonly maintained on inactivated mouse embryonic fibroblast feeder cells in medium supplemented with fetal bovine serum or proprietary replacements. An undefined medium containing unknown quantities of reagents has limited the development of applications for pluripotent cells because of the relative lack of knowledge regarding cell responses to differentiating growth factors. Therefore we developed a serum-free medium, designated ESF7, in which mES cells can be maintained in an undifferentiated state without feeder cells. The medium was tested for culturing miPS cells. The miPS cells have been maintained in ESF7 medium for more than 3 years with an undifferentiated phenotype manifested by the expression of pluripotency marker genes and alkaline phosphatase, and these cells exhibited largely normal karyotypes. Furthermore, we found that fibroblast growth factor-2 (FGF-2) with heparin induced miPS cell differentiation into neuronal cells, both in an adherent monolayer and in embryoid body suspension culture. Moreover, we found that FGF-2 with bone morphogenetic protein 2 induced miPS cell differentiation into cardiomyocytes in embryoid body suspension culture. Furthermore, we transplanted subcutaneously miPS cells maintained in ESF7 into the dorsal flanks of SCID mice; all of the transplants produced tumors with tissues derived from all three embryonic germ layers. As this simple serum-free adherent monoculture system supports the long-term propagation of pluripotent iPS cells in vitro, it will allow us to elucidate cell responses to growth factors under defined conditions, and it should provide useful information for differentiation protocols for human iPS cells.

  7. 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.

  8. Energy efficiency of platinum-free alkaline direct formate fuel cells

    International Nuclear Information System (INIS)

    Wang, L.Q.; Bellini, M.; Filippi, J.; Folliero, M.; Lavacchi, A.; Innocenti, M.; Marchionni, A.; Miller, H.A.; Vizza, F.

    2016-01-01

    Highlights: • Alkaline Platinum-free formate fuel cells top 42% efficiency @ 60 °C. • Palladium efficiently oxidizes formate. • Power density exceeding 250 mW cm −2 @ 60 °C has been achieved. • Performance exceeds that of Alkaline Direct Alcohol Fuel Cell. - Abstract: We report the energy performance of a new platinum-free alkaline direct formate fuel cell, equipped with a commercial anion exchange membrane, a nanostructured Pd/C anode and a Fe–Co/C cathode. The cell was investigated both at room temperature and at 60 °C for the determination of the following parameters: (i) maximum power density, (ii) delivered energy, (iii) faradic (fuel conversion) and energy efficiency. These parameters show a dramatic dependence on fuel composition. The highest energy efficiency is obtained using high energy density fuel (4 M KCOOH and 4 M KOH) and with a maximum operating temperature of 60 °C. This represents a key step in the progress of alkaline platinum-free DFFC technology, demonstrating their potential as power sources for portable electronic devices and remote power generation systems. For example, a fuel load of 750 ml in a DFFC device operating at 60 °C would be able to produce 90 W h of energy, that required to fully charge the battery of a laptop computer.

  9. 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

  10. Synthesis and cell-free cloning of DNA libraries using programmable microfluidics.

    Science.gov (United States)

    Ben Yehezkel, Tuval; Rival, Arnaud; Raz, Ofir; Cohen, Rafael; Marx, Zipora; Camara, Miguel; Dubern, Jean-Frédéric; Koch, Birgit; Heeb, Stephan; Krasnogor, Natalio; Delattre, Cyril; Shapiro, Ehud

    2016-02-29

    Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. 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.

  12. Cell-free biology: exploiting the interface between synthetic biology and synthetic chemistry.

    Science.gov (United States)

    Harris, D Calvin; Jewett, Michael C

    2012-10-01

    Just as synthetic organic chemistry once revolutionized the ability of chemists to build molecules (including those that did not exist in nature) following a basic set of design rules, cell-free synthetic biology is beginning to provide an improved toolbox and faster process for not only harnessing but also expanding the chemistry of life. At the interface between chemistry and biology, research in cell-free synthetic systems is proceeding in two different directions: using synthetic biology for synthetic chemistry and using synthetic chemistry to reprogram or mimic biology. In the coming years, the impact of advances inspired by these approaches will make possible the synthesis of nonbiological polymers having new backbone compositions, new chemical properties, new structures, and new functions. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Biochemical Preparation of Cell Extract for Cell-Free Protein Synthesis without Physical Disruption.

    Directory of Open Access Journals (Sweden)

    Kei Fujiwara

    Full Text Available Cell-free protein synthesis (CFPS is a powerful tool for the preparation of toxic proteins, directed protein evolution, and bottom-up synthetic biology. The transcription-translation machinery for CFPS is provided by cell extracts, which usually contain 20-30 mg/mL of proteins. In general, these cell extracts are prepared by physical disruption; however, this requires technical experience and special machinery. Here, we report a method to prepare cell extracts for CFPS using a biochemical method, which disrupts cells through the combination of lysozyme treatment, osmotic shock, and freeze-thaw cycles. The resulting cell extracts showed similar features to those obtained by physical disruption, and was able to synthesize active green fluorescent proteins in the presence of appropriate chemicals to a concentration of 20 μM (0.5 mg/mL.

  14. Enabling Lead Free Interconnects in DoD Weapon Systems

    Science.gov (United States)

    2017-09-28

    Develop tailored Lead-free webinars for the Program Management and Systems Engineering. These webinars target interconnect reliability and whisker risk to...the needs of the weapon system program managers . Examples of risk will be given and information will be provided on where to access more detailed...Electronics Risk Mitigation: WP-201573T2 Summary Strategic Environmental Research and Development Program (SERDP) Environmental Security Technology

  15. A note on controllability of deterministic context-free systems

    Czech Academy of Sciences Publication Activity Database

    Masopust, Tomáš

    2012-01-01

    Roč. 48, č. 8 (2012), s. 1934-1937 ISSN 0005-1098 R&D Projects: GA ČR(CZ) GPP202/11/P028 Institutional support: RVO:67985840 Keywords : discrete-event systems * controllability * deterministic context-free systems Subject RIV: BA - General Mathematics Impact factor: 2.919, year: 2012 http://www.sciencedirect.com/science/article/pii/S0005109812002543

  16. 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.

  17. Determination of the potency of a novel saw palmetto supercritical CO2 extract (SPSE for 5α-reductase isoform II inhibition using a cell-free in vitro test system

    Directory of Open Access Journals (Sweden)

    Pais P

    2016-04-01

    Full Text Available Pilar Pais, Agustí Villar, Santiago Rull Euromed, Barcelona, Spain Background: The nicotinamide adenine dinucleotide phosphate-dependent membrane protein 5α-reductase catalyses the conversion of testosterone to the most potent androgen – 5α-dihydrotestosterone. Two 5α-reductase isoenzymes are expressed in humans: type I and type II. The latter is found primarily in prostate tissue. Saw palmetto extract (SPE has been used extensively in the treatment of lower urinary tract symptoms secondary to benign prostatic hyperplasia (BPH. The pharmacological effects of SPE include the inhibition of 5α-reductase, as well as anti-inflammatory and antiproliferative effects. Clinical studies of SPE have been inconclusive – some have shown significant results, and others have not – possibly the result of varying bioactivities of the SPEs used in the studies. Purpose: To determine the in vitro potency in a cell-free test system of a novel SP supercritical CO2 extract (SPSE, an inhibitor of the 5α-reductase isoenzyme type II. Materials and methods: The inhibitory potency of SPSE was compared to that of finasteride, an approved 5α-reductase inhibitor, on the basis of the enzymatic conversion of the substrate androstenedione to the 5α-reduced product 5α-androstanedione. Results: By concentration-dependent inhibition of 5α-reductase type II in vitro (half-maximal inhibitory concentration 3.58±0.05 µg/mL, SPSE demonstrated competitive binding toward the active site of the enzyme. Finasteride, the approved 5α-reductase inhibitor tested as positive control, led to 63%–75% inhibition of 5α-reductase type II. Conclusion: SPSE effectively inhibits the enzyme that has been linked to BPH, and the amount of extract required for activity is comparatively low. It can be confirmed from the results of this study that SPSE has bioactivity that promotes prostate health at a level that is superior to that of many other phytotherapeutic extracts. The

  18. 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.

  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. Forced versus free traffic in an automated milking system

    DEFF Research Database (Denmark)

    Munksgaard, Lene; Rushen, J.; de Passillé, A.M.

    2011-01-01

    Cows in automated milking systems with free access to feeders sometimes show a reduced use of the robotic milkers, while forced traffic where cows have to pass through the robot to reach the feeders may reduce feeding time and frequency. We examined two groups of 35 lactating cows. For 21 d, one....... The average number of visits to the robot, either with or without milking, did not differ between the two types of traffic. On average cows lay down for 12.3 h/d, with no significant difference between free and forced traffic. The type of traffic did not affect the duration of time spent in the feeding area....... With both forced and free traffic, there was marked diurnal variation in feeding, with relatively little feeding occurring between 2200 h and 0800 h. Use of forced traffic did not reduce feeding time. It did not improve access to the robot milker or eliminate diurnal variation. Differences between cows...

  1. Unsupervised Calculation of Free Energy Barriers in Large Crystalline Systems

    Science.gov (United States)

    Swinburne, Thomas D.; Marinica, Mihai-Cosmin

    2018-03-01

    The calculation of free energy differences for thermally activated mechanisms in the solid state are routinely hindered by the inability to define a set of collective variable functions that accurately describe the mechanism under study. Even when possible, the requirement of descriptors for each mechanism under study prevents implementation of free energy calculations in the growing range of automated material simulation schemes. We provide a solution, deriving a path-based, exact expression for free energy differences in the solid state which does not require a converged reaction pathway, collective variable functions, Gram matrix evaluations, or probability flux-based estimators. The generality and efficiency of our method is demonstrated on a complex transformation of C 15 interstitial defects in iron and double kink nucleation on a screw dislocation in tungsten, the latter system consisting of more than 120 000 atoms. Both cases exhibit significant anharmonicity under experimentally relevant temperatures.

  2. Animal Cell Expression Systems.

    Science.gov (United States)

    Butler, M; Reichl, U

    2017-10-03

    control. Over the past decade, however, there have been various commercial influenza vaccines made available from cell technology using animal host cells. Analysis of glycosylation control shows that the type of host cell has the greatest influence on the final analyzed glycan profile. Other factors such as the virus strain, the cultivation system, or various process parameters have been shown to have only a minor effect on the glycosylation pattern. We predict that the analysis of glycan profiles in viral vaccines will become increasingly important in the development and consistent manufacturing of safe and potent vaccines. Graphical Abstract.

  3. 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)

  4. 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)

  5. 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.

  6. Cell-Free Production of Protein Biologics Within 24 H.

    Science.gov (United States)

    Sullivan, Challise J; Pendleton, Erik D; Dresios, John

    2018-01-01

    Protein biologics have emerged as a safe and effective group of drug products that can be used in a variety of medical disorders and clinical settings, including treatment of orphan diseases, personalized medicine, and point-of-care applications. However, the full potential of protein biologics for such applications will not be realized until there are methods available for rapid and cost-effective production of small scale products for individual needs. Here, we describe a modular and scalable method for rapid and adaptable production of protein-based medical products at small doses. The method includes cell-free synthesis of the protein target in a reactor module followed by a fluidic process for protein purification. As a proof of concept, we describe the application of this method for expression and purification of a bioactive pharmaceutically relevant protein biologic, recombinant human erythropoietin, at a single dose within 24 h. This method can be applied toward the development of automated platforms for rapid and adaptive production of protein biologics at the point of care in response to specific medical needs.

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

    Directory of Open Access Journals (Sweden)

    Mark R. Openshaw

    2016-02-01

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

  8. Uracil excision repair in Mycobacterium tuberculosis cell-free extracts.

    Science.gov (United States)

    Kumar, Pradeep; Bharti, Sanjay Kumar; Varshney, Umesh

    2011-05-01

    Uracil excision repair is ubiquitous in all domains of life and initiated by uracil DNA glycosylases (UDGs) which excise the promutagenic base, uracil, from DNA to leave behind an abasic site (AP-site). Repair of the resulting AP-sites requires an AP-endonuclease, a DNA polymerase, and a DNA ligase whose combined activities result in either short-patch or long-patch repair. Mycobacterium tuberculosis, the causative agent of tuberculosis, has an increased risk of accumulating uracils because of its G + C-rich genome, and its niche inside host macrophages where it is exposed to reactive nitrogen and oxygen species, two major causes of cytosine deamination (to uracil) in DNA. In vitro assays to study DNA repair in this important human pathogen are limited. To study uracil excision repair in mycobacteria, we have established assay conditions using cell-free extracts of M. tuberculosis and M. smegmatis (a fast-growing mycobacterium) and oligomer or plasmid DNA substrates. We show that in mycobacteria, uracil excision repair is completed primarily via long-patch repair. In addition, we show that M. tuberculosis UdgB, a newly characterized family 5 UDG, substitutes for the highly conserved family 1 UDG, Ung, thereby suggesting that UdgB might function as backup enzyme for uracil excision repair in mycobacteria. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. 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...

  10. Mechanically stable solvent-free lipid bilayers in nano- and micro-tapered apertures for reconstitution of cell-free synthesized hERG channels.

    Science.gov (United States)

    Tadaki, Daisuke; Yamaura, Daichi; Araki, Shun; Yoshida, Miyu; Arata, Kohei; Ohori, Takeshi; Ishibashi, Ken-Ichi; Kato, Miki; Ma, Teng; Miyata, Ryusuke; Tozawa, Yuzuru; Yamamoto, Hideaki; Niwano, Michio; Hirano-Iwata, Ayumi

    2017-12-18

    The self-assembled bilayer lipid membrane (BLM) is the basic component of the cell membrane. The reconstitution of ion channel proteins in artificially formed BLMs represents a well-defined system for the functional analysis of ion channels and screening the effects of drugs that act on them. However, because BLMs are unstable, this limits the experimental throughput of BLM reconstitution systems. Here we report on the formation of mechanically stable solvent-free BLMs in microfabricated apertures with defined nano- and micro-tapered edge structures. The role of such nano- and micro-tapered structures on the stability of the BLMs was also investigated. Finally, this BLM system was combined with a cell-free synthesized human ether-a-go-go-related gene channel, a cardiac potassium channel whose relation to arrhythmic side effects following drug treatment is well recognized. Such stable BLMs as these, when combined with a cell-free system, represent a potential platform for screening the effects of drugs that act on various ion-channel genotypes.

  11. 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.

  12. Efficiency of Free Energy Transduction in Autonomous Systems

    OpenAIRE

    Kawaguchi, Kyogo; Sano, Masaki

    2011-01-01

    We consider the thermodynamics of chemical coupling from the viewpoint of free energy transduction efficiency. In contrast to an external parameter-driven stochastic energetics setup, the dynamic change of the equilibrium distribution induced by chemical coupling, adopted, for example, in biological systems, is inevitably an autonomous process. We found that the efficiency is bounded by the ratio between the non-symmetric and the symmetrized Kullback-Leibler distance, which is significantly l...

  13. Generation of transgene-free lung disease-specific human iPS cells using a single excisable lentiviral stem cell cassette

    OpenAIRE

    Somers, A.; Jean, J.C.; Sommer, C.A.; Omari, A.; Ford, C.C.; Mills, J.A.; Ying, L.; Sommer Gianotti, A.; Jean, J.M.; Smith, B.W.; Lafyatis, R.; Demierre, M.F.; Weiss, D.J.; French, D.L.; Gadue, P.

    2010-01-01

    The development of methods to achieve efficient reprogramming of human cells while avoiding the permanent presence of reprogramming transgenes represents a critical step towards the use of induced pluripotent stem cells (iPSC) for clinical purposes, such as disease modeling or reconstituting therapies. While several methods exist for generating iPSC free of reprogramming transgenes from mouse cells or neonatal normal human tissues, a sufficiently efficient reprogramming system is still needed...

  14. Clinical applications of urinary cell-free DNA in cancer: current insights and promising future.

    Science.gov (United States)

    Lu, Tian; Li, Jinming

    2017-01-01

    Liquid biopsy is gaining significant attention as a tool for unveiling the molecular landscape of tumor and holds great promise for individualized medicine for cancer. Cell-free DNA serves as an extremely important component of liquid biopsy for cancer, and cell-free DNA in urine is even promising due to the remarkable advantage of urine as an ultra-noninvasive sample source over tissue and blood. Compared with the widely studied cell-free DNA in blood, less is known about the role of urinary cell-free DNA. Urinary cell-free DNA has the ability to give comprehensive and crucial information on cancer as it carries genetic messages from cells shedding directly into urine as well as transporting from circulation. As an indispensable component of liquid biopsy, urinary cell-free DNA is believed to have the potential of being a useful and ultra-noninvasive tool for cancer screening, diagnosis, prognosis, and monitoring of cancer progression and therapeutic effect. In this review, we provide the current insights into the clinical applications of urinary cell-free DNA in cancer. We also introduce the basic biological significance and some technical issues in the detection of urinary cell-free DNA.

  15. 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.

  16. 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...

  17. Carcinogenicity, efficiency and biosafety analysis in xeno-free human amniotic stem cells for regenerative medical therapies.

    Science.gov (United States)

    Phermthai, Tatsanee; Thongbopit, Sasiprapa; Pokathikorn, Puttachart; Wichitwiengrat, Suparat; Julavijitphong, Suphakde; Tirawanchai, Nednapis

    2017-08-01

    Human amniotic mesenchymal stromal cells (hAMSCs) are a potent and attractive stem cell source for use in regenerative medicine. However, the safe uses of therapeutic-grade MSCs are equally as important as the efficiency of MSCs. To provide efficient, clinic-compliant (safe for therapeutic use) MSCs, hAMSC lines that completely eliminate the use of animal products and have been characterized for carcinogenicity and biosafety are required. Here, we have efficiently generated 10 hAMSC lines under human umbilical cord blood serum (hUCS)-supplemented medium (xeno-free culture) and fetal bovine serum (FBS)-supplemented medium (standard culture) and investigated carcinogenicity and immunosuppressive properties in the resultant hAMSC lines. All hAMSC lines were examined for efficiency (growth kinetics, cryopreservation, telomere length, phenotypic characterization, differentiation potential), carcinogenicity (proto-oncogene and tumor suppressor gene and epigenomic stability) and safety (immunosuppressive properties). Stem cell characteristics between the xeno-free hAMSC lines and the cell lines generated using the standard culture system showed no differences. Xeno-free hAMSC lines displayed normal growth proliferation potential, morphological, karyotypic, phenotypic differentiation properties and telomere lengths. Additionally, they retained normal immunosuppressive effects. As a marker of carcinogenicity and biosafety, proto-oncogenes expression levels showed no differences in xeno-free hAMSCs, and we detected no SNP mutations on hotspot codons of the P53 tumor suppressor gene and stable epigenomic imprinting in xeno-free hAMSC lines. Xeno-free hAMSC lines retain essential stem cell characteristics, with a high degree of certainty for meeting biosafety and carcinogenicity standards for a xeno-free system supplemented with allogenic hUCS. The cell lines are suitable and valuable for therapeutic purposes. Copyright © 2017 International Society for Cellular Therapy

  18. 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

  19. High pressure gas storage system consumable analyses for ALT flights free flight 1 and free flight 6

    Science.gov (United States)

    Hurst, J. E.

    1977-01-01

    Consumables analyses are presented for the high pressure gas storage oxygen (O2), and hydrogen (H2) reactant systems for orbiter vehicle 101 approach and landing tests, for two flights, free flight 1 and free flight 6 are given. The consumables analyses are based on average power data. The required system and mission data updates were made in order to perform the analyses, notably the thermal environment profiles of the reactant storage cylinders and the power profile for the Electrical power subsystem. No mission-dependent environment profiles were provided; therefore, nominal free flight mission profile was used to generate environment profiles for free flights 1 and 6.

  20. Circulating Cell-Free DNA Differentiates Severity of Inflammation.

    Science.gov (United States)

    Frank, Mayu O

    2016-10-01

    As the U.S. population ages, the incidence of chronic disease will rise. Chronic diseases have been linked to chronic inflammation. The purpose of this review is to summarize the literature on cell-free DNA (cfDNA) in relation to inflammation. PubMed, EMBASE, and Web of Science were searched. Inclusion criteria were noninterventional studies on acute and chronic inflammation, autoimmunity, and infection published in English after 2000, conducted in humans using the fluorescence method of quantifying DNA. Of the 442 articles retrieved, 83 were identified for full-text review and 13 remained after application of inclusion criteria. Of the reviewed studies, three involved acute inflammation, six involved chronic inflammation, and four involved infection. Healthy controls with interpretable results were included in six studies, three of which used the Quant-iT high-sensitivity DNA kit and found cfDNA quantities near 800 ng/ml, while the other three used other fluorescence methods and found quantities below 100 ng/ml. All 13 studies compared groups, and all but 1 found statistically significant differences between them. Among studies using the Quant-iT reagent, levels were higher in infection than in chronic inflammation. Among studies that used other reagents, levels increased from chronic to acute inflammation to severe infection. CfDNA levels were associated with mortality and with clinical outcomes in acute inflammation and infection. Most studies assessed cfDNA's correlation with other inflammation biomarkers and found inconclusive results. There appears to be an association between inflammation and cfDNA. Further research is necessary before cfDNA can be used clinically as a measure of inflammation. © The Author(s) 2016.

  1. 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 photovolta...

  2. Circulating cell-free DNA for non-invasive cancer management.

    Science.gov (United States)

    Stewart, Caitlin M; Tsui, Dana W Y

    2018-03-11

    Cell-free DNA (cfDNA) was first identified in human plasma in 1948 and is thought to be released from cells throughout the body into the circulatory system. In cancer, a portion of the cfDNA originates from tumour cells, referred to as circulating-tumour DNA (ctDNA), and can contain mutations corresponding to the patient's tumour, for instance specific TP53 alleles. Profiling of cfDNA has recently become an area of increasing clinical relevance in oncology, in particular due to advances in the sensitivity of molecular biology techniques and development of next generation sequencing technologies, as this allows tumour mutations to be identified and tracked non-invasively. This has opened up new possibilities for monitoring tumour evolution and acquisition of resistance, as well as for guiding treatment decisions when tumour biopsy tissue is insufficient or unavailable. In this review, we will discuss the biology of cell-free nucleic acids, methods of analysis, and the potential clinical uses of these techniques, as well as the on-going clinical development of ctDNA assays. Copyright © 2018. Published by Elsevier Inc.

  3. A noninvasive technique for the measurement of the energetic state of free-suspension mammalian cells.

    Science.gov (United States)

    Ben-Tchavtchavadze, M; Chen, J; Perrier, Michel; Jolicoeur, Mario

    2010-01-01

    A perfusion small-scale bioreactor allowing on-line monitoring of the cell energetic state was developed for free-suspension mammalian cells. The bioreactor was designed to perform in vivo nuclear magnetic resonance (NMR) spectroscopy, which is a noninvasive and nondestructive method that permits the monitoring of intracellular nutrient concentrations, metabolic precursors and intermediates, as well as metabolites and energy shuttles, such as ATP, ADP, and NADPH. The bioreactor was made of a 10-mm NMR tube following a fluidized bed design. Perfusion flow rate allowing for adequate oxygen supply was found to be above 0.79 mL min(-1) for high-density cell suspensions (10(8) cells). Chinese hamster ovary (CHO) cells were studied here as model system. Hydrodynamic studies using coloration/decoloration and residence time distribution measurements were realized to perfect bioreactor design as well as to determine operating conditions bestowing adequate homogeneous mixing and cell retention in the NMR reading zone. In vivo (31)P NMR was performed and demonstrated the small-scale bioreactor platform ability to monitor the cell physiological behavior for 30-min experiments.

  4. 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.

  5. 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.

  6. Optimizing front metallization patterns: Efficiency with aesthetics in free-form solar cells

    NARCIS (Netherlands)

    Gupta, D.K.; Langelaar, M.; Barink, M.; Keulen, F. van

    2016-01-01

    Free-form solar cells are cells of unconventional shapes (e.g. hexagonal, leaf-shaped etc). Their flexible shape adds to the aesthetics of the surroundings as well as allows to place them over objects where conventional solar cells might not fit. Evidently, these cells need to be efficient as well,

  7. Committee Opinion No. 640: Cell-Free DNA Screening For Fetal Aneuploidy.

    Science.gov (United States)

    2015-09-01

    Noninvasive prenatal screening that uses cell-free DNA from the plasma of pregnant women offers tremendous potential as a screening method for fetal aneuploidy. A number of laboratories have validated different techniques for the use of cell-free DNA as a screening test for fetal aneuploidy. All tests have a high sensitivity and specificity for trisomy 18 and trisomy 21, regardless of which molecular technique is used. Women whose results are not reported, indeterminate, or uninterpretable (a "no call" test result) from cell-free DNA screening should receive further genetic counseling and be offered comprehensive ultrasound evaluation and diagnostic testing because of an increased risk of aneuploidy. Patients should be counseled that cell-free DNA screening does not replace the precision obtained with diagnostic tests, such as chorionic villus sampling or amniocentesis and, therefore, is limited in its ability to identify all chromosome abnormalities. Cell-free DNA screening does not assess risk of fetal anomalies such as neural tube defects or ventral wall defects. Patients who are undergoing cell-free DNA screening should be offered maternal serum alpha-fetoprotein screening or ultrasound evaluation for risk assessment. The cell-free DNA screening test should not be considered in isolation from other clinical findings and test results. Management decisions, including termination of the pregnancy, should not be based on the results of the cell-free DNA screening alone. Patients should be counseled that a negative cell-free DNA test result does not ensure an unaffected pregnancy. Given the performance of conventional screening methods, the limitations of cell-free DNA screening performance, and the limited data on cost-effectiveness in the low-risk obstetric population, conventional screening methods remain the most appropriate choice for first-line screening for most women in the general obstetric population.

  8. Committee Opinion Summary No. 640: Cell-Free DNA Screening For Fetal Aneuploidy.

    Science.gov (United States)

    2015-09-01

    Noninvasive prenatal screening that uses cell-free DNA from the plasma of pregnant women offers tremendous potential as a screening method for fetal aneuploidy. A number of laboratories have validated different techniques for the use of cell-free DNA as a screening test for fetal aneuploidy. All tests have a high sensitivity and specificity for trisomy 18 and trisomy 21, regardless of which molecular technique is used. Women whose results are not reported, indeterminate, or uninterpretable (a "no call" test result) from cell-free DNA screening should receive further genetic counseling and be offered comprehensive ultrasound evaluation and diagnostic testing because of an increased risk of aneuploidy. Patients should be counseled that cell-free DNA screening does not replace the precision obtained with diagnostic tests, such as chorionic villus sampling or amniocentesis and, therefore, is limited in its ability to identify all chromosome abnormalities. Cell-free DNA screening does not assess risk of fetal anomalies such as neural tube defects or ventral wall defects. Patients who are undergoing cell-free DNA screening should be offered maternal serum alpha-fetoprotein screening or ultrasound evaluation for risk assessment. The cell-free DNA screening test should not be considered in isolation from other clinical findings and test results. Management decisions, including termination of the pregnancy, should not be based on the results of the cell-free DNA screening alone. Patients should be counseled that a negative cell-free DNA test result does not ensure an unaffected pregnancy. Given the performance of conventional screening methods, the limitations of cell-free DNA screening performance, and the limited data on cost-effectiveness in the low-risk obstetric population, conventional screening methods remain the most appropriate choice for first-line screening for most women in the general obstetric population.

  9. Generation of Storable Retinal Organoids and Retinal Pigmented Epithelium from Adherent Human iPS Cells in Xeno-Free and Feeder-Free Conditions.

    Science.gov (United States)

    Reichman, Sacha; Slembrouck, Amélie; Gagliardi, Giuliana; Chaffiol, Antoine; Terray, Angélique; Nanteau, Céline; Potey, Anais; Belle, Morgane; Rabesandratana, Oriane; Duebel, Jens; Orieux, Gael; Nandrot, Emeline F; Sahel, José-Alain; Goureau, Olivier

    2017-05-01

    Human induced pluripotent stem cells (hiPSCs) are potentially useful in regenerative therapies for retinal disease. For medical applications, therapeutic retinal cells, such as retinal pigmented epithelial (RPE) cells or photoreceptor precursors, must be generated under completely defined conditions. To this purpose, we have developed a two-step xeno-free/feeder-free (XF/FF) culture system to efficiently differentiate hiPSCs into retinal cells. This simple method, relies only on adherent hiPSCs cultured in chemically defined media, bypassing embryoid body formation. In less than 1 month, adherent hiPSCs are able to generate self-forming neuroretinal-like structures containing retinal progenitor cells (RPCs). Floating cultures of isolated structures enabled the differentiation of RPCs into all types of retinal cells in a sequential overlapping order, with the generation of transplantation-compatible CD73 + photoreceptor precursors in less than 100 days. Our XF/FF culture conditions allow the maintenance of both mature cones and rods in retinal organoids until 280 days with specific photoreceptor ultrastructures. Moreover, both hiPSC-derived retinal organoids and dissociated retinal cells can be easily cryopreserved while retaining their phenotypic characteristics and the preservation of CD73 + photoreceptor precursors. Concomitantly to neural retina, this process allows the generation of RPE cells that can be effortlessly amplified, passaged, and frozen while retaining a proper RPE phenotype. These results demonstrate that simple and efficient retinal differentiation of adherent hiPSCs can be accomplished in XF/FF conditions. This new method is amenable to the development of an in vitro GMP-compliant retinal cell manufacturing protocol allowing large-scale production and banking of hiPSC-derived retinal cells and tissues. Stem Cells 2017;35:1176-1188. © 2017 AlphaMed Press.

  10. Strategies to Suspension Serum-Free Adaptation of Mammalian Cell Lines for Recombinant Glycoprotein Production.

    Science.gov (United States)

    Caron, Angelo Luis; Biaggio, Rafael Tagé; Swiech, Kamilla

    2018-01-01

    Serum-free suspension cultures are preferably required for recombinant protein production due to its readiness in upstream/downstream processing and scale-up, therefore increasing process productivity and competitiveness. This type of culture replaces traditional cell culturing as the presence of animal-derived components may introduce lot-a-lot variability and adventitious pathogens to the process. However, adapting cells to serum-free conditions is challenging, time-consuming, and cell line and medium dependent. In this chapter, we present different approaches that can be used to adapt mammalian cell lines from an anchorage-dependent serum supplemented culture to a suspension serum-free culture.

  11. 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.

  12. Elevated Concentrations of Serum Immunoglobulin Free Light Chains in Systemic Lupus Erythematosus Patients in Relation to Disease Activity, Inflammatory Status, B Cell Activity and Epstein-Barr Virus Antibodies

    DEFF Research Database (Denmark)

    Draborg, Anette H; Lydolph, Magnus; Westergaard, Marie

    2015-01-01

    , FLCs' association with Epstein-Barr virus (EBV) antibodies was examined. METHODS: Using a nephelometric assay, κFLC and λFLC concentrations were quantified in sera from 45 SLE patients and 40 healthy controls. SLE patients with renal insufficiency were excluded in order to preclude high concentrations...... of serum FLCs due to decreased clearance. RESULTS: Serum FLC concentrations were significantly elevated in SLE patients compared to healthy controls (pdisease activity (SLE disease activity......OBJECTIVE: In this study, we examined the concentration of serum immunoglobulin free light chains (FLCs) in systemic lupus erythematosus (SLE) patients and investigated its association with various disease parameters in order to evaluate the role of FLCs as a potential biomarker in SLE. Furthermore...

  13. 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.

  14. Cell-Free Transmission of Human Adenovirus by Passive Mass Transfer in Cell Culture Simulated in a Computer Model

    Science.gov (United States)

    Yakimovich, Artur; Gumpert, Heidi; Burckhardt, Christoph J.; Lütschg, Verena A.; Jurgeit, Andreas; Sbalzarini, Ivo F.

    2012-01-01

    Viruses spread between cells, tissues, and organisms by cell-free and cell-cell transmissions. Both mechanisms enhance disease development, but it is difficult to distinguish between them. Here, we analyzed the transmission mode of human adenovirus (HAdV) in monolayers of epithelial cells by wet laboratory experimentation and a computer simulation. Using live-cell fluorescence microscopy and replication-competent HAdV2 expressing green fluorescent protein, we found that the spread of infection invariably occurred after cell lysis. It was affected by convection and blocked by neutralizing antibodies but was independent of second-round infections. If cells were overlaid with agarose, convection was blocked and round plaques developed around lytic infected cells. Infected cells that did not lyse did not give rise to plaques, highlighting the importance of cell-free transmission. Key parameters for cell-free virus transmission were the time from infection to lysis, the dose of free viruses determining infection probability, and the diffusion of single HAdV particles in aqueous medium. With these parameters, we developed an in silico model using multiscale hybrid dynamics, cellular automata, and particle strength exchange. This so-called white box model is based on experimentally determined parameters and reproduces viral infection spreading as a function of the local concentration of free viruses. These analyses imply that the extent of lytic infections can be determined by either direct plaque assays or can be predicted by calculations of virus diffusion constants and modeling. PMID:22787215

  15. 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.

  16. Actuator prototype system by voice commands using free software

    Directory of Open Access Journals (Sweden)

    Jaime Andrango

    2016-06-01

    Full Text Available This prototype system is a software application that through the use of techniques of digital signal processing, extracts information from the user's speech, which is then used to manage the on/off actuator on a peripheral computer when vowels are pronounced. The method applies spectral differences. The application uses the parallel port as actuator, with the information recorded in the memory address 378H. This prototype was developed using free software tools for its versatility and dynamism, and to allow other researchers to base on it for further studies.

  17. MCF-7 Human Breast Cancer Cells Form Differentiated Microtissues in Scaffold-Free Hydrogels

    Science.gov (United States)

    Vantangoli, Marguerite M.; Madnick, Samantha J.; Huse, Susan M.; Weston, Paula; Boekelheide, Kim

    2015-01-01

    Three-dimensional (3D) cultures are increasing in use because of their ability to represent in vivo human physiology when compared to monolayer two-dimensional (2D) cultures. When grown in 3D using scaffold-free agarose hydrogels, MCF-7 human breast cancer cells self-organize to form directionally-oriented microtissues that contain a luminal space, reminiscent of the in vivo structure of the mammary gland. When compared to MCF-7 cells cultured in 2D monolayer culture, MCF-7 microtissues exhibit increased mRNA expression of luminal epithelial markers keratin 8 and keratin 19 and decreased expression of basal marker keratin 14 and the mesenchymal marker vimentin. These 3D MCF-7 microtissues remain responsive to estrogens, as demonstrated by induction of known estrogen target mRNAs following exposure to 17β-estradiol. Culture of MCF-7 cells in scaffold-free conditions allows for the formation of more differentiated, estrogen-responsive structures that are a more relevant system for evaluation of estrogenic compounds than traditional 2D models. PMID:26267486

  18. 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.

  19. Serum-Free Cryopreservation of Human Amniotic Epithelial Cells

    OpenAIRE

    H. Niknejad; H. Peirovi; B. Jambar Noushin

    2013-01-01

    Introduction & Objective: One of the important issues in long term storage of cells is removal of animal serum from cell culture environments. The aim of this study was to evaluate amni-otic fluid (AF), which is full of growth factors, as substitute for fetal bovine serum (FBS) in the cryopreservation protocol. Materials & Methods: In this experimental study human amniotic epithelial cells were isolated from placentas which were seronegative for microbial infections. The cells were preserved ...

  20. 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.

  1. XUV free-electron laser-based projection lithography systems

    Energy Technology Data Exchange (ETDEWEB)

    Newnam, B.E.

    1990-01-01

    Free-electron laser sources, driven by rf-linear accelerators, have the potential to operate in the extreme ultraviolet (XUV) spectral range with more than sufficient average power for high-volume projection lithography. For XUV wavelengths from 100 nm to 4 nm, such sources will enable the resolution limit of optical projection lithography to be extended from 0.25 {mu}m to 0.05{mu}m and with an adequate total depth of focus (1 to 2 {mu}m). Recent developments of a photoinjector of very bright electron beams, high-precision magnetic undulators, and ring-resonator cavities raise our confidence that FEL operation below 100 nm is ready for prototype demonstration. We address the motivation for an XUV FEL source for commercial microcircuit production and its integration into a lithographic system, include reflecting reduction masks, reflecting XUV projection optics and alignment systems, and surface-imaging photoresists. 52 refs., 7 figs.

  2. Conditions for a high plating efficiency of free cell suspensions of Haplopappus gracilis (Nutt) gray.

    Science.gov (United States)

    Werry, P A; Stoffelsen, K M

    1978-07-01

    Suspensions of Haplopappus gracilis cells, containing about 80% free cells, were obt ained from log-phase cultures by filtration through 3 nylon sieves having decreasing mesh widths from 297, 210 and 88 μm. From the free cell suspensions, 75 to 90% of the cells developed into visible colonies when the plating procedure was divided into two steps: a) plating the cells at high concentration in soft agar on feeder agar; b) replating the resulting aggregations at appropriate concentrations on fresh feeder agar. From the results, it is inferred that, in the replating step, the volume of the inoculum is the deciding factor which influences the resulting plating efficiency.

  3. Improved recovery of bisulphite-treated cell-free DNA in plasma

    DEFF Research Database (Denmark)

    Pedersen, Inge Søkilde; Krarup, H.B.; Thorlacius-Ussing, O.

    Detection of cell-free methylated DNA in plasma is a promising tool for tumour diagnosis and monitoring. Due to the very low amount of cell-free DNA in plasma, sensitivity of the detection methods are of utmost importance. The vast majority of currently available methods for analysing DNA...... of PCR amplifying methylated and umethylated MEST. This procedure allows low levels of DNA to be easily and reliably analysed, a prerequisite for the clinical usefulness of cell-free methylated DNA detection in plasma....

  4. 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

    state, may be of significant benefit for cfDNA research. Methods CfDNA was isolated from the growth medium of C3A spheroid cultures in rotating bioreactors during both normal growth and treatment with acetaminophen. Spheroid growth was monitored via planimetry, lactate dehydrogenase activity and glucose...... environment. Combining 3D culture and cfDNA research could, therefore, optimize both research fields.......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...

  5. Label-free identification of white blood cell using optical diffraction tomography (Conference Presentation)

    Science.gov (United States)

    Yoon, Jonghee; Kim, Kyoohyun; Kim, Min-hyeok; Kang, Suk-Jo; Park, YongKeun

    2016-03-01

    White blood cells (WBC) have crucial roles in immune systems which defend the host against from disease conditions and harmful invaders. Various WBC subsets have been characterized and reported to be involved in many pathophysiologic conditions. It is crucial to isolate a specific WBC subset to study its pathophysiological roles in diseases. Identification methods for a specific WBC population are rely on invasive approaches, including Wright-Gimesa staining for observing cellular morphologies and fluorescence staining for specific protein markers. While these methods enable precise classification of WBC populations, they could disturb cellular viability or functions. In order to classify WBC populations in a non-invasive manner, we exploited optical diffraction tomography (ODT). ODT is a three-dimensional (3-D) quantitative phase imaging technique that measures 3-D refractive index (RI) distributions of individual WBCs. To test feasibility of label-free classification of WBC populations using ODT, we measured four subtypes of WBCs, including B cell, CD4 T cell, CD8 T cell, and natural killer (NK) cell. From measured 3-D RI tomograms of WBCs, we obtain quantitative structural and biochemical information and classify each WBC population using a machine learning algorithm.

  6. 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.

  7. 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)

    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. PMID:24392097

  8. Equation-free model reduction for complex dynamical systems

    International Nuclear Information System (INIS)

    Le Maitre, O. P.; Mathelin, L.; Le Maitre, O. P.

    2010-01-01

    This paper presents a reduced model strategy for simulation of complex physical systems. A classical reduced basis is first constructed relying on proper orthogonal decomposition of the system. Then, unlike the alternative approaches, such as Galerkin projection schemes for instance, an equation-free reduced model is constructed. It consists in the determination of an explicit transformation, or mapping, for the evolution over a coarse time-step of the projection coefficients of the system state on the reduced basis. The mapping is expressed as an explicit polynomial transformation of the projection coefficients and is computed once and for all in a pre-processing stage using the detailed model equation of the system. The reduced system can then be advanced in time by successive applications of the mapping. The CPU cost of the method lies essentially in the mapping approximation which is performed offline, in a parallel fashion, and only once. Subsequent application of the mapping to perform a time-integration is carried out at a low cost thanks to its explicit character. Application of the method is considered for the 2-D flow around a circular cylinder. We investigate the effectiveness of the reduced model in rendering the dynamics for both asymptotic state and transient stages. It is shown that the method leads to a stable and accurate time-integration for only a fraction of the cost of a detailed simulation, provided that the mapping is properly approximated and the reduced basis remains relevant for the dynamics investigated. (authors)

  9. Isolation and functional aspects of free luteal cells

    International Nuclear Information System (INIS)

    Luborsky, J.L.; Berhrman, H.R.

    1985-01-01

    Methods of luteal cell isolation employ enzymatic treatment of luteal tissue with collagenase and deoxyribonuclease. Additional enzymes such as hyaluronidase or Pronase are also used in some instances. Isolated luteal cells retain the morphological characteristics of steroid secreting cells after isolation. They contain mitochondria, variable amounts of lipid droplets, and an extensive smooth endoplasmic reticulum. Isolated luteal cells have been used in numerous studies to examine the regulation of steriodogenesis by luteinizing hormone (LH). LH receptor binding studies were employed to quantitate specific properties of hormone-receptor interaction in relation to cellular function. Binding of [ 125 I]LH to bovine luteal cells and membranes was compared and it was concluded that the enzymatic treatment used to isolate cells did not change the LH receptor binding kinetics

  10. Study on bioactivity of cell-free filtrates from dairy propionibacteria.

    Science.gov (United States)

    Filippone, Alfonso; Sinigaglia, Milena; Altieri, Clelia

    2014-12-01

    In this paper, the bioactivity of cell-free extracts obtained by dairy propionibacteria strains was investigated. Probiotic bifidobacteria and lactobacilli were used as microbial targets. The extracellular filtrates were added as ingredient (1% v/v) into the growth medium; the effect of cell-free filtrates was evaluated through viable count of microbial targets on appropriate media, monitoring the response of target microorganisms both in growth and death phase. The Gompertz equation was used to model the experimental data. Kinetics and time parameters were estimated in order to quantify the effectiveness of cell-free filtrates effect. To emphasize the results about the bioactivity of cell-free extract, supporting a complete picture of response, a new approach was developed: Probiotic Stability Time was calculated. This temporal parameter, defined as the time over that the cell load preserve a living value upper than 10(7) cfu ml(-1), was very useful to evaluate the probiotic capability and effectiveness. A stimulant effect was registered on growth and a positive one was recordered on survival of both bifidobacteria and lactobacilli strains, and the results obtained suggest that a prebiotic activity by dairy propionibacteria cell-free filtrates could be supposed. The cell-free filtrate obtained from Propionibacterium freudenreichii subsp. shermanii was the most effective, in our experimental conditions. Although bifidobacteria were the most sensitive to the effect of cell-free filtrates, lactobacilli have been showed a similar probiotic stability time, showing a high sensitivity to the filtrates. This paper is the first report of a positive bioactivity by propionibacteria cell-free filtrates on lactobacilli. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. A free radical-generating system induces the cholesterol biosynthesis pathway: a role in Alzheimer's disease.

    Science.gov (United States)

    Recuero, María; Vicente, María Carmen; Martínez-García, Ana; Ramos, María C; Carmona-Saez, Pedro; Sastre, Isabel; Aldudo, Jesús; Vilella, Elisabet; Frank, Ana; Bullido, María J; Valdivieso, Fernando

    2009-04-01

    Oxidative stress, which plays a critical role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD), is intimately linked to aging - the best established risk factor for AD. Studies in neuronal cells subjected to oxidative stress, mimicking the situation in AD brains, are therefore of great interest. This paper reports that, in human neuronal cells, oxidative stress induced by the free radical-generating xanthine/xanthine oxidase (X-XOD) system leads to apoptotic cell death. Microarray analyses showed a potent activation of the cholesterol biosynthesis pathway following reductions in the cell cholesterol synthesis caused by the X-XOD treatment; furthermore, the apoptosis was reduced by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) expression with an interfering RNA. The potential importance of this mechanism in AD was investigated by genetic association, and it was found that HMGCR, a key gene in cholesterol metabolism and among those most strongly upregulated, was associated with AD risk. In summary, this work presents a human cell model prepared to mimic the effect of oxidative stress in neurons that might be useful in clarifying the mechanism involved in free radical-induced neurodegeneration. Gene expression analysis followed by genetic association studies indicates a possible link among oxidative stress, cholesterol metabolism and AD.

  12. Ambient pressure fuel cell system

    Science.gov (United States)

    Wilson, Mahlon S.

    2000-01-01

    An ambient pressure fuel cell system is provided with a fuel cell stack formed from a plurality of fuel cells having membrane/electrode assemblies (MEAs) that are hydrated with liquid water and bipolar plates with anode and cathode sides for distributing hydrogen fuel gas and water to a first side of each one of the MEAs and air with reactant oxygen gas to a second side of each one of the MEAs. A pump supplies liquid water to the fuel cells. A recirculating system may be used to return unused hydrogen fuel gas to the stack. A near-ambient pressure blower blows air through the fuel cell stack in excess of reaction stoichiometric amounts to react with the hydrogen fuel gas.

  13. Inhibition of telomerase activity and cell growth by free and ...

    African Journals Online (AJOL)

    found in some plants such as Punica granatum,. Terminalia catappa and Combretum molle [10]. It has inhibitory effect on different kinds of cancer cells including prostate and colon cancer cells. [11,12]. However, the in vivo instability of punicalagin has restricted its use in biomedical research [13]. Previous studies showed ...

  14. Serum-Free Cryopreservation of Human Amniotic Epithelial Cells

    Directory of Open Access Journals (Sweden)

    H. Niknejad

    2013-04-01

    Full Text Available Introduction & Objective: One of the important issues in long term storage of cells is removal of animal serum from cell culture environments. The aim of this study was to evaluate amni-otic fluid (AF, which is full of growth factors, as substitute for fetal bovine serum (FBS in the cryopreservation protocol. Materials & Methods: In this experimental study human amniotic epithelial cells were isolated from placentas which were seronegative for microbial infections. The cells were preserved in 24 different patterns for 12 months in -196 ?C (liquid nitrogen and viability of cells were determined before and after cryopreservation by trypan blue and MTT assay. Moreover, Oct-4 expression was studied to determine pluripotency before and after cryopreservation with immunocytochemistry. Results were compared between groups with ANOVA (Tukey Post-Test. P.value under 0.01 and 0.05 was considered statistically significant. Results: The presence of DMEM, FBS or AF is necessary for amniotic cell cryopreservation. Trypan-blue, MTT and immunocytochemistry showed that there isn’t significant difference between using AF and FBS in viability and pluripotency of cells. Moreover, results showed that DMSO is a better cryoprotectant compared to glycerol. Conclusion : Results showed that amniotic fluid can be a proper substitute for FBS in amniotic epithelial cells cryopreservation. (Sci J Hamadan Univ Med Sci 2013; 20 (1:15-24

  15. Functionalized nanopipettes: toward label-free, single cell biosensors

    OpenAIRE

    Actis, Paolo; Mak, Andy C.; Pourmand, Nader

    2010-01-01

    Nanopipette technology has been proven to be a label-free biosensor capable of identifying DNA and proteins. The nanopipette can include specific recognition elements for analyte discrimination based on size, shape, and charge density. The fully electrical read-out and the ease and low-cost fabrication are unique features that give this technology an enormous potential. Unlike other biosensing platforms, nanopipettes can be precisely manipulated with submicron accuracy and used to study singl...

  16. Advanced free space optics (FSO) a systems approach

    CERN Document Server

    Majumdar, Arun K

    2015-01-01

    This book provides a comprehensive, unified tutorial covering the most recent advances in the technology of free-space optics (FSO). It is an all-inclusive source of information on the fundamentals of FSO as well as up-to-date information on the state-of-the-art in technologies available today. This text is intended for graduate students, and will also be useful for research scientists and engineers with an interest in the field. FSO communication is a practical solution for creating a three dimensional global broadband communications grid, offering bandwidths far beyond what is possible in the Radio Frequency (RF) range. However, the attributes of atmospheric turbulence and scattering impose perennial limitations on availability and reliability of FSO links. From a systems point-of-view, this groundbreaking book provides a thorough understanding of channel behavior, which can be used to design and evaluate optimum transmission techniques that operate under realistic atmospheric conditions. Topics addressed...

  17. 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....

  18. Recent progress of dopant-free organic hole-transporting materials in perovskite solar cells

    Science.gov (United States)

    Dongxue, Liu; Liu, Yongsheng

    2017-01-01

    Organic-inorganic hybrid perovskite solar cells have undergone especially intense research and transformation over the past seven years due to their enormous progress in conversion efficiencies. In this perspective, we review the latest developments of conventional perovskite solar cells with a main focus on dopant-free organic hole transporting materials (HTMs). Regarding the rapid progress of perovskite solar cells, stability of devices using dopant-free HTMs are also discussed to help readers understand the challenges and opportunities in high performance and stable perovskite solar cells. Project supported by the Scientific Research Starting Foundation for Overseas Introduced Talents of College of Chemistry, Nankai University.

  19. 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.

  20. Effect of bacterial cell-free supernatants on infectivity of norovirus surrogates.

    Science.gov (United States)

    Shearer, Adrienne E H; Hoover, Dallas G; Kniel, Kalmia E

    2014-01-01

    Bacterial metabolic products were evaluated for inhibitory effects on viral propagation in cell culture. Cell-free supernatants (CFS) were prepared from growth of Enterococcus faecalis ATCC 19433, Pseudomonas fluorescens ATCC 13525, Escherichia coli 08, Staphylococcus epidermidis ATCC 12228, Bacillus subtilis 168, Bacillus coagulans 185A, B. coagulans 7050, Clostridium sporogenes PA3679, and a commercial probiotic mixture of Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium bifidum, Lactobacillus salivarius, and Streptococcus thermophilus in microbiological medium or milk. The inhibitory effects of CFS on the propagation of murine norovirus 1 and Tulane virus in RAW 264.7 and LLCMK2 cells, respectively, were evaluated in the continuous presence of CFS or after exposure of host cells to CFS. Slight inhibition of viral propagation was observed for murine norovirus and Tulane virus in the continuous presence of CFS of B. subtilis 168 and E. faecalis 19433, respectively. CFS cytotoxicity was also determined by microscopic examination. Virus persisted in the CFS that demonstrated cytotoxic effects, suggesting a lack of direct effect of CFS on virions. The viral propagation indicates a general lack of competitive inhibition by bacterial extracellular products and bears significance in understanding the persistence of virus in food and human systems shared by bacteria that are recognized for their colonization and competitive capabilities.

  1. Recent Development in ITO-free Flexible Polymer Solar Cells

    Directory of Open Access Journals (Sweden)

    Shudi Lu

    2017-12-01

    Full Text Available Polymer solar cells have shown good prospect for development due to their advantages of low-cost, light-weight, solution processable fabrication, and mechanical flexibility. Their compatibility with the industrial roll-to-roll manufacturing process makes it superior to other kind of solar cells. Normally, indium tin oxide (ITO is adopted as the transparent electrode in polymer solar cells, which combines good conductivity and transparency. However, some intrinsic weaknesses of ITO restrict its large scale applications in the future, including a high fabrication price using high temperature vacuum deposition method, scarcity of indium, brittleness and scaling up of resistance with the increase of area. Some substitutes to ITO have emerged in recent years, which can be used in flexible polymer solar cells. This article provides the review on recent progress using other transparent electrodes, including carbon nanotubes, graphene, metal nanowires and nanogrids, conductive polymer, and some other electrodes. Device stability is also discussed briefly.

  2. 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

  3. A free radical-generating system regulates APP metabolism/processing.

    Science.gov (United States)

    Recuero, María; Muñoz, Teresa; Aldudo, Jesús; Subías, Marta; Bullido, María J; Valdivieso, Fernando

    2010-11-19

    Oxidative stress, a risk factor in the pathophysiology of Alzheimer's disease, is intimately associated with aging. We previously reported that the X-XOD free radical generating system acts as a modulator of lipid metabolism and a mild inducer of apoptotic death. Using the same cell model, the present study examines the metabolism/processing of the amyloid precursor protein (APP). Prior to inducing cell death, X-XOD promoted the secretion of α-secretase-cleaved soluble APP (sAPPα) and increased the level of APP carboxy-terminal fragments produced by α and γ secretase (αCTF and γCTF/AICD). In contrast, it reduced the activity of β-secretase and the level of secreted Aβ. The present results indicate that mild oxidative stress maintained throughout culturing regulates APP metabolism/processing in SK-N-MC human neuroblastoma cells. Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  4. 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.

  5. H2 O2 –HBr: A metal-free and organic solvent-free reagent system ...

    Indian Academy of Sciences (India)

    Administrator

    environmental impact. 32. The development of waste-free, non-polluting benign systems and more sustainable strategies is one of the main themes of contemporary organic synthe- sis. 33. So far, our investigations on the oxidation of methylarenes to arylaldehydes consisted in using. Br2/DMSO as a mild catalytic system in ...

  6. Reformate fuel cell system durability

    Energy Technology Data Exchange (ETDEWEB)

    Borup, R. L. (Rodney L.); Inbody, M. A. (Michael A.); Uribe, F. A. (Francisco A.); Tafoya, J. (Jose I.)

    2002-01-01

    The goal of this research is to identify the factors limiting the durability of fuel cells and fuel processors. This includes identifying PEM fuel cell durability issues for operating on pure hydrogen, and those that arise from the fuel processing of liquid hydrocarbons (e.g., gasoline) as a function of fuel composition and impurity content. Benchmark comparisons with the durability of fuel cells operating on pure hydrogen are used to identify limiting factors unique to fuel processing. We describe the design, operation and operational results of the durability system, including the operating conditions for the system, fuel processor sub-section operation over 1000 hours, post-mortem characterization of the catalysts in the fuel processor, and single cell operation.

  7. TUMOR-RELATED METHYLATED CELL-FREE DNA AND CIRCULATING TUMOR CELLS IN MELANOMA

    Directory of Open Access Journals (Sweden)

    Francesca eSalvianti

    2016-01-01

    Full Text Available Solid tumor release into the circulation cell-free DNA (cfDNA and circulating tumor cells (CTCs which represent promising biomarkers for cancer diagnosis. Circulating tumor DNA may be studied in plasma from cancer patients by detecting tumor specific alterations, such as genetic or epigenetic modifications. Ras association domain family 1 isoform A (RASSF1A is a tumor suppressor gene silenced by promoter hypermethylation in a variety of human cancers including melanoma.The aim of the present study was to assess the diagnostic performance of a tumor-related methylated cfDNA marker in melanoma patients and to compare this parameter with the presence of CTCs.RASSF1A promoter methylation was quantified in cfDNA by qPCR in a consecutive series of 84 melanoma patients and 68 healthy controls. In a subset of 68 cases, the presence of CTCs was assessed by a filtration method (Isolation by Size of Epithelial Tumor Cells, ISET as well as by an indirect method based on the detection of tyrosinase mRNA by RT-qPCR. The distribution of RASSF1A methylated cfDNA was investigated in cases and controls and the predictive capability of this parameter was assessed by means of the area under the ROC curve (AUC.The percentage of cases with methylated RASSF1A promoter in cfDNA was significantly higher in each class of melanoma patients (in situ, invasive and metastatic than in healthy subjects (Pearson chi-squared test, p<0.001. The concentration of RASSF1A methylated cfDNA in the subjects with a detectable quantity of methylated alleles was significantly higher in melanoma patients than in controls. The biomarker showed a good predictive capability (in terms of AUC in discriminating between melanoma patients and healthy controls. This epigenetic marker associated to cfDNA did not show a significant correlation with the presence of CTCs, but, when the two parameters are jointly considered, we obtain a higher sensitivity of the detection of positive cases in invasive

  8. Revealing the Differences Between Free and Complexed Enzyme Mechanisms and Factors Contributing to Cell Wall Recalcitrance

    Energy Technology Data Exchange (ETDEWEB)

    Resch, M.

    2014-09-08

    Enzymatic depolymerization of polysaccharides is a key step in the production of fuels and chemicals from lignocellulosic biomass, and discovery of synergistic biomass-degrading enzyme paradigms will enable improved conversion processes. Historically, revealing insights into enzymatic saccharification mechanisms on plant cell walls has been hindered by uncharacterized substrates and low resolution imaging techniques. Also, translating findings between model substrates to intact biomass is critical for evaluating enzyme performance. Here we employ a fungal free enzyme cocktail, a complexed cellulosomal system, and a combination of the two to investigate saccharification mechanisms on cellulose I, II and III along with corn stover from Clean Fractionation (CF), which is an Organosolv pretreatment. The insoluble Cellulose Enriched Fraction (CEF) from CF contains mainly cellulose with minor amounts of residual hemicellulose and lignin, the amount of which depends on the CF pretreatment severity. Enzymatic digestions at both low and high-solids loadings demonstrate that CF reduces the amount of enzyme required to depolymerize polysaccharides relative to deacetylated, dilute acid pretreated corn stover. Transmission and scanning electron microscopy of the biomass provides evidence for the different mechanisms of enzymatic deconstruction between free and complexed enzyme systems, and reveals the basis for the synergistic relationship between the two enzyme paradigms on a process-relevant substrate for the first time. These results also demonstrate that the presence of lignin, rather than cellulose morphology, is more detrimental to cellulosome action than to free cellulases. As enzyme costs are a major economic driver for biorefineries, this study provides key inputs for the evaluation of CF as a pretreatment method for biomass conversion.

  9. Characterization of spontaneous collagen fibrillogenesis in a cell-free and tension-free environment.

    Science.gov (United States)

    Piérard, G E; Hermanns-Lê, T; Delvenne, P; Piérard-Franchimont, C

    2013-06-01

    The collagen fibril packing that forms threads and bundles is poorly defined, despite the fact that it is important for distinct aspects of the adventitial and reticular dermis. The present study explored an in vitro fibrillogenesis model using the property of heat polymerization. The process was performed on glass slides with mixtures of collagen I and III, and the material was viewed by scanning electron microscopy. In all instances, collagen I and III formed fibrils with regular sizes. The formation of threads was influenced by the relative proportions of collagen I and III; increasing the relative proportion of collagen I resulted in the formation of threads showing increasing variations in thickness. These findings are in line with the differential presentation and compositions of the different parts of the dermis. The possible interventions of stromal cells and of other macromoleules of the extracellular matrix were not considered in this study. © The Author(s) CED © 2013 British Association of Dermatologists.

  10. Inhibition of telomerase activity and cell growth by free and ...

    African Journals Online (AJOL)

    Furthermore, the telomerase activity of the nanoliposomal punicalagin-treated cells was significantly inhibited in a time- and dose-dependent manner. Conclusion: Punicalagin shows a novel mechanism of anti-telomerase activity, particularly in the nanoliposomal form, and may provide a basis for the future development of ...

  11. Cytotoxity of cell free filtrates of campylobacter jejuni isolated in ...

    African Journals Online (AJOL)

    Culture filtrates of Campylobacter jejuni strains isolated from clinical specimens in Lagos Nigeria were tested for toxic activity. Two out of five filtrates tested manifested cytopathic effect on BHK cells. The effects were mainly cytotoxic and cytotonic. Toxic activity of C. jejuni filtrates was much lower than toxic activity elicited by ...

  12. Free-energy carriers in human cultured muscle cells

    NARCIS (Netherlands)

    Bolhuis, P. A.; de Zwart, H. J.; Ponne, N. J.; de Jong, J. M.

    1985-01-01

    Creatine phosphate (CrP), adenosine triphosphate (ATP), creatine kinase (CK), adenylate kinase (AK), protein, and DNA were quantified in human muscle cell cultures undergoing transition from dividing myoblasts to multinucleate myotubes. CrP is negligible in cultures grown in commonly applied media

  13. 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.

  14. 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.

  15. Scaffold-Free Fabrication of Osteoinductive Cellular Constructs Using Mouse Gingiva-Derived Induced Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Hiroko Okawa

    2016-01-01

    Full Text Available Three-dimensional (3D cell constructs are expected to provide osteoinductive materials to develop cell-based therapies for bone regeneration. The proliferation and spontaneous aggregation capability of induced pluripotent stem cells (iPSCs thus prompted us to fabricate a scaffold-free iPSC construct as a transplantation vehicle. Embryoid bodies of mouse gingival fibroblast-derived iPSCs (GF-iPSCs were seeded in a cell chamber with a round-bottom well made of a thermoresponsive hydrogel. Collected ball-like cell constructs were cultured in osteogenic induction medium for 30 days with gentle shaking, resulting in significant upregulation of osteogenic marker genes. The constructs consisted of an inner region of unstructured cell mass and an outer osseous tissue region that was surrounded by osteoblast progenitor-like cells. The outer osseous tissue was robustly calcified with elemental calcium and phosphorous as well as hydroxyapatite. Subcutaneous transplantation of the GF-iPSC constructs into immunodeficient mice contributed to extensive ectopic bone formation surrounded by teratoma tissue. These results suggest that mouse GF-iPSCs could facilitate the fabrication of osteoinductive scaffold-free 3D cell constructs, in which the calcified regions and surrounding osteoblasts may function as scaffolds and drivers of osteoinduction, respectively. With incorporation of technologies to inhibit teratoma formation, this system could provide a promising strategy for bone regenerative therapies.

  16. 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

  17. 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

  18. 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.

  19. Phenotyping and comparing the immune cell populations of free-ranging Atlantic bottlenose dolphins (Tursiops truncatus) and dolphins under human care.

    Science.gov (United States)

    Nouri-Shirazi, Mahyar; Bible, Brittany F; Zeng, Menghua; Tamjidi, Saba; Bossart, Gregory D

    2017-03-27

    Studies suggest that free-ranging bottlenose dolphins exhibit a suppressed immune system because of exposure to contaminants or microorganisms. However, due to a lack of commercially available antibodies specific to marine mammal immune cell surface markers, the research has been indecisive. The purpose of this study was to identify cross-reactive terrestrial-specific antibodies in order to assess the changes in the immune cell populations of dolphins under human care and free-ranging dolphins. The blood and PBMC fraction of blood samples from human care and free-ranging dolphins were characterized by H&E staining of cytospin slides and flow cytometry using a panel of terrestrial-specific antibodies. In this study, we show that out of 65 terrestrial-specific antibodies tested, 11 were cross-reactive and identified dolphin immune cell populations within their peripheral blood. Using these antibodies, we found significant differences in the absolute number of cells expressing specific markers within their lymphocyte and monocyte fractions. Interestingly, the peripheral blood mononuclear cell profile of free-ranging dolphins retained an additional population of cells that divided them into two groups showing a low (56%) percentage of smaller cells resembling granulocytes. We found that the cross-reactive antibodies not only identified specific changes in the immune cells of free-ranging dolphins, but also opened the possibility to investigate the causal relationship between immunosuppression and mortality seen in free-ranging dolphins.

  20. Somatic cell reprogramming-free generation of genetically modified pigs.

    Science.gov (United States)

    Tanihara, Fuminori; Takemoto, Tatsuya; Kitagawa, Eri; Rao, Shengbin; Do, Lanh Thi Kim; Onishi, Akira; Yamashita, Yukiko; Kosugi, Chisato; Suzuki, Hitomi; Sembon, Shoichiro; Suzuki, Shunichi; Nakai, Michiko; Hashimoto, Masakazu; Yasue, Akihiro; Matsuhisa, Munehide; Noji, Sumihare; Fujimura, Tatsuya; Fuchimoto, Dai-Ichiro; Otoi, Takeshige

    2016-09-01

    Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs.

  1. Relationships between Cell-Free DNA and Serum Analytes in First and Second Trimesters of Pregnancy

    Science.gov (United States)

    Vora, Neeta L.; Johnson, Kirby L.; Lambert-Messerlian, Geralyn; Tighiouart, Hocine; Peter, Inga; Urato, Adam C.; Bianchi, Diana W.

    2010-01-01

    Objective Circulating cell-free DNA and maternal serum analytes are indicators of fetal and placental condition. Little is known about the relationship of these noninvasive markers to each other, particularly in the first trimester. Our goal was to assess the relationship between first and second trimester cell-free DNA levels and maternal serum screening markers. Methods First and second trimester residual maternal serum samples from 50 women were obtained. First trimester (pregnancy-associated plasma protein A [PAPP-A] and β-hCG), and second trimester serum analytes (β-hCG, alpha-fetoprotein [AFP], unconjugated estriol and inhibin A) had been measured at the time of sample receipt. All fetuses were male, as confirmed by birth records. Cell-free DNA was extracted and measured by real-time quantitative polymerase chain reaction (PCR) amplification using glyceraldehyde phosphate dehydrogenase (GAPDH) and DYS1 as markers of total DNA and fetal DNA, respectively. Determination of linear associations between first and second trimester serum markers and cell-free DNA levels using Pearson correlations was performed. Results Statistically significant correlations between first trimester PAPP-A multiples of the median (MoMs) and both total (r=0.36, p=0.016) and fetal (r= 0.41, p=0.006) DNA in the first trimester were observed. There were no significant correlations between first trimester serum hCG or any second trimester serum marker with DNA levels. Conclusions Correlation between serum PAPP-A and first trimester circulating cell-free fetal and total DNA levels is a novel finding. PAPP-A is a glycoprotein of placental origin, and its correlation to cell-free fetal DNA in maternal serum suggests a common tissue origin, through apoptosis of placental cells. However, since PAPP-A and cell-free DNA were only marginally correlated and cell-free DNA can be reliably detected in the first trimester, the addition of cell-free DNA to serum screening strategies may be helpful in

  2. Label-free isolation of circulating tumor cells in microfluidic devices: Current research and perspectives.

    Science.gov (United States)

    Cima, Igor; Wen Yee, Chay; Iliescu, Florina S; Phyo, Wai Min; Lim, Kiat Hon; Iliescu, Ciprian; Tan, Min Han

    2013-01-01

    This review will cover the recent advances in label-free approaches to isolate and manipulate circulating tumor cells (CTCs). In essence, label-free approaches do not rely on antibodies or biological markers for labeling the cells of interest, but enrich them using the differential physical properties intrinsic to cancer and blood cells. We will discuss technologies that isolate cells based on their biomechanical and electrical properties. Label-free approaches to analyze CTCs have been recently invoked as a valid alternative to "marker-based" techniques, because classical epithelial and tumor markers are lost on some CTC populations and there is no comprehensive phenotypic definition for CTCs. We will highlight the advantages and drawbacks of these technologies and the status on their implementation in the clinics.

  3. 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.

  4. 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.

  5. Concise review: animal substance-free human embryonic stem cells aiming at clinical applications.

    Science.gov (United States)

    Hovatta, Outi; Rodin, Sergey; Antonsson, Liselotte; Tryggvason, Karl

    2014-11-01

    Human embryonic stem cells have been considered the gold standard as a cell source for regenerative medicine since they were first cultured in 1998. They are pluripotent and can form principally all the cells types in the body. They are obtained from supernumerary human in vitro fertilization embryos that cannot be used for infertility treatment. Following studies on factors regulating pluripotency and differentiation, we now have techniques to establish and effectively expand these cells in animal substance-free conditions, even from single cells biopsied from eight-cell stage embryos in chemically defined feeder-free cultures. The genetic stability and absence of tumorigenic mutations can be determined. There are satisfactory animal tests for functionality and safety. The first clinical trials are ongoing for two indications: age-related macular degeneration and spinal cord injury. ©AlphaMed Press.

  6. Control of free-flying space robot manipulator systems

    Science.gov (United States)

    Cannon, Robert H., Jr.

    1990-01-01

    New control techniques for self contained, autonomous free flying space robots were developed and tested experimentally. Free flying robots are envisioned as a key element of any successful long term presence in space. These robots must be capable of performing the assembly, maintenance, and inspection, and repair tasks that currently require human extravehicular activity (EVA). A set of research projects were developed and carried out using lab models of satellite robots and a flexible manipulator. The second generation space robot models use air cushion vehicle (ACV) technology to simulate in 2-D the drag free, zero g conditions of space. The current work is divided into 5 major projects: Global Navigation and Control of a Free Floating Robot, Cooperative Manipulation from a Free Flying Robot, Multiple Robot Cooperation, Thrusterless Robotic Locomotion, and Dynamic Payload Manipulation. These projects are examined in detail.

  7. Mating System of Free-Ranging Dogs (Canis familiaris

    Directory of Open Access Journals (Sweden)

    S. K. Pal

    2011-01-01

    Full Text Available Fourteen females belonging to five groups were selected for the study of mating system in free-ranging domestic dogs (Canis familiaris All the matings occurred between August and December with a peak in late monsoon months (September to November. Both males and females differed in their degree of attractiveness to the opposite sex. The duration of courting association increased with the number of courting males in an association. The females exhibited selectivity by readily permitting some males to mate and avoiding, or even attacking others, if they attempted to mount. Frequency of mounting in courting association increased with the number of males present. There was a positive correlation between the duration of courting association and the frequency of mounting. The young adult males were more likely to copulate successfully than the old adult males. There was a negative correlation between the number of males present in an association and the number of successful copulations. In this study, six types of mating (monogamy, polygyny, promiscuity, polyandry, opportunity and rape were recorded. Mean (±S.E. duration of copulatory ties was 25.65 (±1.43 min. Several natural factors influencing the duration of copulatory ties were identified.

  8. Plasma Cell-Free DNA in Paediatric Lymphomas

    Science.gov (United States)

    Mussolin, Lara; Burnelli, Roberta; Pillon, Marta; Carraro, Elisa; Farruggia, Piero; Todesco, Alessandra; Mascarin, Maurizio; Rosolen, Angelo

    2013-01-01

    Background: Extracellular circulating DNA (cfDNA) can be found in small amounts in plasma of healthy individuals. Increased levels of cfDNA have been reported in patients with cancer of breast, cervix, colon, liver and it was shown that cfDNA can originate from both tumour and non-tumour cells. Objectives: Levels of cfDNA of a large series of children with lymphoma were evaluated and analyzed in relation with clinical characteristics. Methods: plasma cfDNA levels obtained at diagnosis in 201 paediatric lymphoma patients [43 Hodgkin lymphomas (HL), 45 anaplastic large cell lymphomas (ALCL), 88 Burkitt lymphomas (BL), 17 lymphoblastic (LBL), 8 diffuse large B cell lymphoma (DLBCL)] and 15 healthy individuals were determined using a quantitative PCR assay for POLR2 gene and, in addition, for NPM-ALK fusion gene in ALCL patients. Wilcoxon rank sum test was used to compare plasma levels among different patient subgroups and controls and to analyze relationship between levels of cfDNA and clinical characteristics. Results: Levels of cfDNA in lymphoma patients were significantly higher compared with controls (p<0.0001). CfDNA was associated with median age (p=0.01) in HL, and with stage in ALCL (p=0.01). In HL patients high cfDNA levels were correlated with poor prognosis (p=0.03). In ALCL we found that most of the cfDNA (77%) was non-tumor DNA. Conclusion: level of plasma cfDNA might constitute an important non-invasive tool at diagnosis in lymphoma patients' management; in particular in patients with HL, cfDNA seems to be a promising prognostic biomarker. PMID:23678368

  9. Taurine Biosynthesis in a Fish Liver Cell Line (ZFL) Adapted to a Serum-Free Medium

    OpenAIRE

    Chieh-Lun Liu; Aaron M. Watson; Allen R. Place; Rosemary Jagus

    2017-01-01

    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 gr...

  10. Wearable Battery-free Wireless 2-channel EEG Systems Powered by Energy Scavengers

    Directory of Open Access Journals (Sweden)

    Mieke VAN BAVEL

    2008-07-01

    Full Text Available Thermoelectric generators worn on a person’s body have demonstrated their capability to power a variety of wireless sensor nodes that are to improve his/her health or comfort. In this article, the design, fabrication and performance of two prototypes of a battery-free wireless 2-channel electroencephalography (EEG system are presented. The first system is powered solely by a thermoelectric generator that produces 2-2.5mW of power and is worn as a headband. The second system resembles a diadem or headphones and uses a hybrid power supply that combines a thermoelectric generator and photovoltaic cells in one device. This portable EEG headset considerably improves the comfort of patients in clinical as well as in non-clinical environments and opens perspectives for a new range of non-clinical applications.

  11. 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

    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...... 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...... vertical and coplanar interdigitated sensing configuration approaches have been used and compared on the same cell populations....

  12. Cell-free system for synthesizing membrane proteins cell free method for synthesizing membrane proteins

    Science.gov (United States)

    Laible, Philip D; Hanson, Deborah K

    2013-06-04

    The invention provides an in vitro method for producing proteins, membrane proteins, membrane-associated proteins, and soluble proteins that interact with membrane-associated proteins for assembly into an oligomeric complex or that require association with a membrane for proper folding. The method comprises, supplying intracytoplasmic membranes from organisms; modifying protein composition of intracytoplasmic membranes from organism by modifying DNA to delete genes encoding functions of the organism not associated with the formation of the intracytoplasmic membranes; generating appropriate DNA or RNA templates that encode the target protein; and mixing the intracytoplasmic membranes with the template and a transcription/translation-competent cellular extract to cause simultaneous production of the membrane proteins and encapsulation of the membrane proteins within the intracytoplasmic membranes.

  13. Catalyst, Membrane, Free Electrolyte Challenges, and Pathways to Resolutions in High Temperature Polymer Electrolyte Membrane Fuel Cells

    Directory of Open Access Journals (Sweden)

    Timothy Myles

    2017-01-01

    Full Text Available High temperature polymer electrolyte membrane fuel cells (HT-PEMFCs are being studied due to a number of benefits offered versus their low temperature counterparts, including co-generation of heat and power, high tolerance to fuel impurities, and simpler system design. Approximately 90% of the literature on HT-PEM is related to the electrolyte and, for the most part, these electrolytes all use free phosphoric acid, or similar free acid, as the ion conductor. A major issue with using phosphoric acid based electrolytes is the free acid in the electrodes. The presence of the acid on the catalyst sites leads to poor oxygen activity, low solubility/diffusion, and can block electrochemical sites through phosphate adsorption. This review will focus on these issues and the steps that have been taken to alleviate these obstacles. The intention is this review may then serve as a tool for finding a solution path in the community.

  14. 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.

  15. 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

    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......Factor VIII (FVIII) is an important protein in the blood coagulation cascade and dysfunction or deficiency of FVIII causes haemophilia A. Replacement therapy with exogenous recombinant FVIII (rFVIII) works as a substitute for the missing or non-functioning FVIII. The rFVIII protein has been...... 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...

  16. Social Theory and the significance of free will in our system of criminal justice

    NARCIS (Netherlands)

    Schwitters, R.

    2016-01-01

    Free will is a key assumption of our system of criminal justice. However, the assumption of a free will is questioned by the rapidly growing empirical findings of the neuro and the brain sciences. These indicate that human behavior is driven by subconscious forces beyond the free will. In this text

  17. Fabrication of flexible indium tin oxide-free polymer solar cells with silver nanowire transparent electrode

    Science.gov (United States)

    Lin, Ming-Yi; Chen, Tsun-Jui; Xu, Wei-Feng; Hsiao, Li-Jen; Budiawan, Widhya; Tu, Wei-Chen; Chen, Shih-Lun; Chu, Chih-Wei; Wei, Pei-Kuen

    2018-03-01

    Flexible indium tin oxide (ITO)-free poly(3-hexylthiophene):[6,6]-phenyl C61-butyric acid methyl ester (P3HT:PC61BM) solar cells with a spin-coated silver nanowire transparent electrode are demonstrated. The solution-processed silver nanowire thin film not only exhibits high transmission (∼87%), but also shows low sheet resistance R s (∼25 Ω/sq). For solar cells with a conventional structure, the power conversion efficiency (PCE) of devices based on silver nanowires can reach around 2.29%. For the inverted structure, the PCE of devices can reach 3.39%. Conventional and inverted flexible ITO-based P3HT:PC61BM solar cells are also fabricated as a reference for comparison. For both types of solar cells, the PCE of ITO-free devices is very close that of an ITO-based polymer solar cell.

  18. 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.

  19. High-level cell-free production of membrane proteins with nanodiscs.

    Science.gov (United States)

    Roos, Christian; Kai, Lei; Haberstock, Stefan; Proverbio, Davide; Ghoshdastider, Umesh; Ma, Yi; Filipek, Slawomir; Wang, Xiaoning; Dötsch, Volker; Bernhard, Frank

    2014-01-01

    This chapter addresses two major bottlenecks in cell-free membrane protein production. Firstly, we describe the optimization of expression templates for obtaining membrane proteins in preparative scales. We present details for a newly established tag variation screen providing high success rates in improving expression efficiencies while having only minimal impacts on the target protein structure. Secondly, we present protocols for the efficient co-translational insertion of membrane proteins into defined lipid bilayers. We describe the production of nanodiscs and their implementation into cell-free expression reactions for the co-translational reconstitution of membrane proteins. In addition we give guidelines for the loading of nanodiscs with different lipids in order to systematically analyze effects of lipids on the translocation, functional folding, and stability of cell-free expressed membrane proteins.

  20. Lipid nanocarriers based on natural oils with high activity against oxygen free radicals and tumor cell proliferation.

    Science.gov (United States)

    Lacatusu, I; Badea, N; Badea, G; Oprea, O; Mihaila, M A; Kaya, D A; Stan, R; Meghea, A

    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 5mg·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 2.5mg·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. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Prognostic Factors on the Graft-versus-Host Disease-Free and Relapse-Free Survival after Adult Allogeneic Hematopoietic Stem Cell Transplantation

    Directory of Open Access Journals (Sweden)

    Yao-Chung Liu

    2016-01-01

    Full Text Available The cure of hematologic disorders by allogeneic hematopoietic stem cell transplantation (HSCT is often associated with major complications resulting in poor outcome, including graft-versus-host disease (GVHD, relapse, and death. A novel composite endpoint of GVHD-free/relapse-free survival (GRFS in which events include grades 3-4 acute GVHD, chronic GVHD requiring systemic therapy, relapse, or death is censored to completely characterize the survival without mortality or ongoing morbidity. In this regard, studies attempting to identify the prognostic factors of GRFS are quite scarce. Thus, we reviewed 377 adult patients undergoing allogeneic HSCT between 2003 and 2013. The 1- and 2-year GRFS were 40.8% and 36.5%, respectively, significantly worse than overall survival and disease-free survival (log-rank p 2 (p 2 (p<0.001, being male (p=0.028, and hematologic malignancy (p=0.010 were significant for poor outcome. The events between 1-year GRFS and 2-year GRFS predominantly increased in relapsed patients. With prognostic factors of GRFS, we could evaluate the probability of real recovery following HSCT without ongoing morbidity.

  2. Silver nanoparticles inhibit fish gill cell proliferation in protein-free culture medium.

    Science.gov (United States)

    Yue, Yang; Behra, Renata; Sigg, Laura; Schirmer, Kristin

    2016-10-01

    While short-term exposures of vertebrate cells, such as from fish, can be performed in defined, serum-free media, long-term cultures generally require addition of growth factors and proteins, normally supplied with a serum supplement. However, proteins are known to alter nanoparticle properties by binding to nanoparticles. Therefore, in order to be able to study nanoparticle-cell interactions for extended periods, the rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1, was adapted to proliferate in a commercial, serum-free medium, InVitrus VP-6. The newly adapted cell strain was named RTgill-W1-pf (protein free). These cells proliferate at a speed similar to the RTgill-W1 cells cultured in a fully supplemented medium containing 5% fetal bovine serum. As well, they were successfully cryopreserved in liquid nitrogen and fully recovered after thawing. Yet, senescence set in after about 10 passages in InVitrus VP-6 medium, revealing that this medium cannot fully support long-term culture of the RTgill-W1 strain. The RTgill-W1-pf cell line was subsequently applied to investigate the effect of silver nanoparticles (AgNP) on cell proliferation over a period of 12 days. Indeed, cell proliferation was inhibited by 10 μM AgNP. This effect correlated with high levels of silver being associated with the cells. The new cell line, RTgill-W1-pf, can serve as a unique representation of the gill cell-environment interface, offering novel opportunities to study nanoparticle-cell interactions without serum protein interference.

  3. 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.

  4. 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…

  5. Obtention of Free Fatty Acids of Macauba Oil (Acrocomia Aculeata in Organic Solvent Free System

    Directory of Open Access Journals (Sweden)

    Caroline Portilho Trentini

    2014-05-01

    Full Text Available The present study aimed to investigate the enzymatic hydrolysis of oil Macaúba, to obtain a hydrolyzate rich in free fatty acids (FFA for later use in step esterification. The effect of process variables (percentage of catalyst, temperature and water content was evaluated in the FFA yield, using a factorial experimental design 23, where the positive and significant effect of the variables was observed. The results reported yields of 50.5% in FFA in 6 hours of reaction at 60ºC, water percentage of 15 wt% and catalyst percentage of 5 wt%.

  6. 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 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 labor, P = .002 without labor) and interleukin-6 (P 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 methylation ratio and interleukin-6 compared with nonlabored controls. As

  7. Fuel cell-fuel cell hybrid system

    Science.gov (United States)

    Geisbrecht, Rodney A.; Williams, Mark C.

    2003-09-23

    A device for converting chemical energy to electricity is provided, the device comprising a high temperature fuel cell with the ability for partially oxidizing and completely reforming fuel, and a low temperature fuel cell juxtaposed to said high temperature fuel cell so as to utilize remaining reformed fuel from the high temperature fuel cell. Also provided is a method for producing electricity comprising directing fuel to a first fuel cell, completely oxidizing a first portion of the fuel and partially oxidizing a second portion of the fuel, directing the second fuel portion to a second fuel cell, allowing the first fuel cell to utilize the first portion of the fuel to produce electricity; and allowing the second fuel cell to utilize the second portion of the fuel to produce electricity.

  8. Natural zwitterionic l-Carnitine as efficient cryoprotectant for solvent-free cell cryopreservation.

    Science.gov (United States)

    Zhai, Hongwen; Yang, Jing; Zhang, Jiamin; Pan, Chao; Cai, Nana; Zhu, Yingnan; Zhang, Lei

    2017-07-15

    Organic solvents, such as dimethyl sulfoxide (DMSO) and glycerol, have been commonly used as cryoprotectants (CPAs) in cell cryopreservation. However, their cytotoxicity and need of complex freezing protocols have impeded their applications especially in clinical cell therapy and regenerative medicine. Trehalose has been explored as a natural CPA to cryopreserve cells, but its poor cell permeability frequently results in low cryopreservation efficacy. In this work, we presented that a natural zwitterionic molecule-l-carnitine-could serve as a promising CPA for solvent-free cryopreservation. We demonstrated that l-carnitine possessed strong ability to depress water freezing point, and with ultrarapid freezing protocol, we studied the post-thaw survival efficiency of four cell lines (GLC-82 cells, MCF-7 cells, NIH-3T3 cells and Sheep Red Blood Cells) using l-carnitine without addition of any organic solvents. At the optimum l-carnitine concentration, all four cell lines could achieve above 80% survival efficiency, compared with the significantly lower efficiency using organic CPAs and trehalose. After cryopreservation, the recovered cell behaviors including cell attachment and proliferation were found to be similar to the normal cells, indicating that the cell functionalities were not affected. Moreover, l-carnitine showed no observable cytotoxicity, which was superior to the organic CPAs. This work offered an attractive alternative to traditional CPAs and held great promise to revolutionize current cryopreservation technologies, to benefit the patients in various cell-based clinical applications. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Mechatronics in fuel cell systems

    Energy Technology Data Exchange (ETDEWEB)

    Stefanopoulou, Anna G.; Kyungwon Suh [Mechanical Engineering Department, University of Michigan, 1231 Beal Avenue, Ann Arbor, MI 48109, (United States)

    2007-03-15

    Power generation from fuel cells (FCs) requires the integration of chemical, fluid, mechanical, thermal, electrical, and electronic subsystems. This integration presents many challenges and opportunities in the mechatronics field. This paper highlights important design issues and poses problems that require mechatronics solutions. The paper begins by describing the process of designing a toy school bus powered by hydrogen for an undergraduate student project. The project was an effective and rewarding educational activity that revealed complex systems issues associated with FC technology. (Author)

  10. A Quantitative Assessment of Cell-Free DNA Utilizing Several Housekeeping Genes: Measurements from Four Different Cell Lines.

    Science.gov (United States)

    Aucamp, Janine; Bronkhorst, Abel Jacobus; Wentzel, Johannes F; Pretorius, Piet J

    2016-01-01

    Quantitative real-time PCR (qPCR) is regularly used to quantify cell-free nucleic acids (cfNAs) in order to identify biomarkers for various pathologies. However, studies have shown notable housekeeping gene expression variation between healthy and diseased tissues and treated versus untreated cell lines. The release of housekeeping genes by four cell lines was investigated and the housekeeping gene expression between cfNAs and mRNA of the cell lines was observed in order to elucidate their relationship.

  11. Monitoring of organ transplants through genomic analyses of circulating cell-free DNA

    Science.gov (United States)

    de Vlaminck, Iwijn

    Solid-organ transplantation is the preferred treatment for patients with end-stage organ diseases, but complications due to infection and acute rejection undermine its long-term benefits. While clinicians strive to carefully monitor transplant patients, diagnostic options are currently limited. My colleagues and I in the lab of Stephen Quake have found that a combination of next-generation sequencing with a phenomenon called circulating cell-free DNA enables non-invasive diagnosis of both infection and rejection in transplantation. A substantial amount of small fragments of cell-free DNA circulate in blood that are the debris of dead cells. We discovered that donor specific DNA is released in circulation during injury to the transplant organ and we show that the proportion of donor DNA in plasma is predictive of acute rejection in heart and lung transplantation. We profiled viral and bacterial DNA sequences in plasma of transplant patients and discovered that the relative representation of different viruses and bacteria is informative of immunosuppression. This discovery suggested a novel biological measure of a person's immune strength, a finding that we have more recently confirmed via B-cell repertoire sequencing. Lastly, our studies highlight applications of shotgun sequencing of cell-free DNA in the broad, hypothesis free diagnosis of infection.

  12. 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.

  13. Label-free quantitative proteomics of CD133-positive liver cancer stem cells

    Directory of Open Access Journals (Sweden)

    Tsai Sheng-Ta

    2012-11-01

    Full Text Available Abstract Background CD133-positive liver cancer stem cells, which are characterized by their resistance to conventional chemotherapy and their tumor initiation ability at limited dilutions, have been recognized as a critical target in liver cancer therapeutics. In the current work, we developed a label-free quantitative method to investigate the proteome of CD133-positive liver cancer stem cells for the purpose of identifying unique biomarkers that can be utilized for targeting liver cancer stem cells. Label-free quantitation was performed in combination with ID-based Elution time Alignment by Linear regression Quantitation (IDEAL-Q and MaxQuant. Results Initially, IDEAL-Q analysis revealed that 151 proteins were differentially expressed in the CD133-positive hepatoma cells when compared with CD133-negative cells. We then analyzed these 151 differentially expressed proteins by MaxQuant software and identified 10 significantly up-regulated proteins. The results were further validated by RT-PCR, western blot, flow cytometry or immunofluorescent staining which revealed that prominin-1, annexin A1, annexin A3, transgelin, creatine kinase B, vimentin, and EpCAM were indeed highly expressed in the CD133-positive hepatoma cells. Conclusions These findings confirmed that mass spectrometry-based label-free quantitative proteomics can be used to gain insights into liver cancer stem cells.

  14. Speckle noise suppression using a helix-free ferroelectric liquid crystal cell

    Energy Technology Data Exchange (ETDEWEB)

    Andreev, A L; Andreeva, T B; Kompanets, I N [P N Lebedev Physics Institute, Russian Academy of Sciences, Moscow (Russian Federation); Zalyapin, N V [National Research Nuclear University ' ' MEPhI' ' (Russian Federation)

    2014-12-31

    We have studied the method for suppressing speckle noise in patterns produced by a laser based on a fast-response electro-optical cell with a ferroelectric liquid crystal (FLC) in which helicoid is absent, i.e., compensated for. The character of smectic layer deformation in an electric field is considered along with the mechanism of spatially inhomogeneous phase modulation of a laser beam passing through the cell which is accompanied by the destruction of phase relations in the beam. Advantages of a helix-free FLC cell are pointed out as compared to helical crystal cells studied previously. (liquid crystal devices)

  15. 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.

  16. Communication-Free Distributed Coverage for Networked Systems

    KAUST Repository

    Yazicioglu, A. Yasin

    2016-01-15

    In this paper, we present a communication-free algorithm for distributed coverage of an arbitrary network by a group of mobile agents with local sensing capabilities. The network is represented as a graph, and the agents are arbitrarily deployed on some nodes of the graph. Any node of the graph is covered if it is within the sensing range of at least one agent. The agents are mobile devices that aim to explore the graph and to optimize their locations in a decentralized fashion by relying only on their sensory inputs. We formulate this problem in a game theoretic setting and propose a communication-free learning algorithm for maximizing the coverage.

  17. A methanol/air fuel cell system

    Science.gov (United States)

    Asher, W. J.

    1974-01-01

    High power-density, self-regulating fuel cell develops electrical power from catalyzed reaction between methanol and atmospheric oxygen. Cells such as these are of particular interest, because they may one day offer an emission-free, extremely efficient alternative to internal-combustion engines as power source.

  18. 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.

  19. Detection of free immunoglobulin light chains in cerebrospinal fluids of patients with central nervous system lymphomas.

    Science.gov (United States)

    Schroers, Roland; Baraniskin, Alexander; Heute, Christoph; Kuhnhenn, Jan; Alekseyev, Andriy; Schmiegel, Wolff; Schlegel, Uwe; Pels, Hendrik-Johannes

    2010-09-01

    Diagnosis of central nervous system (CNS) lymphoma depends on histopathology of brain biopsies, because no reliable disease marker in the cerebrospinal fluid (CSF) has been identified yet. B-cell lymphomas such as CNS lymphomas are clonally restricted and express either kappa or lambda immunoglobulin light chains. The aim of this study was to find out a potential diagnostic value of free immunoglobulin light chains released into the CSF of CNS lymphoma patients. Kappa (kappa) and lambda (lambda) free immunoglobulin light chains (FLC) were measured in CSF and serum samples collected from 21 patients with primary and secondary CNS lymphomas and 14 control patients with different neurologic disorders. FLC concentrations and ratios were compared between patient groups and were further analyzed in correlation with clinical, cytopathological, and radiological findings. FLC concentrations for all patients were lower in CSF when compared to serum. In patients with CNS lymphoma, the FLC ratios in CSF were higher (range 392-0.3) compared to control patients (range 3.0-0.3). Irrespective of cytopathological proven lymphomatous meningitis, in 11/21 lymphoma CSF samples the FLC ratios were markedly above 3.0 indicating a clonally restricted B-cell population. Increased FLC ratios in CSF were found in those patients showing subependymal lymphoma contact as detected in magnetic resonance imaging. In summary, this is the first report demonstrating that a significant proportion of patients with CNS lymphomas display a markedly increased FLC ratio in the CSF.

  20. 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.

  1. Reductive activity of free and immobilized cells of cyanobacteria toward oxophosphonates-comparative study.

    Science.gov (United States)

    Górak, Monika; Żymańczyk-Duda, Ewa

    2017-01-01

    This report, based on the previous studies, compares the reductive activity of different modes of following photobiocatalysts (on laboratory and preparative scale): Arthrospira maxima , Nostoc cf. muscorum and Nodularia sphaerocarpa , toward diethyl esters of 2-oxopropylphosphonate (1), 2-oxo-2-phenylethylphosphonate (2), and 2-oxobutylphosphonate (3). It was confirmed that immobilization in alginate matrix do not affect the activity and viability of the biocatalysts. Corresponding ( S )-hydroxyphosphonates (1a-3a) were obtained with similar efficiency compared to the free-cell mode with the yield and of the optical purity e.e respectively (e.g., N. sphaerocarpa experiments): (1) yield: 21 %, e.e . 84 %; (2) yield 97 %, e.e . 97; (3) yield 21 %, e.e. 89 %. Scaling up the processes for the best biocatalyst, N. sphaerocarpa , indicated that the use of free-living cells of cyanobacteria is more effective (640 mg of substrate 2, 44 % of yield, 91 % of e . e .), compared to the column bioreactor packed with immobilized cells of this photobiocatalyst (384 mg of substrate 2, 38 % of yield, 86 % of e.e ). In the case of free and immobilized cells of N. cf. muscorum , agitation of the medium was the crucial activity mediator. Shaking culture of free cells of N. cf. muscorum converted the diethyl 2-oxo-2-phenylethylphosphonate (2) with the yield of 43 % (99 % of e.e. ) compared to 18 % (99 % of e.e. , stationary culture). Immobilized cells of this cyanobacterium were also more active toward (2) under shaking conditions (28 % of yield, 99 % of e.e. ) than free ones without agitation.

  2. Long term disease free survival with multimodal therapy in small cell bladder cancer.

    Science.gov (United States)

    Heidegger, Isabel; Tulchiner, Gennadi; Schäfer, Georg; Horninger, Wolfgang; Pichler, Renate

    2016-10-13

    Small cell bladder cancer (SCBC) is an aggressive subtype accounting for less than 1 % of all bladder malignancies associated with rapid progression, early metastases formation and high mortality rates. We present an unusual long term disease free survival of a 60 year-old man who was diagnosed with SCBC two and a half years ago. He underwent four cycles of cisplatin/etoposide chemotherapy as well as a prophylactic whole-brain radiotherapy followed by a radical cystoprostatectomy and ileal neobladder with extended pelvic lymphadenectomy. Since 33 months the patient is now recurrence-free. In this case report, we were able to show that early multimodal therapy results in long term disease free survival, thus we highly recommend neoadjuvant chemotherapy as a part of multimodal management of a primary metastases-free, localized and surgically resectable SCBC.

  3. Disruption of MDCK cell tight junctions by the free-living amoeba Naegleria fowleri.

    Science.gov (United States)

    Shibayama, Mineko; Martínez-Castillo, Moisés; Silva-Olivares, Angélica; Galindo-Gómez, Silvia; Navarro-García, Fernando; Escobar-Herrera, Jaime; Sabanero, Myrna; Tsutsumi, Víctor; Serrano-Luna, Jesús

    2013-02-01

    Naegleria fowleri is the aetiological agent of primary amoebic meningoencephalitis. This parasite invades its host by penetrating the olfactory mucosa. However, the mechanism of epithelium penetration is not well understood. In the present study, we evaluated the effect of N. fowleri trophozoites and the non-pathogenic Naegleria gruberi on Madin-Darby canine kidney (MDCK) tight junction proteins, including claudin-1, occludin and ZO-1, as well as on the actin cytoskeleton. Trophozoites from each of the free-living amoeba species were co-cultured with MDCK cells in a 1 : 1 ratio for 1, 3, 6 or 10 h. Light microscopy revealed that N. fowleri caused morphological changes as early as 3 h post-infection in an epithelial MDCK monolayer. Confocal microscopy analysis revealed that after 10 h of co-culture, N. fowleri trophozoites induced epithelial cell damage, which was characterized by changes in the actin apical ring and disruption of the ZO-1 and claudin-1 proteins but not occludin. Western blot assays revealed gradual degradation of ZO-1 and claudin-1 as early as 3 h post-infection. Likewise, there was a drop in transepithelial electrical resistance that resulted in increased epithelial permeability and facilitated the invasion of N. fowleri trophozoites by a paracellular route. In contrast, N. gruberi did not induce alterations in MDCK cells even at 10 h post-infection. Based on these results, we suggest that N. fowleri trophozoites disrupt epithelial monolayers, which could enable their penetration of the olfactory epithelium and subsequent invasion of the central nervous system.

  4. 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

  5. Heterotrophic free-living and particle-bound bacterial cell size in the ...

    Indian Academy of Sciences (India)

    Regression analysis revealed that 18% of the variation in mean heterotrophic free-living bacterial cell size was due to biological oxygen demand (BOD) in the river Arkavathy, 11% due to surface water velocity (SWV) in the river Cauvery and 11% due to temperature in the river Kapila. Heterotrophic particle-bound bacterial ...

  6. Cell-free plasma microRNA in pancreatic ductal adenocarcinoma and disease controls

    DEFF Research Database (Denmark)

    Carlsen, Anting Liu; Joergensen, Maiken Thyregod; Knudsen, Steen

    2013-01-01

    There are no tumor-specific biochemical markers for pancreatic ductal adenocarcinoma (PDAC). Tissue-specific gene expression including microRNA (miRNA) profiling, however, identifies specific PDAC signatures. This study evaluates associations between circulating, cell-free plasma-miRNA profiles...

  7. Lipid-mediated Wnt protein stabilization enables serum-free culture of human organ stem cells

    NARCIS (Netherlands)

    Tüysüz, Nesrin; van Bloois, Louis|info:eu-repo/dai/nl/304839183; van den Brink, Stieneke; Begthel, Harry; Verstegen, Monique M A; Cruz, Luis J; Hui, Lijian; van der Laan, Luc J W; de Jonge, Jeroen; Vries, Robert; Braakman, Eric; Mastrobattista, Enrico|info:eu-repo/dai/nl/228061105; Cornelissen, Jan J; Clevers, Hans|info:eu-repo/dai/nl/07164282X; Ten Berge, Derk

    2017-01-01

    Wnt signalling proteins are essential for culture of human organ stem cells in organoids, but most Wnt protein formulations are poorly active in serum-free media. Here we show that purified Wnt3a protein is ineffective because it rapidly loses activity in culture media due to its hydrophobic nature,

  8. Evaluation of prenatal RHD typing strategies on cell-free fetal DNA from maternal plasma

    NARCIS (Netherlands)

    Grootkerk-Tax, Martine G. H. M.; Soussan, Aicha Ait; de Haas, Masja; Maaskant-van Wijk, Petra A.; van der Schoot, C. Ellen

    2006-01-01

    BACKGROUND: The discovery of cell-free fetal DNA in maternal plasma led to the development of assays to predict the fetal D status with RHD-specific sequences. Few assays are designed in such a way that the fetus can be typed in RHDpsi mothers and that RHDpsi fetuses are correctly typed. Owing to

  9. Clinical applications of cell-free fetal DNA from maternal plasma

    NARCIS (Netherlands)

    Rijnders, Robbert J. P.; Christiaens, Godelieve C. M. L.; Bossers, Bernadette; van der Smagt, Jasper J.; van der Schoot, C. Ellen; de Haas, Masja

    2004-01-01

    OBJECTIVE: To describe our clinical experience with detection and analysis of cell-free fetal DNA derived from maternal plasma for prenatal sexing and fetal rhesus-D typing. METHODS: Real-time quantitative polymerase chain reactions (PCRs) of rhesus-D sequences and the SRY gene were validated and

  10. Free Energies of Formation Measurements on Solid-State Electrochemical Cells

    Science.gov (United States)

    Rollino, J. A.; Aronson, S.

    1972-01-01

    A simple experiment is proposed that can provide the student with some insight into the chemical properties of solids. It also demonstrates the relationship between the Gibbs free energy of formation of an ionic solid and the emf of an electrochemical cell. (DF)

  11. Maternal vitamin B12 deficiency and abnormal cell-free DNA results in pregnancy

    NARCIS (Netherlands)

    Schuring-Blom, Heleen; Lichtenbelt, Klaske; van Galen, Karin; Elferink, Martin; Weiss, Marjan; Vermeesch, Joris Robert; Page-Christiaens, Lieve

    2016-01-01

    What's Already Known about this Topic? Prenatal testing with cell-free DNA may incidentally identify maternal genetic anomalies and malignancies. What does this Study Add? Profound vitamin B12 deficiency with intramedullary hemolysis may cause abnormal genomic patterns that can be detected by

  12. In-chip fabrication of free-form 3D constructs for directed cell migration analysis

    DEFF Research Database (Denmark)

    Olsen, Mark Holm; Hjortø, Gertrud Malene; Hansen, Morten

    2013-01-01

    Free-form constructs with three-dimensional (3D) microporosity were fabricated by two-photon polymerization inside the closed microchannel of an injection-molded, commercially available polymer chip for analysis of directed cell migration. Acrylate constructs were produced as woodpile topologies...

  13. 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

  14. 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

  15. 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.

  16. Fuel cell system blower configuration

    Energy Technology Data Exchange (ETDEWEB)

    Patel, Kirtikumar H.; Saito, Kazuo

    2017-11-28

    An exemplary fuel cell system includes a cell stack assembly having a plurality of cathode components and a plurality of anode components. A first reactant blower has an outlet situated to provide a first reactant to the cathode components. A second reactant blower has an outlet situated to provide a second reactant to the anode components. The second reactant blower includes a fan portion that moves the second reactant through the outlet. The second reactant blower also includes a motor portion that drives the fan portion and a bearing portion associated with the fan portion and the motor portion. The motor portion has a motor coolant inlet coupled with the outlet of the first reactant blower to receive some of the first reactant for cooling the motor portion.

  17. Fluorescent multiplex cell flow systems and methods

    KAUST Repository

    Merzaban, Jasmeen

    2017-06-01

    Systems and methods are provided for simultaneously assaying cell adhesion or cell rolling for multiple cell specimens. One embodiment provides a system for assaying adhesion or cell rolling of multiple cell specimens that includes a confocal imaging system containing a parallel plate flow chamber, a pump in fluid communication with the parallel plate flow chamber via a flow chamber inlet line and a cell suspension in fluid communication with the parallel plate flow chamber via a flow chamber outlet line. The system also includes a laser scanning system in electronic communication with the confocal imaging system, and a computer in communication with the confocal imaging system and laser scanning system. In certain embodiments, the laser scanning system emits multiple electromagnetic wavelengths simultaneously it cause multiple fluorescent labels having different excitation wavelength maximums to fluoresce. The system can simultaneously capture real-time fluorescence images from at least seven cell specimens in the parallel plate flow chamber.

  18. Taurine Biosynthesis in a Fish Liver Cell Line (ZFL Adapted to a Serum-Free Medium

    Directory of Open Access Journals (Sweden)

    Chieh-Lun Liu

    2017-05-01

    Full Text Available 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.

  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. Inhibition of phagocytic activity of ARPE-19 cells by free radical mediated oxidative stress.

    Science.gov (United States)

    Olchawa, Magdalena M; Pilat, Anna K; Szewczyk, Grzegorz M; Sarna, Tadeusz Jan

    2016-08-01

    Oxidative stress is a main factor responsible for key changes leading to the onset of age-related macular degeneration (ARMD) that occur in the retinal pigment epithelium (RPE), which is involved in phagocytosis of photoreceptor outer segments (POS). In this study, hydrogen peroxide (H2O2), H2O2 and iron ions (Fe) or rose Bengal (RB) in the presence of NADH and Fe were used to model free radical mediated oxidative stress to test if free radicals and singlet oxygen have different efficiency to inhibit phagocytosis of ARPE-19 cells. Free radical mediated oxidative stress was confirmed by HPLC-EC(Hg) measurements of cholesterol hydroperoxides in treated cells. Electron paramagnetic resonance (EPR) spin trapping was employed to detect superoxide anion. Cell survival was analyzed by the MTT assay. Specific phagocytosis of fluorescein-5-isothiocyanate-labeled POS and non-specific phagocytosis of fluorescent beads were measured by flow cytometry. HPLC analysis of cells photosensitized with RB in the presence of NADH and Fe indicated substantial increase in formation of free radical-dependent 7α/7β-hydroperoxides. EPR spin trapping confirmed the photogeneration of superoxide anion in samples enriched with RB, NADH and Fe. For all three protocols sub-lethal oxidative stress induced significant inhibition of the specific phagocytosis of POS. In contrast, non-specific phagocytosis was inhibited only by H2O2 or H2O2 and Fe treatment. Inhibition of phagocytosis was transient and recoverable by 24 h. These results suggest that free radicals may exert similar to singlet oxygen efficiency in inhibiting phagocytosis of RPE cells, and that the effect depends on the location where initial reactive species are formed.

  1. Optimal isolation and xeno-free culture conditions for limbal stem cell function.

    Science.gov (United States)

    Stasi, Kalliopi; Goings, DaVida; Huang, Jiayan; Herman, Lindsay; Pinto, Filipa; Addis, Russell C; Klein, Dahlia; Massaro-Giordano, Giacomina; Gearhart, John D

    2014-01-20

    To preserve limbal stem cell (LSC) function in vitro with xenobiotic-free culture conditions. Limbal epithelial cells were isolated from 139 donors using 15 variations of three dissociation solutions. All culture conditions were compared to the baseline condition of murine 3T3-J3 feeders with xenobiotic (Xeno) keratinocyte growth medium at 20% O2. Five Xeno and Xeno-free media with increasing concentrations of calcium and epidermal growth factor (EGF) were evaluated at 5%, 14%, and 20% O2. Human MRC-5, dermal (fetal, neonatal, or adult), and limbal stromal fibroblasts were compared. Statistical analysis was performed on the number of maximum serial weekly passages, percentage of aborted colonies, colony-forming efficiency (CFE), p63α(bright) cells, and RT-PCR ratio of p63α/K12. Immunocytochemistry and RT-PCR for p63α, ABCG2, Bmi1, C/EBPδ , K12, and MUC1 were performed to evaluate phenotype. Dispase/TrypLE was the isolation method that consistently showed the best yield, viability, and CFE. On 3T3-J2 feeders, Xeno-free medium with calcium 0.1 mM and EGF 10 ng/mL at 20% O2 supported more passages with equivalent percentage of aborted colonies, p63α(bright) cells, and p63α/K12 RT-PCR ratio compared to baseline Xeno-media. With this Xeno-free medium, MRC-5 feeders showed the best performance, followed by fetal, neonatal, adult HDF, and limbal fibroblasts. MRC-5 feeders supported serial passages with sustained high expression of progenitor cell markers at levels as robust as the baseline condition without significant difference between 20% and 5% O2. The LSC function can be maintained in vitro under appropriate Xeno-free conditions.

  2. Enzyme-free cell detachment mediated by resonance vibration with temperature modulation.

    Science.gov (United States)

    Kurashina, Yuta; Hirano, Makoto; Imashiro, Chikahiro; Totani, Kiichiro; Komotori, Jun; Takemura, Kenjiro

    2017-10-01

    Cell detachment is an essential process in adherent cell culture. However, trypsinization, which is the most popular detachment technique used in culture, damages cellular membranes. Reducing cellular membrane damage during detachment should improve the quality of cell culture. In this article, we propose an enzyme-free cell detachment method based on resonance vibration with temperature modulation. We developed a culture device that can excite a resonance vibration and control temperature. We then evaluated the cell detachment ratio and the growth response, observed the morphology, and analyzed the cellular protein of the collected cells-mouse myoblast cell line (C2C12). With the temperature of 10°C and the maximum vibration amplitude of 2 μm, 77.9% of cells in number were successfully detached compared with traditional trypsinization. The 72-h proliferation ratio of the reseeded cells was similar to that with trypsinization, whereas the proliferation ratio of proposed method was 12.6% greater than that of trypsinization after freezing and thawing. Moreover, the cells can be collected relatively intact and both intracellular and cell surface proteins in the proposed method were less damaged than in trypsinization. These results show that this method has definite advantages over trypsinization, which indicates that it could be applied to subcultures of cells that are more susceptible to trypsin damage for mass culture of sustainable clinical use. Biotechnol. Bioeng. 2017;114: 2279-2288. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  3. Immune bioactivity in shellfish toward serum-free cultured human cell lines.

    Science.gov (United States)

    Kong, Z L; Chiang, L C; Fang, F; Shinohara, K; Pan, P

    1997-01-01

    The biologically functional effect of eight kinds of hot-water extracts of shellfish on cultured human cell lines was examined in a serum-free medium model. Meretrix lusoria and Sinonovacula constricta extracts enhanced IgM secretion of both hybridoma HB4C5 and SI102 cells when cultured with the respective extracts. The purified principle exhibited remarked activity in the adsorbed fraction in hydroxyapatite and Concanavalin A columns. The extracts of Corbicula fluminea, Crassostreas gigas, Meretrix lusoria, Anadara granosa, and Sinonovacula constricta enhanced in nitroblue tetrazolium (NBT)-reducing ability of macrophage U-M cells. Meretrix lusoria, Anadara granosa, and Sinonovacula constricta were specifically cytotoxic to both cultures of MCF-7 breast cancer cells and HuH-6KK hepatoblastoma. These findings imply that the extracts of shellfish that were examined exhibited a differential effect on immune cells and tumor cells.

  4. 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.

  5. 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

  6. 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

    III and IV. Multivariate analysis identified DAPK1 as an independent prognostic factor for OS with a hazard ratio of 8.9 (95 % CI 2.7-29.3, P circulating DNA at time of diagnosis, who became long-term survivors, lost the aberrant methylation after......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...... treatment initiation. Conversely, patients that maintained or regained aberrant DAPK1 methylation died soon thereafter. CONCLUSIONS: Aberrant promoter methylation of cell-free circulating DNA can be detected in plasma from DLBCL patients and hold promise as an easily accessible marker for evaluating...

  7. Bacterial production and growth rate estimation from ( sup 3 H)thymidine incorporation for attached and free-living bacteria in aquatic systems

    Energy Technology Data Exchange (ETDEWEB)

    Iriberri, J.; Unanue, M.; Ayo, B.; Barcina, I., Egea, L. (Univ. del Pais Vasco, Bilbao (Spain))

    1990-02-01

    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-{sup 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 {times} 10{sup 11} and 8.678 {times} 10{sup 11} {mu}g of C mol{sup {minus}1} for free-living and attached bacteria in the freshwater system, and 1.276 {times} 10{sup 11} and 1.354 {times} 10{sup 11} {mu}g of C mol{sup {minus}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.

  8. 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

  9. A simple and highly stable free-flow electrophoresis device with thermoelectric cooling system.

    Science.gov (United States)

    Yan, Jian; Guo, Cheng-Gang; Liu, Xiao-Ping; Kong, Fan-Zhi; Shen, Qiao-Yi; Yang, Cheng-Zhang; Li, Jun; Cao, Cheng-Xi; Jin, Xin-Qiao

    2013-12-20

    Complex assembly, inconvenient operations, poor control of Joule heating and leakage of solution are still fundamental issues greatly hindering application of free-flow electrophoresis (FFE) for preparative purpose in bio-separation. To address these issues, a novel FFE device was developed based on our previous work. Firstly, a new mechanical structure was designed for compact assembly of separation chamber, fast removal of air bubble, and good anti-leakage performance. Secondly, a highly efficient thermoelectric cooling system was used for dispersing Joule heating for the first time. The systemic experiments revealed the three merits: (i) 3min assembly without any liquid leakage, 80 times faster than pervious FFE device designed by us or commercial device (4h); (ii) 5s removing of air bubble in chamber, 1000-fold faster than a normal one (2h or more) and (iii) good control of Joule heating by the cooling system. These merits endowed the device high stable thermo- and hydro-dynamic flow for long-term separation even under high electric field of 63V/cm. Finally, the developed device was used for up to 8h continuous separation of 5mg/mL fuchsin acid and purification of three model proteins of phycocyanin, myoglobin and cytochrome C, demonstrating the applicability of FFE. The developed FFE device has evident significance to the studies on stem cell, cell or organelle proteomics, and protein complex as well as micro- or nano-particles. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. 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.

  11. Label-free isolation and deposition of single bacterial cells from heterogeneous samples for clonal culturing

    OpenAIRE

    J. Riba; T. Gleichmann; S. Zimmermann; R. Zengerle; P. Koltay

    2016-01-01

    The isolation and analysis of single prokaryotic cells down to 1??m and less in size poses a special challenge and requires micro-engineered devices to handle volumes in the picoliter to nanoliter range. Here, an advanced Single-Cell Printer (SCP) was applied for automated and label-free isolation and deposition of bacterial cells encapsulated in 35?pl droplets by inkjet-like printing. To achieve this, dispenser chips to generate micro droplets have been fabricated with nozzles 20??m in size....

  12. Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

    Science.gov (United States)

    Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei

    2014-09-01

    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.

  13. 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.

  14. In-Situ Observation of Membrane Protein Folding during Cell-Free Expression.

    Directory of Open Access Journals (Sweden)

    Axel Baumann

    Full Text Available Proper insertion, folding and assembly of functional proteins in biological membranes are key processes to warrant activity of a living cell. Here, we present a novel approach to trace folding and insertion of a nascent membrane protein leaving the ribosome and penetrating the bilayer. Surface Enhanced IR Absorption Spectroscopy selectively monitored insertion and folding of membrane proteins during cell-free expression in a label-free and non-invasive manner. Protein synthesis was performed in an optical cell containing a prism covered with a thin gold film with nanodiscs on top, providing an artificial lipid bilayer for folding. In a pilot experiment, the folding pathway of bacteriorhodopsin via various secondary and tertiary structures was visualized. Thus, a methodology is established with which the folding reaction of other more complex membrane proteins can be observed during protein biosynthesis (in situ and in operando at molecular resolution.

  15. 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

  16. 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)

  17. 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.

  18. Algebraic Method in the Analysis of Decoherence-Free Subspaces in Open Quantum Systems

    Science.gov (United States)

    Kamizawa, Takeo

    2018-01-01

    In open quantum systems, a subspace which is not affected by the environmental noise is called a decoherence-free subspace. Such a subspace plays an important role in applications such as quantum information transmissions. In the literature, several "definitions" of decoherence-free subspaces were proposed, but they are model-dependent and slightly different. In this paper, we will study a general framework of decoherence-free subspaces and provide a criterion for the existence of a decoherence-free subspace in open quantum systems.

  19. Generation of induced pluripotent stem cells from virus-free in vivo reprogramming of BALB/c mouse liver cells.

    Science.gov (United States)

    de Lázaro, Irene; Bussy, Cyrill; Yilmazer, Açelya; Jackson, Maj Simonsen; Humphreys, Neil E; Kostarelos, Kostas

    2014-09-01

    The in vivo cell reprogramming of terminally differentiated somatic cells to a pluripotent state by the ectopic expression of defined transcription factors has been previously shown in the BALB/c mouse liver upon plasmid DNA injection with no teratoma formation in the host tissue. Here, we hypothesized that the reprogrammed cells could be extracted from the tissue and cultured in vitro. We called these cells in vivo induced pluripotent stem (i(2)PS) cells because they showed pluripotent characteristics equivalent to a standard mouse ES cell line (E14TG2A). The pluripotent character of i(2)PS cells was determined by a battery of morphological, molecular and functional assays, including their contribution to adult tissues of chimeric mice upon blastocyst injection. These observations further confirm that terminally differentiated somatic cells in wild type, adult animals can be reprogrammed in vivo using virus-free methodologies. The reprogrammed cells can generate in vitro stem cell colonies that exhibit pluripotency similar to ES cells with numerous implications for the application of in vivo reprogramming for tissue regenerative purposes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. H 2 O 2-HBr: A metal-free and organic solvent-free reagent system ...

    Indian Academy of Sciences (India)

    A novel, practical and environmentally benign approach has been developed for the oxidation of methylarenes using H2O2-HBr system in water. Arylaldehydes containing electon-withdrawing groups are isolated in good to high yields. Methylarenes containing electon-donating groups, in contrast, are transformed into ...

  1. Fuel-cell engine stream conditioning system

    Science.gov (United States)

    DuBose, Ronald Arthur

    2002-01-01

    A stream conditioning system for a fuel cell gas management system or fuel cell engine. The stream conditioning system manages species potential in at least one fuel cell reactant stream. A species transfer device is located in the path of at least one reactant stream of a fuel cell's inlet or outlet, which transfer device conditions that stream to improve the efficiency of the fuel cell. The species transfer device incorporates an exchange media and a sorbent. The fuel cell gas management system can include a cathode loop with the stream conditioning system transferring latent and sensible heat from an exhaust stream to the cathode inlet stream of the fuel cell; an anode humidity retention system for maintaining the total enthalpy of the anode stream exiting the fuel cell related to the total enthalpy of the anode inlet stream; and a cooling water management system having segregated deionized water and cooling water loops interconnected by means of a brazed plate heat exchanger.

  2. 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

  3. Non-invasive stem cell tracking in hindlimb ischemia animal model using bio-orthogonal copper-free click chemistry.

    Science.gov (United States)

    Lee, Si Yeon; Lee, Sangmin; Lee, Jangwook; Yhee, Ji Young; Yoon, Hwa In; Park, Soon-Jung; Koo, Heebeom; Moon, Sung-Hwan; Lee, Hyukjin; Cho, Yong Woo; Kang, Sun Woong; Lee, Sang-Yup; Kim, Kwangmeyung

    2016-10-28

    Labeling of stem cells aims to distinguish transplanted cells from host cells, understand in vivo fate of transplanted cells, particularly important in stem cell therapy. Adipose-derived mesenchymal stem cells (ASCs) are considered as an emerging therapeutic option for tissue regeneration, but much remains to be understood regarding the in vivo evidence. In this study, a simple and efficient cell labeling method for labeling and tracking of stem cells was developed based on bio-orthogonal copper-free click chemistry, and it was applied in a mouse hindlimb ischemia model. The human ASCs were treated with tetra-acetylated N-azidoacetyl-d-mannosamine (Ac 4 ManNAz) to generate glycoprotein with unnatural azide groups on the cell surface, and the generated azide groups were fluorescently labeled by specific binding of dibenzylcyclooctyne-conjugated Cy5 (DBCO-Cy5). The safe and long-term labeling of the hASCs by this method was first investigated in vitro. Then the DBCO-Cy5-hASCs were transplanted into the hindlimb ischemia mice model, and we could monitor and track in vivo fate of the cells using optical imaging system. We could clearly observe the migration potent of the hASCs toward the ischemic lesion. This approach to design and tailor new method for labeling of stem cells may be useful to provide better understanding on the therapeutic effects of transplanted stem cells into the target diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Determination of Free and Total Sulfites in Wine using an Automatic Flow Injection Analysis System with Voltammetric Detection

    OpenAIRE

    Gonçalves, Luís Moreira; Pacheco, João Grosso; Magalhães, Paulo Jorge; Rodrigues, José António; Barros, Aquiles Araújo

    2009-01-01

    Abstract An automated Flow Injection Analysis (FIA) system based on a initial analyte separation by gas-diffusion and subsequent determination by square-wave voltammetry (SWV) in a flow cell is proposed for the determination of total and free content of sulphur dioxide (SO2) in wine. The proposed method was compared with two iodometric methodologies (the Ripper method and the simplified method commonly used by the wine industry). The developed method shown repeatability (RSD lower ...

  5. 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.

  6. Uptake of label-free graphene oxide by Caco-2 cells is dependent on the cell differentiation status.

    Science.gov (United States)

    Kucki, Melanie; Diener, Liliane; Bohmer, Nils; Hirsch, Cordula; Krug, Harald F; Palermo, Vincenzo; Wick, Peter

    2017-06-21

    Understanding the interaction of graphene-related materials (GRM) with human cells is a key to the assessment of their potential risks for human health. There is a knowledge gap regarding the potential uptake of GRM by human intestinal cells after unintended ingestion. Therefore the aim of our study was to investigate the interaction of label-free graphene oxide (GO) with the intestinal cell line Caco-2 in vitro and to shed light on the influence of the cell phenotype given by the differentiation status on cellular uptake behaviour. Internalisation of two label-free GOs with different lateral size and thickness by undifferentiated and differentiated Caco-2 cells was analysed by scanning electron microscopy and transmission electron microscopy. Semi-quantification of cells associated with GRM was performed by flow cytometry. Undifferentiated Caco-2 cells showed significant amounts of cell-associated GRM, whereas differentiated Caco-2 cells exhibited low adhesion of GO sheets. Transmission electron microscopy analysis revealed internalisation of both applied GO (small and large) by undifferentiated Caco-2 cells. Even large GO sheets with lateral dimensions up to 10 µm, were found internalised by undifferentiated cells, presumably by macropinocytosis. In contrast, no GO uptake could be found for differentiated Caco-2 cells exhibiting an enterocyte-like morphology with apical brush border. Our results show that the internalisation of GO is highly dependent on the cell differentiation status of human intestinal cells. During differentiation Caco-2 cells undergo intense phenotypic changes which lead to a dramatic decrease in GRM internalisation. The results support the hypothesis that the cell surface topography of differentiated Caco-2 cells given by the brush border leads to low adhesion of GO sheets and sterical hindrance for material uptake. In addition, the mechanical properties of GRM, especially flexibility of the sheets, seem to be an important factor for

  7. Differential mapping of the free barbed and pointed ends of actin filaments in cells.

    Science.gov (United States)

    Ofer, Noa; Abu Shah, Enas; Keren, Kinneret

    2014-06-01

    The actin cytoskeleton plays a pivotal role in many cellular processes. Detailed analysis of the architecture of cellular actin networks provides valuable insight into the dynamic self-organization underlying these processes. In particular, since most of the actin turnover occurs at the tips of actin filaments, it is insightful to map the distribution of filament ends. Here we report a method for differentially labeling the pointed and the barbed ends of actin filaments in cellular networks by permeabilizing cells, following a brief fixation, and introducing labeled actin monomers in the presence or absence of capping protein, respectively. This method quantitatively maps the distributions of free barbed ends and free pointed ends in adherent cells, providing information on the polarity of cytoskeletal structures and mapping active sites available for actin assembly or disassembly. We demonstrate the use of this method by mapping the distribution of actin filament ends in motile fish epithelial keratocytes and in several mammalian cell lines, and show that free barbed ends are enriched near the tip of protruding lamellipodia while free pointed ends concentrate toward the rear. © 2014 Wiley Periodicals, Inc.

  8. Simulation of the microbunching instability in beam delivery systems for free electron lasers

    International Nuclear Information System (INIS)

    Pogorelov, Ilya; Qiang, Ji; Ryne, Rob; Venturini, Marco; Zholents, Alexander; Warnock, Robert

    2007-01-01

    In this paper, we examine the growth of the microbunching instability in the electron beam delivery system of a free electron laser(FEL). We present the results of two sets of simulations, one conducted using a direct Vlasov solver, the other using a particle-in-cell code Impact-Z with the number of simulation macroparticles ranging up to 100million. Discussion is focused on the details of longitudinal dynamics and on numerical values of uncorrelated (slice) energy spread at different points in the lattice. In particular, we assess the efficacy of laser heater in suppression of the instability, and look at the interplay between physical and numerical noise in particle-based simulations

  9. 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.

  10. Acute Toxicity of Silver Free and Encapsulated in Nanosized Zeolite for Eukaryotic Cells.

    Science.gov (United States)

    Anfray, Clément; Dong, Biao; Komaty, Sarah; Mintova, Svetlana; Valable, Samuel

    2017-04-26

    The potential toxicity of encapsulated silver in EMT-type nanosized zeolites on prokaryotic cells, human tumor cell lines from various origins, and primary cultures of neurons and astrocytes was investigated. Silver in cationic form (Ag + ) was encapsulated in EMT-type nanosized zeolites via an ion exchange process (Ag + -EMT) and compared with the reduced silver (Ag 0 ) in the zeolite (Ag 0 -EMT). As reference samples for the toxicity measurements, pure EMT-type zeolite and silver perchlorate were used. Cells were exposed to silver-containing zeolites (50, 100, and 400 μg/mL) for 24 and 48 h. After exposure to Ag + -EMT (50 μg/mL) for 24 h, a loss in cell viability independent of the cell type was observed, ranging from -34.37 ± 23.90% for astrocytes to -99.5 ± 0.24% for U87-MG cells. These results were comparable with the toxicity for silver perchlorate. The Ag 0 -EMT sample showed lower toxicity on human cell lines in comparison to that of Ag + -EMT. A decrease in cell viability, i.e., -73.46 ± 20.78% and -62.3 ± 17.96% for U87-MG and HEK 293 cells, respectively, under exposure only to high concentration of Ag 0 -EMT (400 μg/mL) for 24 h was measured. However, the Ag 0 -EMT was as toxic as the Ag + -EMT for astrocytes and neurons (-97.95 ± 3.31% and -100 ± 1.11%, respectively, after exposure to 50 μg/mL for 24 h). No decrease in cell viability exposed to pure EMT zeolite was found. The results demonstrate the severe toxicity of silver cations, either free or encapsulated, in comparison to reduced silver encapsulated in zeolite nanocrystals. Therefore, silver cations, either free or encapsulated, must be used with great caution regarding their toxicity on eukaryotic cells.

  11. Protective effect of aqueous extract from Spirulina platensis against cell death induced by free radicals

    Science.gov (United States)

    2010-01-01

    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 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. PMID:20858231

  12. Improved site-specific recombinase-based method to produce selectable marker- and vector-backbone-free transgenic cells

    Science.gov (United States)

    Yu, Yuan; Tong, Qi; Li, Zhongxia; Tian, Jinhai; Wang, Yizhi; Su, Feng; Wang, Yongsheng; Liu, Jun; Zhang, Yong

    2014-02-01

    PhiC31 integrase-mediated gene delivery has been extensively used in gene therapy and animal transgenesis. However, random integration events are observed in phiC31-mediated integration in different types of mammalian cells; as a result, the efficiencies of pseudo attP site integration and evaluation of site-specific integration are compromised. To improve this system, we used an attB-TK fusion gene as a negative selection marker, thereby eliminating random integration during phiC31-mediated transfection. We also excised the selection system and plasmid bacterial backbone by using two other site-specific recombinases, Cre and Dre. Thus, we generated clean transgenic bovine fetal fibroblast cells free of selectable marker and plasmid bacterial backbone. These clean cells were used as donor nuclei for somatic cell nuclear transfer (SCNT), indicating a similar developmental competence of SCNT embryos to that of non-transgenic cells. Therefore, the present gene delivery system facilitated the development of gene therapy and agricultural biotechnology.

  13. 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.

  14. Biological Synthesis of Silver Nanoparticles by Cell-Free Extract of Spirulina platensis

    Directory of Open Access Journals (Sweden)

    Gaurav Sharma

    2015-01-01

    Full Text Available The present study explores biological synthesis of silver nanoparticles (AgNPs using the cell-free extract of Spirulina platensis. Biosynthesised AgNPs were characterised by UV-Vis spectroscopy, SEM, TEM, and FTIR analysis and finally evaluated for antibacterial activity. Extracellular synthesis using aqueous extract of S. platensis showed the formation of well scattered, highly stable, spherical AgNPs with an average size of 30–50 nm. The size and morphology of the nanoparticles were confirmed by SEM and TEM analysis. FTIR and UV-Vis spectra showed that biomolecules, proteins and peptides, are mainly responsible for the formation and stabilisation of AgNPs. Furthermore, the synthesised nanoparticles exhibited high antibacterial activity against pathogenic Gram-negative, that is, Escherichia coli, MTCC-9721; Proteus vulgaris, MTCC-7299; Klebsiella pneumoniae, MTCC-9751, and Gram-positive, that is, Staphylococcus aureus, MTCC-9542; S. epidermidis, MTCC-2639; Bacillus cereus, MTCC-9017, bacteria. The AgNPs had shown maximum zone of inhibition (ZOI that is 31.3±1.11 in P. vulgaris. Use of such a microalgal system provides a simple, cost-effective alternative template for the biosynthesis of nanomaterials of silver in a large scale that could be of great use in biomedical applications.

  15. Platinum Group Metal-free Catalysts for Hydrogen Evolution Reaction in Microbial Electrolysis Cells.

    Science.gov (United States)

    Yuan, Heyang; He, Zhen

    2017-07-01

    Hydrogen gas is a green energy carrier with great environmental benefits. Microbial electrolysis cells (MECs) can convert low-grade organic matter to hydrogen gas with low energy consumption and have gained a growing interest in the past decade. Cathode catalysts for the hydrogen evolution reaction (HER) present a major challenge for the development and future applications of MECs. An ideal cathode catalyst should be catalytically active, simple to synthesize, durable in a complex environment, and cost-effective. A variety of noble-metal free catalysts have been developed and investigated for HER in MECs, including Nickel and its alloys, MoS 2 , carbon-based catalysts and biocatalysts. MECs in turn can serve as a research platform to study the durability of the HER catalysts. This personal account has reviewed, analyzed, and discussed those catalysts with an emphasis on synthesis and modification, system performance and potential for practical applications. It is expected to provide insights into the development of HER catalysts towards MEC applications. © 2017 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Coupled cell-free synthesis, segregation, and core glycosylation of a secretory protein.

    Science.gov (United States)

    Lingappa, V R; Lingappa, J R; Prasad, R; Ebner, K E; Blobel, G

    1978-05-01

    mRNA from rat mammary glands 13-15 days post partum was translated in a wheat germ cell-free system either in the absence or in the presence of ribosome-denuded membranes prepared from isolated rough microsomes of dog pancreas. Newly synthesized alpha-lactalbumin was identified by immunoprecipitation with a monospecific rabbit antiserum against rat alpha-lactalbumin and was characterized by partial amino-terminal sequence determination and by lectin affinity chromatography. In the absence of membranes a presumably unglycosylated form of alpha-lactalbumin was synthesized that bound neither to concanavalin A-Sepharose nor to Ricinus communis lectin-agarose and that contained an amino-terminal signal peptide region comprising 19 amino acid residues. In the presence of membranes a processed form was synthesized that lacked the signal peptide portion and that had an amino-terminal sequence identical to that of mature alpha-lactalbumin. Furthermore, this processed form was found to be segregated, presumably within the microsomal vesicles, because it was resistant to post-translational proteolysis. It was also found to be glycosylated, and because it bound to concanavalin A-Sepharose, from which it could be eluted specifically by alpha-methyl mannoside, but not to R. communis lectin-agarose, it was presumably core-glycosylated. Processing, segregation, and core glycosylation were observed to proceed only when membranes were present during translation and not when they were added after translation.

  17. Robust production of recombinant phosphoproteins using cell-free protein synthesis.

    Science.gov (United States)

    Oza, Javin P; Aerni, Hans R; Pirman, Natasha L; Barber, Karl W; ter Haar, Charlotte M; Rogulina, Svetlana; Amrofell, Matthew B; Isaacs, Farren J; Rinehart, Jesse; Jewett, Michael C

    2015-09-09

    Understanding the functional and structural consequences of site-specific protein phosphorylation has remained limited by our inability to produce phosphoproteins at high yields. Here we address this limitation by developing a cell-free protein synthesis (CFPS) platform that employs crude extracts from a genomically recoded strain of Escherichia coli for site-specific, co-translational incorporation of phosphoserine into proteins. We apply this system to the robust production of up to milligram quantities of human MEK1 kinase. Then, we recapitulate a physiological signalling cascade in vitro to evaluate the contributions of site-specific phosphorylation of mono- and doubly phosphorylated forms on MEK1 activity. We discover that only one phosphorylation event is necessary and sufficient for MEK1 activity. Our work sets the stage for using CFPS as a rapid high-throughput technology platform for direct expression of programmable phosphoproteins containing multiple phosphorylated residues. This work will facilitate study of phosphorylation-dependent structure-function relationships, kinase signalling networks and kinase inhibitor drugs.

  18. 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.

  19. 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.

  20. Application of mixed-organic-cation for high performance hole-conductor-free perovskite solar cells.

    Science.gov (United States)

    Xiao, Meng; Zhao, Li; Wei, Shoubin; Li, Yanyan; Dong, Binghai; Xu, Zuxun; Wan, Li; Wang, Shimin

    2018-01-15

    ABX 3 -type organic lead halide perovskites have gained increasing attention as light harvester for solar cells due to their high power conversion efficiency (PCE). Recently, it has become a trend to avoid the use of expensive hole-transport materials (HTMs) and precious metals, such as Au, to be competitive in future commercial development. In this study, we fabricated mixed-cation perovskite-based solar cells through one-step spin-coating using methylammonium (CH 3 NH 3 + ) and formamidinium (HN=CHNH 3 + ) cations to extend the optical absorption range into the red region and enhance the utilization of solar light. The synthesized hole-conductor-free cells with carbon electrode and mixed cations exhibited increased short-circuit current, outperforming the cells prepared with pure methylammonium, and PCE of 10.55%. This paper proposes an efficient approach for fabricating high-performance and low-cost perovskite solar cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. 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

  2. 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

  3. Label-free recognition of drug resistance via impedimetric screening of breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Bilge Eker

    Full Text Available We present a novel study on label-free recognition and distinction of drug resistant breast cancer cells (MCF-7 DOX from their parental cells (MCF-7 WT via impedimetric measurements. Drug resistant cells exhibited significant differences in their dielectric properties compared to wild-type cells, exerting much higher extracellular resistance (Rextra . Immunostaining revealed that MCF-7 DOX cells gained a much denser F-actin network upon acquiring drug resistance indicating that remodeling of actin cytoskeleton is probably the reason behind higher Rextra , providing stronger cell architecture. Moreover, having exposed both cell types to doxorubicin, we were able to distinguish these two phenotypes based on their substantially different drug response. Interestingly, impedimetric measurements identified a concentration-dependent and reversible increase in cell stiffness in the presence of low non-lethal drug doses. Combined with a profound frequency analysis, these findings enabled distinguishing distinct cellular responses during drug exposure within four concentration ranges without using any labeling. Overall, this study highlights the possibility to differentiate drug resistant phenotypes from their parental cells and to assess their drug response by using microelectrodes, offering direct, real-time and noninvasive measurements of cell dependent parameters under drug exposure, hence providing a promising step for personalized medicine applications such as evaluation of the disease progress and optimization of the drug treatment of a patient during chemotherapy.

  4. The Shipboard Employment of a Free Electron Laser Weapon System

    National Research Council Canada - National Science Library

    Allgaier, Gregory

    2003-01-01

    .... Considerations for this weapon system include employment, design, and stability. In order to reach a MW class laser, system parameters must be optimized and the high power optical beam must be appropriately managed...

  5. 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.

  6. 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.

  7. Free – bass accordion in modern educational systems

    OpenAIRE

    Algin, Sergei

    2012-01-01

    The purpose of the work – to prove the advantages of the introduction of accordion with system of Kravtsov in modern educational systems. Are used practical methods. Conclusion: The accordion with a system of Kravtsov anticipates an interesting perspective in piano accordion – bayan culture and education.

  8. Battery Cell Balancing System and Method

    Science.gov (United States)

    Davies, Francis J. (Inventor)

    2014-01-01

    A battery cell balancing system is operable to utilize a relatively small number of transformers interconnected with a battery having a plurality of battery cells to selectively charge the battery cells. Windings of the transformers are simultaneously driven with a plurality of waveforms whereupon selected battery cells or groups of cells are selected and charged. A transformer drive circuit is operable to selectively vary the waveforms to thereby vary a weighted voltage associated with each of the battery cells.

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

    Science.gov (United States)

    Paesen, Rik; Gyselaers, Wilfried; Stinissen, Piet

    2016-01-01

    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. PMID:27746820

  11. 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 mitochondrial DNA (cutoff: 19.84 raw Ct; sensitivity: 84%; specificity: 100%; p 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.

  12. 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

  13. Anchor-free NEMS non-volatile memory cell for harsh environment data storage

    International Nuclear Information System (INIS)

    Singh, Pushpapraj; Chua, Geng Li; Liang, Ying Shun; Jayaraman, Karthik Gopal; Do, Anh Tuan; Kim, Tony Tae-Hyoung

    2014-01-01

    This work demonstrates a novel anchor-free nano-electromechanical (NEMS) based non-volatile memory cell, suitable for high temperature (T ≤ 300 °C) and radiation prone harsh environment applications. The anchor-free circular metal beam is actuated by electrostatic force and is held in one of the bi-stable memory states by adhesion force between two smooth metal surfaces in contact. Smooth metal layers form strong van der Waals stiction between two surfaces in contact and memory detection (Logic-‘1’ / Logic-‘0’) is obtained by detecting the conductance between two fixed contacts. This anchor-free design offers highest density (9F 2 footprint) compared to other mechanical memory devices reported to date. (paper)

  14. CD30 cell graphs of Hodgkin lymphoma are not scale-free--an image analysis approach.

    Science.gov (United States)

    Schäfer, Hendrik; Schäfer, Tim; Ackermann, Jörg; Dichter, Norbert; Döring, Claudia; Hartmann, Sylvia; Hansmann, Martin-Leo; Koch, Ina

    2016-01-01

    Hodgkin lymphoma (HL) is a type of B-cell lymphoma. To diagnose the subtypes, biopsies are taken and immunostained. The slides are scanned to produce high-resolution digital whole slide images (WSI). Pathologists manually inspect the spatial distribution of cells, but little is known on the statistical properties of cell distributions in WSIs. Such properties would give valuable information for the construction of theoretical models that describe the invasion of malignant cells in the lymph node and the intercellular interactions. In this work, we define and discuss HL cell graphs. We identify CD30(+) cells in HL WSIs, bringing together the fields of digital imaging and network analysis. We define special graphs based on the positions of the immunostained cells. We present an automatic analysis of complete WSIs to determine significant morphological and immunohistochemical features of HL cells and their spatial distribution in the lymph node tissue under three different medical conditions: lymphadenitis (LA) and two types of HL. We analyze the vertex degree distributions of CD30 cell graphs and compare them to a null model. CD30 cell graphs show higher vertex degrees than expected by a random unit disk graph, suggesting clustering of the cells. We found that a gamma distribution is suitable to model the vertex degree distributions of CD30 cell graphs, meaning that they are not scale-free. Moreover, we compare the graphs for LA and two subtypes of HL. LA and classical HL showed different vertex degree distributions. The vertex degree distributions of the two HL subtypes NScHL and mixed cellularity HL (MXcHL) were similar. The CellProfiler pipeline used for cell detection is available at https://sourceforge.net/projects/cellgraphs/. ina.koch@bioinformatik.uni-frankfurt.de Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  15. 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.

  16. 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...

  17. Synthetic Biology for Cell-Free Biosynthesis: Fundamentals of Designing Novel In Vitro Multi-Enzyme Reaction Networks.

    Science.gov (United States)

    Morgado, Gaspar; Gerngross, Daniel; Roberts, Tania M; Panke, Sven

    2018-01-01

    Cell-free biosynthesis in the form of in vitro multi-enzyme reaction networks or enzyme cascade reactions emerges as a promising tool to carry out complex catalysis in one-step, one-vessel settings. It combines the advantages of well-established in vitro biocatalysis with the power of multi-step in vivo pathways. Such cascades have been successfully applied to the synthesis of fine and bulk chemicals, monomers and complex polymers of chemical importance, and energy molecules from renewable resources as well as electricity. The scale of these initial attempts remains small, suggesting that more robust control of such systems and more efficient optimization are currently major bottlenecks. To this end, the very nature of enzyme cascade reactions as multi-membered systems requires novel approaches for implementation and optimization, some of which can be obtained from in vivo disciplines (such as pathway refactoring and DNA assembly), and some of which can be built on the unique, cell-free properties of cascade reactions (such as easy analytical access to all system intermediates to facilitate modeling).

  18. The cell-free fetal DNA fraction in maternal blood decreases after physical activity

    DEFF Research Database (Denmark)

    Schlütter, Jacob Mørup; Hatt, Lotte; Bach, Cathrine

    2014-01-01

    OBJECTIVE: If noninvasive prenatal testing using next generation sequencing is to be effective for pregnant women, a cell-free fetal DNA (cffDNA) fraction above 4% is essential unless the depth of sequencing is increased. This study's objective is to determine whether physical activity has...... an effect on the proportion of cell-free DNA (cfDNA) arising from the fetus (fetal fraction). METHODS: Nine pregnant women carrying male fetuses at gestational age 12(+0)  weeks to 14(+6)  weeks were included. Plasma from nine pregnant women was drawn prior to, immediately after, and 30 min after 30 min...... of cycling with a pulse-rate of 150 beats per minute. The concentrations of cffDNA (DYS14) and cfDNA (RASSF1A) were assessed using quantitative real-time polymerase chain reaction. RESULTS: The fetal fraction decreased significantly in all participants after physical activity (p 

  19. 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 3 NH 3 PbI 3 ) perovskite. Dopant-free perovskite solar cells have been fabricated using CH 3 NH 3 PbI 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)

  20. Prognostic importance of cell-free DNA in chemotherapy resistant ovarian cancer treated with bevacizumab

    DEFF Research Database (Denmark)

    Steffensen, Karina Dahl; Madsen, Christine Vestergaard; Andersen, Rikke Fredslund

    2014-01-01

    of EOC in combination with chemotherapy. However, only a minor subgroup will benefit from the treatment and there is an obvious need for new markers to select such patients. The purpose of this study was to investigate the effect of single-agent bevacizumab in multiresistant EOC and the importance...... of circulating cell-free DNA (cfDNA) in predicting treatment response. METHODS: One hundred and forty-four patients with multi-resistant EOC were treated with single-agent bevacizumab 10mg/kg every three weeks. Baseline plasma samples were analysed for levels of cfDNA by real-time polymerase chain reaction (PCR......-agent bevacizumab treatment in multiresistant EOC appears to be a valuable treatment option with acceptable side-effects. Cell-free DNA showed independent prognostic importance in patients treated with bevacizumab and could be applied as an adjunct for treatment selection....

  1. 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.

  2. Chip based electroanalytical systems for cell analysis

    DEFF Research Database (Denmark)

    Spegel, C.; Heiskanen, A.; Skjolding, L.H.D.

    2008-01-01

    ' measurements of processes related to living cells, i.e., systems without lysing the cells. The focus is on chip based amperometric and impedimetric cell analysis systems where measurements utilizing solely carbon fiber microelectrodes (CFME) and other nonchip electrode formats, such as CFME for exocytosis...

  3. 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.

  4. 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.

  5. 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

  6. Generation of Footprint-Free Induced Pluripotent Stem Cells from Human Fibroblasts Using Episomal Plasmid Vectors.

    Science.gov (United States)

    Ovchinnikov, Dmitry A; Sun, Jane; Wolvetang, Ernst J

    2015-01-01

    Human induced pluripotent stem cells (hiPSCs) have provided novel insights into the etiology of disease and are set to transform regenerative medicine and drug screening over the next decade. The generation of human iPSCs free of a genetic footprint of the reprogramming process is crucial for the realization of these potential uses. Here we describe in detail the generation of human iPSC from control and disease-carrying individuals' fibroblasts using episomal plasmids.

  7. 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.

  8. Does dietary fibre stimulate intestinal epithelial cell proliferation in germ free rats?

    OpenAIRE

    Goodlad, R A; Ratcliffe, B; Fordham, J P; Wright, N A

    1989-01-01

    The aim of the present experiment was to investigate the role of hind gut fermentation in the proliferative response of the intestinal epithelium to dietary fibre. We have previously shown that refeeding starved rats with an elemental diet supplemented with fermentable dietary fibre (but not inert bulk) is capable of stimulating intestinal epithelial cell proliferation throughout the gastrointestinal tract. Three groups of 10 germ free (GF) rats and three groups of 10 conventional (CV) rats, ...

  9. Recent advances in cell-free PrPSc amplification technique.

    Science.gov (United States)

    Atarashi, Ryuichiro

    2009-01-01

    The development of amplification technology for abnormal forms of prion protein in vitro has had a great impact on the field of prion research. This novel technology has generated new possibilities for understanding the molecular basis of prions and for developing an early diagnostic test for prion diseases. This review provides an overview of recent progress in cell-free PrPSc amplification techniques.

  10. Free and Open Source Management Information Systems and ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    What is the impact of introducing an automated management information system within a microfinance institution or savings and credit cooperative on the employees, the clients and the business processes? The project will involve deployment of an open source management information system (Mifos Vanilla) in two ...

  11. 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

  12. Detection value of free cancer cells in peritoneal washing in gastric cancer: a systematic review and meta-analysis.

    Science.gov (United States)

    Tustumi, Francisco; Bernardo, Wanderley Marques; Dias, Andre Roncon; Ramos, Marcus Fernando Kodama Pertille; Cecconello, Ivan; Zilberstein, Bruno; Ribeiro-Júnior, Ulysses

    2016-12-01

    Intraperitoneal free cancer cells in gastric adenocarcinoma are associated with a poor outcome. However, the true prognostic value of intraperitoneal free cancer cells is still unclear, leading to a lack of consensus in the management of gastric cancer. The aim of the present study is to perform a systematic review and meta-analysis to analyze intraperitoneal free cancer cells-positive patients with regard to tumor oncologic stage, recurrence, grade of cellular differentiation, and survival rates and to analyze the clinical significance of intraperitoneal free cancer cells with regard to prognosis. Databases were searched up to January 2016 for prognostic factors associated with intraperitoneal free cancer cells, including oncologic stage, depth of neoplasm invasion, lymph nodal spread, differentiation grade of the tumor, and recurrence and survival rates. A total of 100 studies were identified. Meta-analysis revealed a clear association between intraperitoneal free cancer cells and a poor prognosis. intraperitoneal free cancer cells -positive patients had higher rates of nodal spread (risk difference: 0.29; pcancer cells are associated with a poor outcome in gastric cancer. This surrogate biomarker should be used to guide therapy both prior to and after surgery.

  13. 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.

  14. 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.

  15. Glial Cells: The Other Cells of the Nervous System

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 7; Issue 1. Glial Cells: The Other Cells of the Nervous System - An Introduction to Glial Cells. Medha S Rajadhyaksha Yasmin Khan. Series Article Volume 7 Issue 1 January 2002 pp 4-10 ...

  16. A critical synopsis: Continuous growth of proximal tubular kidney epithelial cells in hormone-supplemented serum-free medium

    Science.gov (United States)

    Chuman, L. M.; FINE; COHEN; Saier, M. H.

    1985-01-01

    The kidney forms urine and reabsorbs electrolytes and water. Kidney cell lines and hormone supplemented serum free medium were used for growth. The hormones were insulin, transferrin, vasopressin, cholesterol, prostaglandins, hydrocortisone, and triidothyronine. Epithelial cell lines are polar and form hemicysts. The Madin-Darby canine kidney(MDCK) cell line used is distal tubulelike. LLC-PK sub 1 cells are derived from pig kidneys and have the properties of different kidney segments. The LLC-PK sub 1 cells with proximal tubule properties were maintained in hormone-supplemented serum free medium. Seven factors (the aforementioned homrones and selenium) were needed for growth. Hormone-defined medium supported LLC-PK sub 1 cell growth, allowed transport (as seen by hemicyst formation), and influenced cell morphology. Vasopressin (used for growth and morphology) could be partially replaced by isobutylmethylxanthine or dibutyryl cAMP. The defined medium was used to isolate rabbit proximal tubule kidney epithelial cells free of fibroblasts.

  17. Label-Free Raman Hyperspectral Imaging of Single Cells Cultured on Polymer Substrates.

    Science.gov (United States)

    Sinjab, Faris; Sicilia, Giovanna; Shipp, Dustin W; Marlow, Maria; Notingher, Ioan

    2017-12-01

    While Raman hyperspectral imaging has been widely used for label-free mapping of biomolecules in cells, these measurements require the cells to be cultured on weakly Raman scattering substrates. However, many applications in biological sciences and engineering require the cells to be cultured on polymer substrates that often generate large Raman scattering signals. Here, we discuss the theoretical limits of the signal-to-noise ratio in the Raman spectra of cells in the presence of polymer signals and how optical aberrations may affect these measurements. We show that Raman spectra of cells cultured on polymer substrates can be obtained using automatic subtraction of the polymer signals and demonstrate the capabilities of these methods in two important applications: tissue engineering and in vitro toxicology screening of drugs. Apart from their scientific and technological importance, these applications are examples of the two most common measurement configurations: (1) cells cultured on an optically thick polymer substrate measured using an immersion/dipping objective; and (2) cells cultured on a transparent polymer substrate and measured using an inverted optical microscope. In these examples, we show that Raman hyperspectral data sets with sufficient quality can be successfully acquired to map the distribution of common biomolecules in cells, such as nucleic acids, proteins, and lipids, as well as detecting the early stages of apoptosis. We also discuss strategies for further improvements that could expand the application of Raman hyperspectral imaging on polymer substrates even further in biomedical sciences and engineering.

  18. Degradation of h-acid by free and immobilized cells of Alcaligenes latus

    Directory of Open Access Journals (Sweden)

    M.S. Usha

    2010-12-01

    Full Text Available Alcaligenes latus, isolated from industrial effluent, was able to grow in mineral salts medium with 50 ppm (0.15 mM of H-acid as a sole source of carbon. Immobilization of Alcaligenes latus in Ca-alginate and polyurethane foam resulted in cells embedded in the matrices. When free cells and immobilized cells were used for biodegradation studies at concentration ranging from 100 ppm (0.3 mM to 500 ppm (1.15 mM degradation rate was enhanced with immobilized cells. Cells immobilized in polyurethane foam showed 100% degradation up to 350 ppm (1.05 mM and 57% degradation at 500 ppm (1.5 mM. Degradation rate of Ca-alginate immobilized cells was less as compared to that of polyurethane foam immobilized cells. With Ca-alginate immobilized cells 100% degradation was recorded up to 200 ppm (0.6 mM of H-acid and only 33% degradation was recorded at 500 ppm (1.5 mM of H-acid. Spectral analysis of the products after H-acid utilization showed that the spent medium did not contain any aromatic compounds indicating H-acid degradation by A. latus.

  19. 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

  20. 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....

  1. A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors

    Directory of Open Access Journals (Sweden)

    Satoko eMatsunaga

    2015-10-01

    Full Text Available Due to their high frequency of genomic mutations, human retroviruses often develop resistance to antiretroviral drugs. The emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1 is a significant obstacle to the effective long-term treatment of HIV infection. The development of a rapid and versatile drug-susceptibility assay would enable acquisition of phenotypic information and facilitate determination of the appropriate choice of antiretroviral agents. In this study, we developed a novel in vitro method, termed the Cell-Free Drug Susceptibility Assay (CFDSA, for monitoring phenotypic information regarding the drug resistance of HIV-1 protease (PR. The CFDSA utilizes a wheat germ cell-free protein production system to synthesize enzymatically active HIV-1 PRs directly from PCR products amplified from HIV-1 molecular clones or clinical isolates in a rapid one-step procedure. Enzymatic activity of PRs can be readily measured by AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen in the presence or absence of clinically used protease inhibitors (PIs. CFDSA measurement of drug resistance was based on the fold resistance to the half-maximal inhibitory concentration (IC50 of various protease inhibitors (PIs. The CFDSA could serve as a non-infectious, rapid, accessible, and reliable alternative to infectious cell-based phenotypic assays for evaluation of PI-resistant HIV-1.

  2. Nanogel antigenic protein-delivery system for adjuvant-free intranasal vaccines

    Science.gov (United States)

    Nochi, Tomonori; Yuki, Yoshikazu; Takahashi, Haruko; Sawada, Shin-Ichi; Mejima, Mio; Kohda, Tomoko; Harada, Norihiro; Kong, Il Gyu; Sato, Ayuko; Kataoka, Nobuhiro; Tokuhara, Daisuke; Kurokawa, Shiho; Takahashi, Yuko; Tsukada, Hideo; Kozaki, Shunji; Akiyoshi, Kazunari; Kiyono, Hiroshi

    2010-07-01

    Nanotechnology is an innovative method of freely controlling nanometre-sized materials. Recent outbreaks of mucosal infectious diseases have increased the demands for development of mucosal vaccines because they induce both systemic and mucosal antigen-specific immune responses. Here we developed an intranasal vaccine-delivery system with a nanometre-sized hydrogel (`nanogel') consisting of a cationic type of cholesteryl-group-bearing pullulan (cCHP). A non-toxic subunit fragment of Clostridium botulinum type-A neurotoxin BoHc/A administered intranasally with cCHP nanogel (cCHP-BoHc/A) continuously adhered to the nasal epithelium and was effectively taken up by mucosal dendritic cells after its release from the cCHP nanogel. Vigorous botulinum-neurotoxin-A-neutralizing serum IgG and secretory IgA antibody responses were induced without co-administration of mucosal adjuvant. Importantly, intranasally administered cCHP-BoHc/A did not accumulate in the olfactory bulbs or brain. Moreover, intranasally immunized tetanus toxoid with cCHP nanogel induced strong tetanus-toxoid-specific systemic and mucosal immune responses. These results indicate that cCHP nanogel can be used as a universal protein-based antigen-delivery vehicle for adjuvant-free intranasal vaccination.

  3. Experimental self-etching HEMA-free adhesive systems: cytotoxicity and degree of conversion.

    Science.gov (United States)

    Barbosa, Marília Oliveira; de Carvalho, Rodrigo Varella; Demarco, Flávio Fernando; Ogliari, Fabrício Aulo; Zanchi, Cesar Henrique; Piva, Evandro; da Silva, Adriana Fernandes

    2015-01-01

    The aim of this study was to evaluate the effect of replacing 2-hydroxyethyl methacrylate (HEMA) by methacrylate surfactant monomers on the cytotoxicity and degree of conversion of two-step self-etching dentin adhesive systems. Five HEMA-free adhesive systems were tested: Bis-EMA 10, Bis-EMA 30, PEG400, PEG400UDMA, PEG1000, and a HEMA group was used as positive control. The cytotoxicity of the experimental primers, with different monomer concentrations (2 or 20 wt%), and bond resins, containing 25 wt% surfactant, was assessed using murine fibroblast cell line 3T3 and the tetrazolium assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)). The degree of conversion of the bond resins was analyzed using Fourier transform infrared spectroscopy. The data were submitted to statistical analysis using level of significance set at P adhesive system formulations since they showed lower cytotoxicity and similar degree of conversion when compared with the HEMA-containing group.

  4. Fundamentals of fuel cell system integration

    Science.gov (United States)

    Krumpelt, Michael; Kumar, Romesh; Myles, Kevin M.

    1994-04-01

    Fuel cells are theoretically very efficient energy conversion devices that have the potential of becoming a commercial product for numerous uses in the civilian economy. We have analyzed several fuel cell system designs with regard to thermal and chemical integration of the fuel cell stack into the rest of the system. Thermal integration permits the use of the stack waste heat for the endothermic steps of fuel reforming. Chemical integration provides the steam needed for fuel reforming from the water produced by the electrochemical cell reaction. High-temperature fuel cells, such as the molten carbonate and the solid oxide fuel cells, permit this system integration in a relatively simple manner. Lower temperature fuel cells, such as the polymer electrolyte and phosphoric acid systems, require added system complexity to achieve such integration. The system economics are affected by capital and fuel costs and technical parameters, such as electrochemical fuel utilization, current density, and system complexity. At today's low fuel prices and the high fuel cell costs (in part, because of the low rates of production of the early prototypes), fuel cell systems are not cost competitive with conventional power generation. With the manufacture and sale of larger numbers of fuel cell systems, the total costs will decrease from the current several thousand dollars per kW, to perhaps less than $100 per kW as production volumes approa ch a million units per year.

  5. Role of free radicals in an adriamycin-resistant human small cell lung cancer cell line

    NARCIS (Netherlands)

    Meijer, C.; Mulder, N H; Timmer-Bosscha, H; Zijlstra, J G; de Vries, E G

    1987-01-01

    In two Adriamycin (Adr) resistant sublines (GLC4-Adr1 and GLC4-Adr2) of a human small cell lung carcinoma cell line, GLC4, cross-resistance for radiation was found. GLC4-Adr1 has an acquired Adr resistance factor of 44 after culturing without Adr for 20 days and GLC4-Adr2, the same subline cultured

  6. 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.

  7. Cell boundary fault detection system

    Science.gov (United States)

    Archer, Charles Jens [Rochester, MN; Pinnow, Kurt Walter [Rochester, MN; Ratterman, Joseph D [Rochester, MN; Smith, Brian Edward [Rochester, MN

    2009-05-05

    A method determines a nodal fault along the boundary, or face, of a computing cell. Nodes on adjacent cell boundaries communicate with each other, and the communications are analyzed to determine if a node or connection is faulty.

  8. Glial Cells: The Other Cells of the Nervous System ...

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 7; Issue 6. Glial Cells: The Other Cells of the Nervous System - Oligodendrocytes – Ensheathers of the CNS. Yasmin Khan Medha S Rajadhyaksha. Series Article Volume 7 Issue 6 June 2002 pp 6-13 ... Keywords. Glia; myelin; central nervous system.

  9. The impact of residential demand response on the costs of a fossil-free system reserve

    DEFF Research Database (Denmark)

    Katz, Jonas; Balyk, Olexandr; Hevia Koch, Pablo Alejandro

    2016-01-01

    In order to achieve a better understanding of the system value of residential demand response, we study the potential impact of flexible demand on the costs of system reserves in a fossil-free electricity supply. Comparing these costs with traditional means of regulation our analysis aims...... to contribute to determining the least-cost options for regulation in a fossil-free power system. We extend an existing energy system model with demand response and reserve modelling and analyse the impact for the case of Denmark in 2035 to reflect a system based on renewable resources for electricity...

  10. 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.

  11. Cyclic Heating-Annealing and Boltzmann Distribution of Free Energies in a Spin-Glass System

    International Nuclear Information System (INIS)

    Zhou Haijun

    2007-01-01

    Ergodicity of a spin-glass is broken at low temperatures; the system is trapped in one of many ergodic configurational domains. Transitions between different ergodic domains are achievable through a heating-annealing procedure. If this experiment is repeated infinite times, all ergodic configurational domains will be visited with frequences that decreasing exponentially with their free energies. The mean free energy density of a spin-glass system on a random graph is calculated based on this free energy Boltzmann distribution in the present work, by means of the cavity approach.

  12. 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 methods: 52 of SLE ... C-reactive protein (CRP), routine autoantibodies for autoimmune diseases, complements (C3 &. C4), anti-nucleosome antibodies ... Supported by theory and observation, two major sources of cf-DNA ...

  13. Aged Gut Microbiota Contributes to Systemical Inflammaging after Transfer to Germ-Free Mice

    Directory of Open Access Journals (Sweden)

    Floris Fransen

    2017-11-01

    Full Text Available Advanced age is associated with chronic low-grade inflammation, which is usually referred to as inflammaging. Elderly are also known to have an altered gut microbiota composition. However, whether inflammaging is a cause or consequence of an altered gut microbiota composition is not clear. In this study, gut microbiota from young or old conventional mice was transferred to young germ-free (GF mice. Four weeks after gut microbiota transfer immune cell populations in spleen, Peyer’s patches, and mesenteric lymph nodes from conventionalized GF mice were analyzed by flow cytometry. In addition, whole-genome gene expression in the ileum was analyzed by microarray. Gut microbiota composition of donor and recipient mice was analyzed with 16S rDNA sequencing. Here, we show by transferring aged microbiota to young GF mice that certain bacterial species within the aged microbiota promote inflammaging. This effect was associated with lower levels of Akkermansia and higher levels of TM7 bacteria and Proteobacteria in the aged microbiota after transfer. The aged microbiota promoted inflammation in the small intestine in the GF mice and enhanced leakage of inflammatory bacterial components into the circulation was observed. Moreover, the aged microbiota promoted increased T cell activation in the systemic compartment. In conclusion, these data indicate that the gut microbiota from old mice contributes to inflammaging after transfer to young GF mice.

  14. Two-dimensional Al hydroxide interaction with cancerous cell membrane building units: Complexed free energy and orientation analysis

    Science.gov (United States)

    Tsukanov, A. A.; Psakhie, S. G.

    2017-09-01

    The application of hierarchical nanoparticles based on metal hydroxides in biomedicine, including anticancer therapy and medical imaging, is a rapidly developing field. Low-dimensional aluminum oxyhydroxide nanomaterials (AlOOH-NM) are quite promising base to develop hybrid theranostic nano-agents with core-shell architecture, which is determined by AlOOH-NMs physicochemical properties such as: large specific surface area, pH-dependent charge, amphoteric behavior, high surface density of polar groups capable to form non-covalent bonds, low or null cytotoxicity and biocompatibility. Characterization of the system behavior within interface between NM and plasmatic membrane is crucial for the understanding of nano-agent—cell interaction. In the present work the complex in silico study including the free energy estimation and orientation analysis of phosphatidylcholine (POPC) and phosphatidylethanolamine (POPE) lipids interacting with AlOOH nanosheet was conducted to understand the effect of such nanomaterial on cancerous cell plasmatic membrane.

  15. 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.

  16. Advances in Derivative-Free State Estimation for Nonlinear Systems

    DEFF Research Database (Denmark)

    Nørgaard, Magnus; Poulsen, Niels Kjølstad; Ravn, Ole

    In this paper we show that it involves considerable advantages to use polynomial approximations obtained with an interpolation formula for derivation of state estimators for nonlinear systems. The estimators become more accurate than estimators based on Taylor approximations, and yet the implemen...

  17. Photosensitization by Diaziquone: Correlation Between Diaziquone Cytotoxicity and Photoinduced Free Radicals in MCF-7 Cells

    Science.gov (United States)

    Al-Nabulsi, Isaf

    The ability of visible light to enhance the activity of diaziquone (AZQ) was evaluated in MCF-7 human breast cancer cells. Exponentially growing monolayers of MCF -7 cells were incubated for 1 hr with AZQ (IC_ {90}, 0.05 muM, IC50, 0.3 muM, or various concentrations of AZQ) prior to variable time intervals of visible light irradiation. Irradiations were performed using a 100W quartz-halogen lamp or 100W mercury arc lamp with a dose rate of 30 or 170 mW/m ^2, respectively. The effect of visible light and/or AZQ on cellular growth was determined by clonogenic assay. The results show that MCF-7 cells were sensitive to growth inhibition by AZQ. Without AZQ, visible light irradiation had no effect on cell survival, while with AZQ, visible light potentiated its cytotoxicity by a factor of 1.6 at 10% survival. This potentiation of AZQ activity is correlated with the formation of free radicals (hydroxyl radicals and AZQ semiquinone) and with the production of DNA strand breaks as measured by electron paramagnetic resonance and gel electrophoresis, respectively. These results support the hypothesis that free radical formation is part of the mechanism of action of AZQ. Moreover, they indicate that visible light irradiation can increase the activity of AZQ and may allow its use in the treatment of tumor in human patients.

  18. Label-free investigation of the effects of lithium niobate polarization on cell adhesion

    Science.gov (United States)

    Mandracchia, B.; Gennari, O.; Paturzo, M.; Grilli, S.; Ferraro, P.

    2017-06-01

    The determination of contact area is pivotal to understand how biomaterials properties influence cell adhesion. In particular, the influence of surface charges is well-known but still controversial, especially when new functional materials and methods are introduced. Here, we use for the first time Holographic Total Internal Reflection Microscopy (HoloTIRM) to study the influence of the spontaneous polarization of ferroelectric lithium niobate (LN) on the adhesion properties of fibroblast cells. The selective illumination of a very thin region directly above the substrate, achieved by Total Internal Reflection, provides high-contrast images of the contact regions. Holographic recording, on the other hand, allows for label-free quantitative phase imaging of the contact areas between cells and LN. Phase signal is more sensitive in the first 100nm and, thus more reliable in order to locate focal contacts. This work shows that cells adhering on negatively polarized LN present a significant increase of the contact area in comparison with cells adhering on the positively polarized LN substrate, as well as an intensification of contact vicinity. This confirms the potential of LN as a platform for investigating the role of charges on cellular processes. The similarity of cell adhesion behavior on negatively polarized LN and glass control also confirms the possibility to use LN as an active substrate without impairing cell behavior.

  19. Chromosomal instability in cell-free DNA is a serum biomarker for prostate cancer.

    Science.gov (United States)

    Schütz, Ekkehard; Akbari, Mohammad R; Beck, Julia; Urnovitz, Howard; Zhang, William W; Bornemann-Kolatzki, Kirsten; Mitchell, William M; Nam, Robert K; Narod, Steven A

    2015-01-01

    Genomic instability resulting in copy number variation is a hallmark of malignant transformation and may be identified through massive parallel sequencing. Tumor-specific cell free DNA (cfDNA) present in serum and plasma provides a real-time, easily accessible surrogate. DNA was extracted from serum of 204 patients with prostate cancer (Gleason score 2-10), 207 male controls, and patients with benign hyperplasia (n = 10) and prostatitis (n = 10). DNA was amplified by use of random primers, tagged with molecular identifiers, sequenced on a SOLID system, and aligned to the human genome. We evaluated the number of sequence reads of cfDNA in sliding 100-kbp intervals for variation from controls. We used chromosomal regions with significant variations in alignment hits for their ability to segregate patients and matched controls. Using ROC curves to assess diagnostic performance, we evaluated the number of regions in a first subset (n = 177), with variations in alignment hits alone, provided an area under the curve (AUC) of 0.81 (95% CI 0.7-0.9, P prostate cancer from controls with an AUC of 0.92 (0.87-0.95), reaching a diagnostic accuracy of 83%. Both benign prostatic hypertrophy and prostatitis could be distinguished from prostate cancer by use of cfDNA, with an accuracy of 90%. Assessment of a limited number of chromosomal structural instabilities by use of massive parallel sequencing of cfDNA was sufficient to distinguish between prostate cancer and controls. This large cohort demonstrates the utility of cfDNA in prostate cancer recently established in other malignant neoplasms. © 2014 American Association for Clinical Chemistry.

  20. Treatment of osteochondral lesions in the knee using a cell-free scaffold.

    Science.gov (United States)

    Verdonk, P; Dhollander, A; Almqvist, K F; Verdonk, R; Victor, J

    2015-03-01

    The treatment of osteochondral lesions is of great interest to orthopaedic surgeons because most lesions do not heal spontaneously. We present the short-term clinical outcome and MRI findings of a cell-free scaffold used for the treatment of these lesions in the knee. A total of 38 patients were prospectively evaluated clinically for two years following treatment with an osteochondral nanostructured biomimetic scaffold. There were 23 men and 15 women; the mean age of the patients was 30.5 years (15 to 64). Clinical outcome was assessed using the Knee Injury and Osteoarthritis Outcome Score (KOOS), the Tegner activity scale and a Visual Analgue scale for pain. MRI data were analysed based on the Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) scoring system at three, 12 and 24 months post-operatively. There was a continuous significant clinical improvement after surgery. In two patients, the scaffold treatment failed (5.3%) There was a statistically significant improvement in the MOCART precentage scores. The repair tissue filled most of the defect sufficiently. We found subchondral laminar changes in all patients. Intralesional osteophytes were found in two patients (5.3%). We conclude that this one-step scaffold-based technique can be used for osteochondral repair. The surgical technique is straightforward, and the clinical results are promising. The MRI aspects of the repair tissue continue to evolve during the first two years after surgery. However, the subchondral laminar and bone changes are a concern. ©2015 The British Editorial Society of Bone & Joint Surgery.

  1. Facilitating Constructive Alignment in Power Systems Engineering Education Using Free and Open-Source Software

    Science.gov (United States)

    Vanfretti, L.; Milano, F.

    2012-01-01

    This paper describes how the use of free and open-source software (FOSS) can facilitate the application of constructive alignment theory in power systems engineering education by enabling the deep learning approach in power system analysis courses. With this aim, this paper describes the authors' approach in using the Power System Analysis Toolbox…

  2. Recognizing Cursive Typewritten Text Using Segmentation-Free System

    Directory of Open Access Journals (Sweden)

    Mohammad S. Khorsheed

    2015-01-01

    Full Text Available Feature extraction plays an important role in text recognition as it aims to capture essential characteristics of the text image. Feature extraction algorithms widely range between robust and hard to extract features and noise sensitive and easy to extract features. Among those feature types are statistical features which are derived from the statistical distribution of the image pixels. This paper presents a novel method for feature extraction where simple statistical features are extracted from a one-pixel wide window that slides across the text line. The feature set is clustered in the feature space using vector quantization. The feature vector sequence is then injected to a classification engine for training and recognition purposes. The recognition system is applied to a data corpus which includes cursive Arabic text of more than 600 A4-size sheets typewritten in multiple computer-generated fonts. The system performance is compared to a previously published system from the literature with a similar engine but a different feature set.

  3. Systems Biology and Stem Cell Pluripotency

    DEFF Research Database (Denmark)

    Mashayekhi, Kaveh; Hall, Vanessa Jane; Freude, Kristine

    2016-01-01

    Recent breakthroughs in stem cell biology have accelerated research in the area of regenerative medicine. Over the past years, it has become possible to derive patient-specific stem cells which can be used to generate different cell populations for potential cell therapy. Systems biological...... improve systems biology and its uses in the field. In this chapter, we first give a general background on stem cell biology and regenerative medicine. Stem cell potency is introduced together with the hierarchy of stem cells ranging from pluripotent embryonic stem cells (ESCs) and induced pluripotent stem...... modeling of stem cell pluripotency and differentiation have largely been based on prior knowledge of signaling pathways, gene regulatory networks, and epigenetic factors. However, there is a great need to extend the complexity of the modeling and to integrate different types of data, which would further...

  4. P2X-selective purinergic antagonists are strong inhibitors of HIV-1 fusion during both cell-to-cell and cell-free infection.

    Science.gov (United States)

    Swartz, Talia H; Esposito, Anthony M; Durham, Natasha D; Hartmann, Boris M; Chen, Benjamin K

    2014-10-01

    Human immunodeficiency virus type 1 (HIV-1) infection is chronic and presently still incurable. Antiretroviral drugs effectively suppress replication; however, persistent activation of inflammatory pathways remains a key cause of morbidity. Recent studies proposed that purinergic signaling is required for HIV-1 infection. Purinergic receptors are distributed throughout a wide variety of tissue types and detect extracellular ATP as a danger signal released from dying cells. We have explored how these pathways are involved in the transmission of HIV-1 from cell to cell through virological synapses. Infection of CD4+ T lymphocytes with HIV-1 in the presence of an inhibitor of P2X receptors effectively inhibited HIV-1 infection through both cell-free and cell-to-cell contact in a dose-dependent manner. Inhibition of direct cell-to-cell infection did not affect the formation of virological synapses or the subsequent cell-to-cell transfer of HIV-1. During both cell-free and cell-to-cell CD4+ T lymphocyte infection, purinergic antagonists blocked infection at the level of viral membrane fusion. During cell-to-cell transmission, we observed CXCR4 colocalization with the newly internalized virus particles within target lymphocytes and found that the purinergic antagonists did not impair the recruitment of the coreceptor CXCR4 to the site of Gag internalization in the target cell. In a screen of a library of purinergic antagonists, we found that the most potent inhibitors of HIV-1 fusion were those that target P2X receptors, while P2Y-selective receptor antagonists or adenosine receptor antagonists were ineffective. Our results suggest that P2X receptors may provide a therapeutic target and that purinergic antagonists may have potent activity against viral infection of CD4+ T lymphocytes by both cell-free and cell-to-cell transmission. This study identifies purinergic antagonists to be potent inhibitors of HIV-1 cell-free and cell-to-cell-mediated infection and provides a

  5. Free fatty acid palmitate impairs the vitality and function of cultured human bladder smooth muscle cells.

    Directory of Open Access Journals (Sweden)

    Andreas Oberbach

    Full Text Available BACKGROUND: Incidence of urinary tract infections is elevated in patients with diabetes mellitus. Those patients show increased levels of the saturated free fatty acid palmitate. As recently shown metabolic alterations induced by palmitate include production and secretion of the pro-inflammatory cytokine interleukine-6 (IL-6 in cultured human bladder smooth muscle cells (hBSMC. Here we studied the influence of palmitate on vital cell properties, for example, regulation of cell proliferation, mitochondrial enzyme activity and antioxidant capacity in hBSMC, and analyzed the involvement of major cytokine signaling pathways. METHODOLOGY/PRINCIPAL FINDINGS: HBSMC cultures were set up from bladder tissue of patients undergoing cystectomy and stimulated with palmitate. We analyzed cell proliferation, mitochondrial enzyme activity, and antioxidant capacity by ELISA and confocal immunofluorescence. In signal transduction inhibition experiments we evaluated the involvement of NF-κB, JAK/STAT, MEK1, PI3K, and JNK in major cytokine signaling pathway regulation. We found: (i palmitate decreased cell proliferation, increased mitochondrial enzyme activity and antioxidant capacity; (ii direct inhibition of cytokine receptor by AG490 even more strongly suppressed cell proliferation in palmitate-stimulated cells, while counteracting palmitate-induced increase of antioxidant capacity; (iii in contrast knockdown of the STAT3 inhibitor SOCS3 increased cell proliferation and antioxidant capacity; (iv further downstream JAK/STAT3 signaling cascade the inhibition of PI3K or JNK enhanced palmitate induced suppression of cell proliferation; (v increase of mitochondrial enzyme activity by palmitate was enhanced by inhibition of PI3K but counteracted by inhibition of MEK1. CONCLUSIONS/SIGNIFICANCE: Saturated free fatty acids (e.g., palmitate cause massive alterations in vital cell functions of cultured hBSMC involving distinct major cytokine signaling pathways. Thereby

  6. Assessment of EGFR mutation status using cell-free DNA from bronchoalveolar lavage fluid.

    Science.gov (United States)

    Park, Sojung; Hur, Jae Young; Lee, Kye Young; Lee, Jae Cheol; Rho, Jin Kyung; Shin, Sun Hwa; Choi, Chang-Min

    2017-08-28

    Much attention has been focused on epidermal growth factor receptor (EGFR) mutation testing since the introduction of EGFR-tyrosine kinase inhibitors have improved survival in EGFR-positive lung cancer patients. Liquid biopsy using circulating tumor cells or cell-free DNA (cfDNA) has enabled less invasive testing, but requires a highly sensitive method. To date, liquid biopsy using bronchoalveolar lavage (BAL) fluid has rarely been used. From 20 patients with lung adenocarcinoma, we isolated cfDNA from 20 samples of cell-free BAL fluid and 19 cell-free bronchial washing samples. cfDNA was examined for EGFR mutations using peptide nucleic acid (PNA)-mediated PCR clamping method. In cases where the results from the tumor biopsy and BAL-derived cfDNA test were not consistent, PANAMutyper™ R EGFR kit was used along with PNA clamping-assisted fluorescence melting curve analysis. We included 17 patients with advanced stage disease and three with non-advanced stage disease. Tumor biopsy detected EGFR mutations in 12 of the patients. One patient had a p.L858R mutation and a de novo p.T790M mutation. The results from PNA-mediated PCR clamping were 75.0% (9/12) concordant with the tumor biopsy results for EGFR mutation status. PANAMutyper with fluorescence melting curve analysis was performed in three cases, which detected EGFR mutations in two more patients (11/12, 91.7%). EGFR mutations were detected in the cfDNA extracted from two bronchial washing samples. cfDNA from BAL fluid could be used for molecular testing of EGFR mutations and identification of p.T790M mutations, with an easily applicable method.

  7. 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.

  8. Label-Free Imaging and Biochemical Characterization of Bovine Sperm Cells

    Directory of Open Access Journals (Sweden)

    Maria Antonietta Ferrara

    2015-04-01

    Full Text Available 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.

  9. Compact Layer Free Perovskite Solar Cells with a High-Mobility Hole-Transporting Layer.

    Science.gov (United States)

    Zhu, Qianqian; Bao, Xichang; Yu, Jianhua; Zhu, Dangqiang; Qiu, Meng; Yang, Renqiang; Dong, Lifeng

    2016-02-03

    A high-mobility diketopyrrolopyrrole-based copolymer (P) was employed in compact layer free CH3NH3PbI3 perovskite solar cells as a hole-transporting layer (HTL). By using the P-HTL, the 6.62% device efficiency with conventional poly-3-hexylthiophene was increased to 10.80% in the simple device configuration (ITO/CH3NH3PbI3/HTL/MoO3/Ag). With improved short circuit current density, open circuit voltage, and fill factor, the higher power conversion efficiency of P-HTL device is ascribed to the higher carrier mobility, more suitable energy level, and lower interfacial charge recombination. Advantages of applying P-HTL to perovskite solar cells, such as low cost, low-temperature processing, and excellent performance with simple cell structure, exhibit a possibility for commercial applications.

  10. 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

  11. Examination by EPR spectroscopy of free radicals in melanins isolated from A-375 cells exposed on valproic acid and cisplatin.

    Science.gov (United States)

    Chodurek, Ewa; Zdybel, Magdalena; Pilawa, Barbara; Dzierzewicz, Zofia

    2012-01-01

    Drug binding by melanin biopolymers influence the effectiveness of the chemotherapy, radiotherapy and photodynamic therapy. Free radicals of melanins take part in formation of their complex with drugs. The aim of this work was to determine the effect of the two compounds: valproic acid (VPA) and cisplatin (CPT) on free radicals properties of melanin isolated from A-375 melanoma cells. Free radicals were examined by an X-band (9.3 GHz) electron paramagnetic resonance (EPR) spectroscopy. EPR spectra were measured for the model synthetic eumelanin - DOPA-melanin, the melanin isolated from the control A-375 cells and these cells treated by VPA, CPT and both VPA and CPT. For all the examined samples broad EPR lines (deltaBpp: 0.48-0.68 mT) with g-factors of 2.0045-2.0060 characteristic for o-semiquinone free radicals were observed. Free radicals concentrations (N) in the tested samples, g-factors, amplitudes (A), integral intensities (I) and linewidths (deltaBpp) of the EPR spectra, were analyzed. The EPR lines were homogeneously broadened. Continuous microwave saturation of the EPR spectra indicated that slow spin-lattice relaxation processes existed in all the tested melanin samples. The relatively slowest spin-lattice relaxation processes characterized melanin isolated from A-375 cells treated with both VPA and CPT. The changes of the EPR spectra with increasing microwave power in the range of 2.2-70 mW were evaluated. Free radicals concentrations in the melanin from A-375 cells were higher than in the synthetic DOPA-melanin. The strong increase of free radicals concentration in the melanin from A-375 cells was observed after their treating by VPA. CPT also caused the increase of free radicals concentrations in the examined natural melanin. The free radicals concentration in melanin isolated from A-375 cells treated with both VPA and CPT was slightly higher than those in melanin from the control cells.

  12. Sex steroid production associated with puberty is absent in germ cell-free salmon

    NARCIS (Netherlands)

    Kleppe, Lene; Andersson, Eva; Skaftnesmo, Kai Ove; Edvardsen, Rolf B; Fjelldal, Per Gunnar; Norberg, Birgitta; Bogerd, Jan; Schulz, Rüdiger W; Wargelius, Anna

    2017-01-01

    In all vertebrates studied so far, germ cells are not required for pubertal maturation of the gonadal steroidogenic system, subsequent development of secondary sex characteristics and reproductive behavior. To explore if the absence of germ cells affects puberty or growth in Atlantic salmon, germ

  13. Imposed Environmental Stresses Facilitate Cell-Free Nanoparticle Formation by Deinococcus radiodurans

    Science.gov (United States)

    2017-01-01

    ABSTRACT The biological synthesis of metal nanoparticles has been examined in a wide range of organisms, due to increased interest in green synthesis and environmental remediation applications involving heavy metal ion contamination. Deinococcus radiodurans is particularly attractive for environmental remediation involving metal reduction, due to its high levels of resistance to radiation and other environmental stresses. However, few studies have thoroughly examined the relationships between environmental stresses and the resulting effects on nanoparticle biosynthesis. In this work, we demonstrate cell-free nanoparticle production and study the effects of metal stressor concentrations and identity, temperature, pH, and oxygenation on the production of extracellular silver nanoparticles by D. radiodurans R1. We also report the synthesis of bimetallic silver and gold nanoparticles following the addition of a metal stressor (silver or gold), highlighting how production of these particles is enabled through the application of environmental stresses. Additionally, we found that both the morphology and size of monometallic and bimetallic nanoparticles were dependent on the environmental stresses imposed on the cells. The nanoparticles produced by D. radiodurans exhibited antimicrobial activity comparable to that of pure silver nanoparticles and displayed catalytic activity comparable to that of pure gold nanoparticles. Overall, we demonstrate that biosynthesized nanoparticle properties can be partially controlled through the tuning of applied environmental stresses, and we provide insight into how their application may affect nanoparticle production in D. radiodurans during bioremediation. IMPORTANCE Biosynthetic production of nanoparticles has recently gained prominence as a solution to rising concerns regarding increased bacterial resistance to antibiotics and a desire for environmentally friendly methods of bioremediation and chemical synthesis. To date, a range of

  14. Water Maintenance-Free Inorganic Proton Conductors for PEM Fuel Cells

    National Research Council Canada - National Science Library

    Mackay, Richard

    2001-01-01

    .... This technology could be of significant economic and technical benefit to the Army and industries by providing low cost, high reliability, high efficiency, low temperature fuel cells without water management systems...

  15. Cell free phosphorylation method to assess the utility of new nucleotides as nucleoside reserve transcriptase inhibitors

    CSIR Research Space (South Africa)

    Lebea, Phiyani J

    2007-06-01

    Full Text Available The acyclic nucleoside phosphonates such as cidofovir, adefovir and tenofovir have proved to be effective in vitro (cell culture systems) and in vivo (animal models, clinical studies) against a wide variety of DNA virus and retrovirus infections...

  16. Cell-free supernatants from probiotic Lactobacillus casei and Lactobacillus rhamnosus GG decrease colon cancer cell invasion in vitro.

    Science.gov (United States)

    Escamilla, Juanita; Lane, Michelle A; Maitin, Vatsala

    2012-08-01

    Probiotics have been shown to have a preventative role in colorectal carcinogenesis but research concerning their prophylactic potential in the later stages of colorectal cancer, specifically metastasis is limited. This study explored the potential of cell-free supernatants (CFS) from 2 probiotic Lactobacillus sp., Lactobacillus casei and Lactobacillus rhamnosus GG, to inhibit colon cancer cell invasion by influencing matrix metalloproteinase-9 (MMP-9) activity and levels of the tight junction protein zona occludens-1 (ZO-1) in cultured metastatic human colorectal carcinoma cells. HCT-116 cells were treated with CFS from L. casei, L. rhamnosus, or Bacteroides thetaiotaomicron (a gut commensal); or with uninoculated bacterial growth media. Treatment with CFS from both Lactobacillus sp. decreased colorectal cell invasion but treatment with CFS from B. thetaiotaomicron did not. CFS from both Lactobacillus sp. decreased MMP-9 and increased ZO-1 protein levels. L. rhamnosus CFS also lowered MMP-9 activity. To begin elucidating the secreted bacterial factor conveying these responses, Lactobacillus sp. CFS were fractionated into defined molecular weight ranges and cell invasion assessed. Fractionation revealed that the inhibitory activity was contained primarily in the >100 kDa and 50-100 kDa fractions, suggesting the inhibitory compound may be a macromolecule such as a protein, nucleic acid, or a polysaccharide.

  17. Systems, methods and computer readable media for estimating capacity loss in rechargeable electrochemical cells

    Science.gov (United States)

    Gering, Kevin L.

    2013-06-18

    A system includes an electrochemical cell, monitoring hardware, and a computing system. The monitoring hardware periodically samples charge characteristics of the electrochemical cell. The computing system periodically determines cell information from the charge characteristics of the electrochemical cell. The computing system also periodically adds a first degradation characteristic from the cell information to a first sigmoid expression, periodically adds a second degradation characteristic from the cell information to a second sigmoid expression and combines the first sigmoid expression and the second sigmoid expression to develop or augment a multiple sigmoid model (MSM) of the electrochemical cell. The MSM may be used to estimate a capacity loss of the electrochemical cell at a desired point in time and analyze other characteristics of the electrochemical cell. The first and second degradation characteristics may be loss of active host sites and loss of free lithium for Li-ion cells.

  18. 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...

  19. 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.

  20. Bioreduction of Cu(II) by cell-free copper reductase from a copper resistant Pseudomonas sp. NA.

    Science.gov (United States)

    Andreazza, Robson; Okeke, Benedict C; Pieniz, Simone; Brandelli, Adriano; Lambais, Mácio R; Camargo, Flávio A O

    2011-11-01

    Environmental copper contamination is a serious human health problem. Copper reductase is produced by microorganisms to facilitate copper uptake by ATPases into the cells increasing copper biosorption. This study assessed the reduction of Cu(II) by cell-free extracts of a highly copper-resistant bacterium, Pseudomonas sp. strain NA, isolated from vineyard soil contaminated with copper. Both intact cells and cell-free extract of Pseudomonas sp. strain NA displayed substantial reduction of Cu(II). Intact cells reduced more then 80 mg L(-1) of Cu(II) from medium amended with 200 mg L(-1) of copper after 24 h of incubation. Cell-free extract of the isolate reduced more than 65% of the Cu(II) at initial copper concentration of 200 mg L(-1) after 24 h. Soluble protein production was high at 72 h of incubation at 100 mg L(-1) of copper, with more then 60 μg L(-1) of total soluble protein in cell-free extract recorded. Cu(II) reduction by isolate NA was increased when copper concentration increased for both intact cells and cell-free extract. Results indicate that Pseudomonas sp. strain NA produces copper reductase enzyme as the key mechanism of copper biotransformation.

  1. 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.

  2. Circulating cell-free DNA levels increase variably following chorionic villus sampling.

    Science.gov (United States)

    Vora, Neeta L; Johnson, Kirby L; Peter, Inga; Tighiouart, Hocine; Ralston, Steven J; Craigo, Sabrina D; Bianchi, Diana W

    2010-04-01

    Cell-free fetal DNA (cffDNA) in maternal plasma results from degradation of fetal and/or placental cells. Our objective was to determine if chorionic villus sampling (CVS) causes increased release of fetal and/or maternal DNA. Fifty-two pregnant women were recruited prior to CVS, performed for clinical indications, at 10 5/7 to 13 2/7 weeks. Maternal blood was collected before and within 15 min after CVS. cffDNA was extracted from plasma. Real-time polymerase chain reaction (PCR) amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the Y chromosome sequence DYS1 were used as measures of total and fetal DNA, respectively. All samples were analyzed in triplicate without knowledge of fetal gender. Sensitivity of DYS1 detection in male fetuses was 100% (n = 30); specificity in female fetuses was 100% (n = 22). While a majority of women had > 50% post-procedure increases in both fetal and total DNA, some showed post-procedure decreases. However, overall median proportional increases were not statistically significant. Gestational age (GA), placental location, and individual CVS operator did not correlate with changes in DNA levels. While there were no statistically significant overall changes in DNA levels after CVS, as-yet undiscovered variables may influence the extent of post-procedure release of cell-free DNA in the circulation of pregnant women. Copyright (c) 2010 John Wiley & Sons, Ltd.

  3. Red blood cell deformability in patients with claudication after pain-free treadmill training.

    Science.gov (United States)

    Mika, Piotr; Spodaryk, Krzysztof; Cencora, Andrzej; Mika, Anna

    2006-07-01

    To assess the effect of pain-free treadmill training on red blood cell deformability and walking distance in patients with claudication. Randomized-controlled trial of exercise training. Patients were recruited from the primary care, vascular outpatient clinic. A total of 60 patients with peripheral arterial occlusive disease (stage II according to Leriche-Fontaine) were randomized into the treadmill program or a control group. Fifty-five patients completed the study (27 in the exercising group and 28 in the control group). Patients in the exercising group were walking on the treadmill 3 times a week for 3 months. Each session consisted of 1 hour repetitive walking [performed to 85% of the pain-free walking time (PFWT)] was supervised by a qualified physiotherapist. Changes in erythrocyte deformability and treadmill walking performance (PFWT, maximal walking time) were assessed in both groups before the study and after 3 months. After 3 months of treadmill training, red blood cell deformability in the exercising group significantly increased (Ptraining is associated with a significant increase in red cell deformability in patients with claudication.

  4. 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.

  5. Mesenchymal Stem Cell Secretome: Toward Cell-Free Therapeutic Strategies in Regenerative Medicine.

    Science.gov (United States)

    Vizoso, Francisco J; Eiro, Noemi; Cid, Sandra; Schneider, Jose; Perez-Fernandez, Roman

    2017-08-25

    Earlier research primarily attributed the effects of mesenchymal stem cell (MSC) therapies to their capacity for local engrafting and differentiating into multiple tissue types. However, recent studies have revealed that implanted cells do not survive for long, and that the benefits of MSC therapy could be due to the vast array of bioactive factors they produce, which play an important role in the regulation of key biologic processes. Secretome derivatives, such as conditioned media or exosomes, may present considerable advantages over cells for manufacturing, storage, handling, product shelf life and their potential as a ready-to-go biologic product. Nevertheless, regulatory requirements for manufacturing and quality control will be necessary to establish the safety and efficacy profile of these products. Among MSCs, human uterine cervical stem cells (hUCESCs) may be a good candidate for obtaining secretome-derived products. hUCESCs are obtained by Pap cervical smear, which is a less invasive and painful method than those used for obtaining other MSCs (for example, from bone marrow or adipose tissue). Moreover, due to easy isolation and a high proliferative rate, it is possible to obtain large amounts of hUCESCs or secretome-derived products for research and clinical use.

  6. FREE FLAVONOID CONTENT AND ANTIOXIDANT ACTIVITY OF WINTER WHEAT IN SUSTAINABLE FARMING SYSTEMS

    Directory of Open Access Journals (Sweden)

    Magdaléna Lacko - Bartošová

    2013-02-01

    Full Text Available The objective of this work was to evaluate the free flavonoid content and antioxidant activity of winter wheat white flour, whole grain flour and bran in ecological and integrated farming system in the years 2009-2010. The experiment was established on a scientific research base Dolná Malanta in western Slovakia during the years 2009 and 2010. Content of free flavonoids and antioxidant activity of white flour and whole grain flour was not affected by farming systems. Antioxidant activity of whole grain flour was more effected by a year and by a forecrop. White flour contain two times less free flavonoids and antioxidant activity than whole grain flour. By milling process most of the free flavonoids and other compounds with higher antioxidant activity remain in outer layers of grain.

  7. 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.

  8. Innovative High Temperature Fuel Cell systems

    NARCIS (Netherlands)

    Au, Siu Fai

    2003-01-01

    The world's energy consumption is growing extremely rapidly. Fuel cell systems are of interest by researchers and industry as the more efficient alternative to conventional thermal systems for power generation. The principle of fuel cell conversion does not involve thermal combustion and hence in

  9. Global existence and blowup for free boundary problems of coupled reaction-diffusion systems

    Directory of Open Access Journals (Sweden)

    Jianping Sun

    2014-05-01

    Full Text Available This article concerns a free boundary problem for a reaction-diffusion system modeling the cooperative interaction of two diffusion biological species in one space dimension. First we show the existence and uniqueness of a local classical solution, then we study the asymptotic behavior of the free boundary problem. Our results show that the free boundary problem admits a global solution if the inter-specific competitions are strong, while, if the inter-specific competitions are weak, there exist the blowup solution and a global fast solution.

  10. Utility of KRAS mutation detection using circulating cell-free DNA from patients with colorectal cancer.

    Science.gov (United States)

    Yamada, Takeshi; Iwai, Takuma; Takahashi, Goro; Kan, Hayato; Koizumi, Michihiro; Matsuda, Akihisa; Shinji, Seiichi; Yamagishi, Aya; Yokoyama, Yasuyuki; Tatsuguchi, Atsushi; Kawagoe, Tatsuro; Kitano, Shiro; Nakayama, Masato; Matsumoto, Satoshi; Uchida, Eiji

    2016-07-01

    In this study, we evaluated the clinical utility of detecting KRAS mutations in circulating cell-free (ccf)DNA of metastatic colorectal cancer patients. We prospectively recruited 94 metastatic colorectal cancer patients. Circulating cell-free DNA was extracted from plasma samples and analyzed for the presence of seven KRAS point mutations. Using the Invader Plus assay with peptide nucleic acid clamping method and digital PCR, KRAS mutations were detected in the ccfDNA in 35 of 39 patients previously determined to have primary tumors containing KRAS mutations using the Luminex method, and in 5 of 55 patients with tumors containing wild-type KRAS. Curative resection was undertaken in 7 of 34 patients with primary and ccfDNA KRAS mutations, resulting in the disappearance of the mutation from the cell-free DNA in five of seven patients. Three of these patients had tumor recurrence and KRAS mutations in their ccfDNA reappeared. Epidermal growth factor receptor blockade was administered to 24 of the KRAS tumor wild-type patients. Of the 24 patients with wild-type KRAS in their primary tumors, three patients had KRAS mutations in their ccfDNA and did not respond to treatment with epidermal growth factor receptor (EGFR) blockade. We also detected a new KRAS mutation in five patients during chemotherapy with EGFR blockade, before disease progression was detectable with imaging. The detection of KRAS mutations in ccfDNA is an attractive approach for predicting both treatment response and acquired resistance to EGFR blockade, and for detecting disease recurrence. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  11. Pre-analytical conditions in non-invasive prenatal testing of cell-free fetal RHD.

    Directory of Open Access Journals (Sweden)

    Frederik Banch Clausen

    Full Text Available Non-invasive prenatal testing of cell-free fetal DNA (cffDNA in maternal plasma can predict the fetal RhD type in D negative pregnant women. In Denmark, routine antenatal screening for the fetal RhD gene (RHD directs the administration of antenatal anti-D prophylaxis only to women who carry an RhD positive fetus. Prophylaxis reduces the risk of immunization that may lead to hemolytic disease of the fetus and the newborn. The reliability of predicting the fetal RhD type depends on pre-analytical factors and assay sensitivity. We evaluated the testing setup in the Capital Region of Denmark, based on data from routine antenatal RHD screening.Blood samples were drawn at gestational age 25 weeks. DNA extracted from 1 mL of plasma was analyzed for fetal RHD using a duplex method for exon 7/10. We investigated the effect of blood sample transportation time (n = 110 and ambient outdoor temperatures (n = 1539 on the levels of cffDNA and total DNA. We compared two different quantification methods, the delta Ct method and a universal standard curve. PCR pipetting was compared on two systems (n = 104.The cffDNA level was unaffected by blood sample transportation for up to 9 days and by ambient outdoor temperatures ranging from -10 °C to 28 °C during transport. The universal standard curve was applicable for cffDNA quantification. Identical levels of cffDNA were observed using the two automated PCR pipetting systems. We detected a mean of 100 fetal DNA copies/mL at a median gestational age of 25 weeks (range 10-39, n = 1317.The setup for real-time PCR-based, non-invasive prenatal testing of cffDNA in the Capital Region of Denmark is very robust. Our findings regarding the transportation of blood samples demonstrate the high stability of cffDNA. The applicability of a universal standard curve facilitates easy cffDNA quantification.

  12. Drug-Free Approach To Study the Unusual Cell Cycle of Giardia intestinalis

    Science.gov (United States)

    Horlock-Roberts, Kathleen; Reaume, Chase; Dayer, Guillem; Ouellet, Christine; Cook, Nicholas

    2017-01-01

    differentiation of trophozoite to cyst. Here, we developed counterflow centrifugal elutriation as a drug-free method to obtain fractions of giardia cultures enriched in cells from the G1, S, and G2 stages of the cell cycle. Analysis of these fractions showed that the cells do not show side effects associated with the drugs used for synchronization of giardia cultures. Therefore, counterflow centrifugal elutriation would advance studies on key regulatory events during the giardia cell cycle and identify potential drug targets to block giardia proliferation and transmission. PMID:28959734

  13. 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.

  14. A review on channel models in free space optical communication systems

    Science.gov (United States)

    Anbarasi, K.; Hemanth, C.; Sangeetha, R. G.

    2017-12-01

    Free Space Optical communication (FSO) is a wireless communication technology which uses light to transmit the data in free space. FSO has advantages like unlicensed spectrum and higher bandwidth. In this paper FSO system merits and demerits, challenges in FSO, and various channel models are discussed. To mitigate the turbulence in FSO the mitigation techniques like relaying, diversity schemes and adopting different modulation techniques used in different channels are discussed and its performance comparison is given.

  15. Phase field modelling of precipitate morphologies in systems with tetragonal interfacial free energy anisotropy

    OpenAIRE

    Roy, Arijit; Gururajan, M P

    2017-01-01

    A wide variety of morphologies arise due to the tetragonal anisotropy in interfacial free energy. In this paper, we report on a family of Extended Cahn-Hilliard (ECH) models for incorporating tetragonal anisotropy in interfacial free energy. We list the non-zero and independent parameters that are introduced in our model and list the constraints on them. For appropriate choice of these parameters, our model can produce a many of the morphologies seen in tetragonal systems such as di-pyramids,...

  16. Free fatty acid palmitate activates unfolded protein response pathway and promotes apoptosis in meniscus cells.

    Science.gov (United States)

    Haywood, J; Yammani, R R

    2016-05-01

    Obesity is the major risk factor for the development of osteoarthritis (OA); however, the mechanisms involved are not clearly understood. Obesity is associated with increased production of adipokine and elevated levels of circulating free fatty acids (FFA). A recent study has shown that saturated fatty acid palmitate induced pro-inflammatory and pro-apoptotic pathways in chondrocytes. Meniscus has been shown to be more susceptible than articular cartilage to catabolic stimuli. Thus, the aim of this study was to determine the effect of FFA (specifically, palmitate) on meniscus cells. Cultured primary porcine meniscus cells were stimulated with 500 μM FFA (palmitate and oleate) for 24 h to induce endoplasmic reticulum (ER) stress. After treatment, cell lysates were prepared and immunoblotted for C/EBP homologous protein (CHOP). To determine the activation of unfolded protein response (UPR) signaling, cell lysates were probed for cJun n-terminal kinase (JNK), cleaved caspase -3 and Xbp-1s, an alternative mRNA splicing product generated due to Ire1α activation. Treatment of isolated primary meniscus cells with palmitate but not oleate induced expression of CHOP and Xbp-1s. Palmitate treatment of meniscus cells also activated JNK and increased expression of caspase-3, thus promoting apoptosis in meniscus cells. Palmitate induces ER stress and promotes apoptotic pathways in meniscus cells. This is the first study to establish ER stress as a key metabolic mechanistic link between obesity and OA, in addition to (or operating with) biomechanical factors. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  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. Single-Cell Optical Distortion Correction and Label-Free 3D Cell Shape Reconstruction on Lattices of Nanostructures.

    Science.gov (United States)

    Stephan, Jürgen; Keber, Felix; Stierle, Valentin; Rädler, Joachim O; Paulitschke, Philipp

    2017-12-13

    Imaging techniques can be compromised by aberrations. Especially when imaging through biological specimens, sample-induced distortions can limit localization accuracy. In particular, this phenomenon affects localization microscopy, traction force measurements, and single-particle tracking, which offer high-resolution insights into biological tissue. Here we present a method for quantifying and correcting the optical distortions induced by single, adherent, living cells. The technique uses periodically patterned gold nanostructures as a reference framework to quantify optically induced displacements with micrometer-scale sampling density and an accuracy of a few nanometers. The 3D cell shape and a simplified geometrical optics approach are then utilized to remap the microscope image. Our experiments reveal displacements of up to several hundred nanometers, and in corrected images these distortions are reduced by a factor of 3. Conversely, the relationship between cell shape and distortion provides a novel method of 3D cell shape reconstruction from a single image, enabling label-free 3D cell analysis.

  19. Performance evaluation and channel modeling of MIMO free space optical communication system

    Science.gov (United States)

    Deng, Tianping; Lu, Yimin; Lu, Gang; Peng, Kai

    2005-11-01

    Free space optical communication systems represent one of the most promising approaches for addressing the emerging broadband access market, it can provide high bandwidth with no physical contact, but are hampered by signal fading effects due to particulate scattering caused by atmospheric turbulence. In this paper, we propose a new channel model of MIMO free space optical communication system. The physics meaning of this model is very clear, and its format is very simple. Mathematic results show that MIMO is a very effective way for intensity fluctuation reduction induced by turbulence, thus reduce the bit-error-rate of the system.

  20. PRODUCTIVITY OF LAYERS AND EGG QUALITY IN FREE RANGE AND CAGE SYSTEM OF HOUSING

    OpenAIRE

    Đ. Senčić; Danijela Butko

    2006-01-01

    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 mor...

  1. Simulative Analysis of Various Parameters on Free Space Optical Communication System

    Science.gov (United States)

    Kumar, Naresh; Rana, Ashwani Kumar

    2013-09-01

    The Free Space Optical (FSO) communication system provides a high bandwidth, small size, light weight, low power and low cost alternative to present microwave systems. In this paper, we have designed a model of FSO system using OPTISYSTEM™ simulator to establish an FSO link by a range of 5 km with BER ˜ 10-6 achieved and reported analysis of different parameter that play important role in FSO communication system.

  2. Free Electron Laser For The Siberian Centre For Photochemical Research The Control System

    CERN Document Server

    Avlasov, Y K; Miginskaya, E G; Oreshkov, A D; Ovchar, A V; Salikova, T V; Selivanov, P A; Serednyakov, S I; Tararyshkin, S V; Tribendis, A G; Vinokurov, N A

    2004-01-01

    This article describes the software for the control system of the Free Electron Laser for the Siberian Center for photochemical research. The main components of subsystems composing the control system as well as their hardware and software components are considered. Also main features of each of the subsystems and of the whole control system are mentioned. The prospects of control system development to meet the future requirements are discussed.

  3. Microgravity and clinorotation cause redistribution of free calcium in sweet clover columella cells

    Science.gov (United States)

    Hilaire, E.; Paulsen, A. Q.; Brown, C. S.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

    1995-01-01

    In higher plants, calcium redistribution is believed to be crucial for the root to respond to a change in the direction of the gravity vector. To test the effects of clinorotation and microgravity on calcium localization in higher plant roots, sweet clover (Melilotus alba L.) seedlings were germinated and grown for two days on a slow rotating clinostat or in microgravity on the US Space Shuttle flight STS-60. Subsequently, the tissue was treated with a fixative containing antimonate (a calcium precipitating agent) during clinorotation or in microgravity and processed for electron microscopy. In root columella cells of clinorotated plants, antimonate precipitates were localized adjacent to the cell wall in a unilateral manner. Columella cells exposed to microgravity were characterized by precipitates mostly located adjacent to the proximal and lateral cell wall. In all treatments some punctate precipitates were associated with vacuoles, amyloplasts, mitochondria, and euchromatin of the nucleus. A quantitative study revealed a decreased number of precipitates associated with the nucleus and the amyloplasts in columella cells exposed to microgravity as compared to ground controls. These data suggest that roots perceive a change in the gravitational field, as produced by clinorotation or space flights, and respond respectively differently by a redistribution of free calcium.

  4. The Falsehood of a Single Appraisal System: The Case of the Free ...

    African Journals Online (AJOL)

    This paper focuses on the appraisal system, popularly called the Employee Performance Management and Development System (EPMDS) of the Free State Health Department. The objective of the study was to establish gaps in the successful application of performance appraisals, and identify the major factors required for ...

  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. Low-Complexity Interference-Free Downlink Channel Assignment with Improved Performance in Coordinated Small Cells

    KAUST Repository

    Radaydeh, Redha M.

    2015-05-01

    This paper proposes a low-complexity interference-free channel assignment scheme with improved desired downlink performance in coordinated multi-antenna small-coverage access points (APs) that employ the open-access control strategy. The adopted system treats the case when each user can be granted an access to one of the available channels at a time. Moreover, each receive terminal can suppress a limited number of resolvable interfering sources via its highly-correlated receive array. On the other hand, the operation of the deployed APs can be coordinated to serve active users, and the availability of multiple physical channels and the use of uncorrelated transmit antennas at each AP are exploited to improve the performance of supported users. The analysis provides new approaches to use the transmit antenna array at each AP, the multiple physical channels, the receive antenna array at each user in order to identify interference-free channels per each user, and then to select a downlink channel that provides the best possible improved performance. The event of concurrent interference-free channel identification by different users is also treated to further improve the desired link associated with the scheduled user. The analysis considers the practical scenario of imperfect identification of interference-free channel by an active user and/or the imperfectness in scheduling concurrent users requests on the same channel. The developed formulations can be used to study any performance metric and they are applicable for any statistical and geometric channel models. © 2015 IEEE.

  7. The current status and clinical value of circulating tumor cells and circulating cell-free tumor DNA in bladder cancer.

    Science.gov (United States)

    Riethdorf, Sabine; Soave, Armin; Rink, Michael

    2017-12-01

    Urothelial carcinoma of the bladder (UCB) is a complex disease, which is associated with highly aggressive tumor biologic behavior, especially in patients with muscle-invasive and advanced tumors. Despite multimodal therapy options including surgery, radiotherapy and chemotherapy, UCB patients frequently suffer from poor clinical outcome. Indeed, the potential of diverse opportunities for modern targeted therapies is not sufficiently elucidated in UCB yet. To improve the suboptimal treatment situation in UCB, biomarkers are urgently needed that help detecting minimal residual disease (MRD), predicting therapy response and subsequently prognosis as well as enabling patient stratification for further therapies and therapy monitoring, respectively. To date, decision making regarding treatment planning is mainly based on histopathologic evaluation of biopsies predominantly derived from the primary tumors and on clinical staging. However, both methods are imperfect for sufficient outcome prediction. During disease progression, individual disseminated tumor cells and consecutively metastases can acquire characteristics that do not match those of the corresponding primary tumors, and often are only hardly assessable for further evaluation. Therefore, during recent years, strong efforts were directed to establish non-invasive biomarkers from liquid biopsies. Urine cytology and serum tumor markers have been established for diagnostic purposes, but are still insufficient as universal biomarkers for decision-making and treatment of UCB patients. To date, the clinical relevance of various newly established blood-based biomarkers comprising circulating tumor cells (CTCs), circulating cell-free nucleic acids or tumor-educated platelets is being tested in cancer patients. In this review we summarize the current state and clinical application of CTCs and circulating cell-free tumor DNA originating from blood as biomarkers in patients with different UCB stages.

  8. The current status and clinical value of circulating tumor cells and circulating cell-free tumor DNA in bladder cancer

    Science.gov (United States)

    Soave, Armin; Rink, Michael

    2017-01-01

    Urothelial carcinoma of the bladder (UCB) is a complex disease, which is associated with highly aggressive tumor biologic behavior, especially in patients with muscle-invasive and advanced tumors. Despite multimodal therapy options including surgery, radiotherapy and chemotherapy, UCB patients frequently suffer from poor clinical outcome. Indeed, the potential of diverse opportunities for modern targeted therapies is not sufficiently elucidated in UCB yet. To improve the suboptimal treatment situation in UCB, biomarkers are urgently needed that help detecting minimal residual disease (MRD), predicting therapy response and subsequently prognosis as well as enabling patient stratification for further therapies and therapy monitoring, respectively. To date, decision making regarding treatment planning is mainly based on histopathologic evaluation of biopsies predominantly derived from the primary tumors and on clinical staging. However, both methods are imperfect for sufficient outcome prediction. During disease progression, individual disseminated tumor cells and consecutively metastases can acquire characteristics that do not match those of the corresponding primary tumors, and often are only hardly assessable for further evaluation. Therefore, during recent years, strong efforts were directed to establish non-invasive biomarkers from liquid biopsies. Urine cytology and serum tumor markers have been established for diagnostic purposes, but are still insufficient as universal biomarkers for decision-making and treatment of UCB patients. To date, the clinical relevance of various newly established blood-based biomarkers comprising circulating tumor cells (CTCs), circulating cell-free nucleic acids or tumor-educated platelets is being tested in cancer patients. In this review we summarize the current state and clinical application of CTCs and circulating cell-free tumor DNA originating from blood as biomarkers in patients with different UCB stages. PMID:29354496

  9. Detection of copy number alterations in cell-free tumor DNA from plasma

    DEFF Research Database (Denmark)

    Østrup, Olga; Ahlborn, Lise Barlebo; Lassen, Ulrik

    2017-01-01

    purposes, however specify and reliability of methods have to be tested. METHODS: SNP microarrays (Affymetrix) were used to generate whole-genome copy number profiles from plasma ccfDNA (OncoScan) and paired tumor biopsies (CytoScan) from ten patients with metastatic cancers. Numerical, segmental and focal......BACKGROUND: Somatic copy number alterations (SCNAs) occurring in tumors can provide information about tumor classification, patient's outcome or treatment targets. Liquid biopsies, incl. plasma samples containing circulating cell-free tumor DNA (ccfDNA) can be used to assess SCNAs for clinical...... of SCNAs changes during the treatment course of one patient also indicated that apoptosis/necrosis of non-cancerous cells presumably induced by treatment can influence ccfDNA composition and introduce false-negative findings into the analysis of liquid biopsies. CONCLUSIONS: Genomic alterations detected...

  10. Levels of cell-free DNA and plasma KRAS during treatment of advanced NSCLC

    DEFF Research Database (Denmark)

    Dowler Nygaard, Anneli; Spindler, Karen-Lise Garm; Pallisgaard, Niels

    2014-01-01

    be analysed in plasma and may increase the scientific use of such measurements. In the present study, we investigated: i) the dynamics of cfDNA and plasma mutated KRAS (pmKRAS) during the treatment of patients with advanced NSCLC; and ii) the prognostic value of baseline cfDNA and pmKRAS. Sixty‑nine patients......Non-small cell lung cancer (NSCLC) is one of the most common malignant tumours in the western world and is associated with a poor prognosis. Biomarkers predicting prognosis and therapeutic effects are highly required, and cell-free DNA (cfDNA) may be a feasible option. Genetic mutations can...... were included in a prospective biomarker trial. Inclusion criteria included advanced NSCLC, candidate for first-line treatment, no previous cancer within the five years prior to this study. Blood samples were drawn at baseline, day 8 and at progression. Analyses of cfDNA and KRAS mutations in plasma...

  11. The Study of Lung Cancer Personalized Medicine Through Circulating Cell Free DNA Test

    DEFF Research Database (Denmark)

    Ye, Mingzhi

    Dels, CNVs, and SV in 145 genes with FFPE clinical specimens. While, couple with relatively geological distributions of different subtypes, tumor microenvironment might contribute more to genetic instability and thus tumor evolutions. As for the therapy and rejection monitoring of lung cancer patients, cell...... mutation and clonal expansion in human blood was prevalent in cancer patients and cf-DNA somatic mutation seems to be ideal cancer early diagnosis biomarkers due to its advantages. By using cell-free tumor DNA and peripheral nodule ultra-deep sequencing (>10,000 fold), mutations from nodule tissues had......According to the serious situation of lung cancer in Chinese cancer incidence and mortality, better prognosis and early diagnosis are the key problems. These works are around of lung cancer genetic profiling, pathway signaling and tumor evolution, targeted therapy and transplant monitoring...

  12. Enhanced photocurrent density of HTM-free perovskite solar cells by carbon quantum dots

    Science.gov (United States)

    Zou, Haiyuan; Guo, Daipeng; He, Bowen; Yu, Jiaguo; Fan, Ke

    2018-02-01

    Full-printable and hole transport material (HTM)-free perovskite solar cells (PSCs) with carbon counter electrodes feature high stability and low cost. However, the perovskite film prepared by conventional one-step solution-coating method always shows a relatively poor coverage on the substrate, leading to the limit of the photocurrent density. In this study, we incorporated carbon quantum dots (CQDs) in the perovskite films, and investigated their effects on the performance of TiO2 nanosheet-based and HTM-free PSCs. It was found that the addition of CQDs to the perovskite film can enhance the photocurrent density of PSCs, and the optimal PSC with 0.1% CQDs evolved 60% higher photocurrent density than the pristine one. The improved photocurrent density was attributed to the heterogeneous nuclei derived from CQDs during perovskite crystallization, which would increase amount of perovskite nuclei and form a fine perovskite grain, leading to a better coverage on the substrate. Moreover, due to the excellent conductivity, CQDs in perovskite films could efficiently transport the photo-excited electrons, accelerating the separation and mobilization of charge carriers. This study presents the incorporation of CQDs in perovskite as an efficient approach to promote the performance of HTM-free PSCs.

  13. Indium-Free Perovskite Solar Cells Enabled by Impermeable Tin-Oxide Electron Extraction Layers.

    Science.gov (United States)

    Hu, Ting; Becker, Tim; Pourdavoud, Neda; Zhao, Jie; Brinkmann, Kai Oliver; Heiderhoff, Ralf; Gahlmann, Tobias; Huang, Zengqi; Olthof, Selina; Meerholz, Klaus; Többens, Daniel; Cheng, Baochang; Chen, Yiwang; Riedl, Thomas

    2017-07-01

    Corrosive precursors used for the preparation of organic-inorganic hybrid perovskite photoactive layers prevent the application of ultrathin metal layers as semitransparent bottom electrodes in perovskite solar cells (PVSCs). This study introduces tin-oxide (SnO x ) grown by atomic layer deposition (ALD), whose outstanding permeation barrier properties enable the design of an indium-tin-oxide (ITO)-free semitransparent bottom electrode (SnO x /Ag or Cu/SnO x ), in which the metal is efficiently protected against corrosion. Simultaneously, SnO x functions as an electron extraction layer. We unravel the spontaneous formation of a PbI 2 interfacial layer between SnO x and the CH 3 NH 3 PbI 3 perovskite. An interface dipole between SnO x and this PbI 2 layer is found, which depends on the oxidant (water, ozone, or oxygen plasma) used for the ALD growth of SnO x . An electron extraction barrier between perovskite and PbI 2 is identified, which is the lowest in devices based on SnO x grown with ozone. The resulting PVSCs are hysteresis-free with a stable power conversion efficiency (PCE) of 15.3% and a remarkably high open circuit voltage of 1.17 V. The ITO-free analogues still achieve a high PCE of 11%. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Cancer stem cells of the digestive system.

    Science.gov (United States)

    Colvin, Hugh S; Nishida, Naohiro; Koseki, Jun; Konno, Masamitsu; Kawamoto, Koichi; Tsunekuni, Kenta; Doki, Yuichiro; Mori, Masaki; Ishii, Hideshi

    2014-12-01

    Stem cells of the digestive system are ideal in many ways for research, given they are abundant, highly proliferative and have a uniform structural arrangement. This in turn has enormously aided the research of cancer stem cells of the digestive system, which is now shaping our understanding of cancer stem cells. In this review, the recent advances in the understanding of cancer stem cells of the digestive system have been summarized, including aspects such as their identification, origin, cell-cycle dormancy, relationship with epithelial-mesenchymal transition, cellular metabolism and the underlying molecular mechanisms. Newly acquired knowledge concerning cancer stem cells have led to the development of novel cancer therapeutics with provisional yet encouraging results. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  15. Effect of exogenous adipose-derived stem cells in the early stages following free fat transplantation.

    Science.gov (United States)

    Yuan, Y I; Gao, Jianhua; Lu, Feng

    2015-09-01

    Cotransplantation of adipose-derived stem cells (ASCs) is an effective therapeutic approach for enhancing the survival of transplanted fat tissue; however, the role of ASCs in free fat transplantation remains unclear. In the present study, fat harvested from C57BL/6 mice expressing green fluorescent protein was injected subcutaneously into the back of C57BL/6 mice, who also received ASCs (group A) or received the fat tissue only (group B). The grafts were harvested at days 1, 4, 7, 14, 30 and 90 following the transplantation. Graft volume and histology were evaluated, and the secretion levels of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were quantified using enzyme-linked immunosorbent assays. No statistically significant difference was identified between groups A and B in the graft survival rate up to day 14 following the aspirated fat transplantation; however, the graft survival rate decreased during the following 14-90 days. Initially, group =A exhibited a higher graft survival rate and a greater degree of angiogenesis compared with group B. The ratio of dead cells was not significantly different between the two groups on day 1; however, group A had a greater number of living interstitial cells compared with group B at the later time points. The secretion of VEGF by the ASCs had an earlier peak time in group A (day 4) compared with group B (day 7). In addition, the secretion of HGF in group A was greater compared with group B. Therefore, the role of exogenous ASCs in free fat transplantation may not directly participate in angiogenesis and adipogenesis, but may promote the survival ratio of the graft-resident interstitial cells, which are involved in angiogenesis and adipogenesis, via a paracrine effect.

  16. Chemically Defined and Xeno-Free Cryopreservation of Human Adipose-Derived Stem Cells

    Science.gov (United States)

    López, Melany; Bollag, Roni J.; Yu, Jack C.; Isales, Carlos M.; Eroglu, Ali

    2016-01-01

    The stromal compartment of adipose tissue harbors multipotent cells known as adipose-derived stem cells (ASCs). These cells can differentiate into various lineages including osteogenic, chrondrogenic, adipogenic, and neurogenic; this cellular fraction may be easily obtained in large quantities through a clinically safe liposuction procedure. Therefore, ASCs offer exceptional opportunities for tissue engineering and regenerative medicine. However, current practices involving ASCs typically use fetal bovine serum (FBS)-based cryopreservation solutions that are associated with risks of immunological reactions and of transmitting infectious diseases and prions. To realize clinical applications of ASCs, serum- and xeno-free defined cryopreservation methods are needed. To this end, an animal product-free chemically defined cryopreservation medium was formulated by adding two antioxidants (reduced glutathione and ascorbic acid 2-phosphate), two polymers (PVA and ficoll), two permeating cryoprotectants (ethylene glycol and dimethylsulfoxide), a disaccharide (trehalose), and a calcium chelator (EGTA) to HEPES-buffered DMEM/F12. To limit the number of experimental groups, the concentration of trehalose, both polymers, and EGTA was fixed while the presence of the permeating CPAs and antioxidants was varied. ASCs suspended either in different versions of the defined medium or in the conventional undefined cryopreservation medium (10% dimethylsulfoxide+10% DMEM/F12+80% serum) were cooled to -70°C at 1°C/min before being plunged into liquid nitrogen. Samples were thawed either in air or in a water bath at 37°C. The presence of antioxidants along with 3.5% concentration of each penetrating cryoprotectant improved the freezing outcome to the level of the undefined cryopreservation medium, but the plating efficiency was still lower than that of unfrozen controls. Subsequently, increasing the concentration of both permeating cryoprotectants to 5% further improved the plating

  17. 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…

  18. Metal-free Phtalocyanine and 5-Aminolevulenic Acid in Photodynamic Treatment of Human Vascular Cells

    Science.gov (United States)

    Udartseva, Olga O.; Andreeva, Elena R.; Buravkova, Ludmila B.; Tararak, Eduard M.

    2010-05-01

    Originally developed as a tumor therapy, now photodynamic therapy (PDT) may become a useful tool for treatment of cardiovascular diseases. Different cell types are involved in this vascular pathology, and these cells possess different susceptibility to PDT. In this study we screened the effects of two new photosensitizers (PtS and ALA) on human vascular cells. Human macrophages (Mph), aorta endothelial (HAEC) and smooth muscle (SMC) cells were obtained and cultured as described elsewhere. 2-10 ug/ml PtS was added to culture medium 24 h before PDT. ALA was added in 2-10 mM concentration in serum-free culture medium. Then cells were washed carefully and illuminated with 692-nm (PtS) or 633-nm (ALA) light. Cellular viability was measured with MTT-test. Except the case of use 5-10 mM ALA, either photosensitizer accumulation alone or laser illumination alone did not affect cells. Illumination of PtS or ALA-loaded cells (1-20 J/cm2) impaired cellular viability in dose-dependent manner. LD90 for different vascular cells with PtS were as follows: HAEC -1 J/cm2, SMC -2 J/cm2, Mph -5 J/cm2. HAEC and some Mph were unsusceptible to ALA-PDT. SMC LD90 with ALA was 20 J/cm2. Effects of ALA-PDT depended on protoporphyrin IX (PpIX) formation in cells. HAEC didn't accumulate PpIX and were non-sensitive to ALA-PDT. PpIX formation in Mph changed individually according to donor. Illumination of ALA-loaded Mph with low PpIX formation did not affect cells. However LD90 for Mph with high PpIX formation comprised 20 J/cm2. All cell types were more susceptible to PtS-PDT compared to ALA-PDT. Among tested photosensitizers PtS was the most effective one. HAEC were the most susceptible to PtS-PDT.

  19. A Hybrid Nanomaterial for the Controlled Generation of Free Radicals and Oxidative Destruction of Hypoxic Cancer Cells.

    Science.gov (United States)

    Shen, Song; Zhu, Chunlei; Huo, Da; Yang, Miaoxin; Xue, Jiajia; Xia, Younan

    2017-07-17

    Anticancer modalities based on oxygen free radicals, including photodynamic therapy and radiotherapy, have emerged as promising treatments in the clinic. However, the hypoxic environment in tumor tissue prevents the formation of oxygen free radicals. Here we introduce a novel strategy that employs oxygen-independent free radicals generated from a polymerization initiator for eradicating cancer cells. The initiator is mixed with a phase-change material and loaded into the cavities of gold nanocages. Upon irradiation by a near-infrared laser, the phase-change material is melted due to the photothermal effect of gold nanocages, leading to the release and decomposition of the loaded initiator to generate free radicals. The free radicals produced in this way are highly effective in inducing apoptosis in hypoxic cancer cells. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Selective analysis of cell-free DNA in maternal blood for evaluation of fetal trisomy.

    Science.gov (United States)

    Sparks, Andrew B; Wang, Eric T; Struble, Craig A; Barrett, Wade; Stokowski, Renee; McBride, Celeste; Zahn, Jacob; Lee, Kevin; Shen, Naiping; Doshi, Jigna; Sun, Michel; Garrison, Jill; Sandler, Jay; Hollemon, Desiree; Pattee, Patrick; Tomita-Mitchell, Aoy; Mitchell, Michael; Stuelpnagel, John; Song, Ken; Oliphant, Arnold

    2012-01-01

    To develop a novel prenatal assay based on selective analysis of cell-free DNA in maternal blood for evaluation of fetal Trisomy 21 (T21) and Trisomy 18 (T18). Two hundred ninety-eight pregnancies, including 39 T21 and seven T18 confirmed fetal aneuploidies, were analyzed using a novel, highly multiplexed assay, termed digital analysis of selected regions (DANSR™). Cell-free DNA from maternal blood samples was analyzed using DANSR assays for loci on chromosomes 21 and 18. Products from 96 separate patients were pooled and sequenced together. A standard Z-test of chromosomal proportions was used to distinguish aneuploid samples from average-risk pregnancy samples. DANSR aneuploidy discrimination was evaluated at various sequence depths. At the lowest sequencing depth, corresponding to 204,000 sequencing counts per sample, average-risk cases where distinguished from T21 and T18 cases, with Z statistics for all cases exceeding 3.6. Increasing the sequencing depth to 410,000 counts per sample substantially improved separation of aneuploid and average-risk cases. A further increase to 620,000 counts per sample resulted in only marginal improvement. This depth of sequencing represents less than 5% of that required by massively parallel shotgun sequencing approaches. Digital analysis of selected regions enables highly accurate, cost efficient, and scalable noninvasive fetal aneuploidy assessment. © 2012 John Wiley & Sons, Ltd.

  1. Non-invasive prenatal diagnosis using cell-free fetal DNA technology: applications and implications.

    Science.gov (United States)

    Hall, Alison; Bostanci, A; Wright, C F

    2010-01-01

    Cell-free fetal DNA and RNA circulating in maternal blood can be used for the early non-invasive prenatal diagnosis (NIPD) of an increasing number of genetic conditions, both for pregnancy management and to aid reproductive decision-making. Here we present a brief review of the scientific and clinical status of the technology, and an overview of key ethical, legal and social issues raised by the analysis of cell-free fetal DNA for NIPD. We suggest that the less invasive nature of the technology brings some distinctive issues into focus, such as the possibility of broader uptake of prenatal diagnosis and access to the technology directly by the consumer via the internet, which have not been emphasised in previous work in this area. We also revisit significant issues that are familiar from previous debates about prenatal testing. Since the technology seems to transect existing distinctions between screening and diagnostic tests, there are important implications for the form and process involved in obtaining informed consent or choice. This analysis forms part of the work undertaken by a multidisciplinary group of experts which made recommendations about the implementation of this technology within the UK National Health Service. Copyright 2010 S. Karger AG, Basel.

  2. 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

  3. Cell-free antigens of Sporothrix brasiliensis: antigenic diversity and application in an immunoblot assay.

    Science.gov (United States)

    Almeida-Paes, Rodrigo; Bailão, Alexandre Melo; Pizzini, Cláudia Vera; Reis, Rosani Santos; Soares, Célia Maria de Almeida; Peralta, José Mauro; Gutierrez-Galhardo, Maria Clara; Zancopé-Oliveira, Rosely Maria

    2012-11-01

    Sporotrichosis is a subcutaneous mycosis diagnosed by isolation of the fungus in culture. Serological tests for help in diagnosis in general do not use purified or recombinant antigens, because there is a paucity of described immunoreactive proteins, especially for the new described Sporothrix species, such as Sporothrix brasiliensis. This study aims to characterise antigens from S. brasiliensis and verify their application in serodiagnosis of sporotrichosis. An immunoblot assay allied with computer-based analysis was used to identify putative antigenic molecules in a cell-free extracts of both morphological phases of this fungus, and to delineate antigenic polymorphism among seven S. brasiliensis isolates and one S. schenckii Brazilian strain. The mycelial and yeast phase of the fungus originated 14 and 23 reactive bands, respectively, which were variable in intensity. An 85 kDa antigen, verified in the yeast phase of the fungus, was observed in all strains used and the immunodominant protein was identified. This protein, however, cross-react with serum samples from patients infected with other pathogens. The results show that the S. brasiliensis cell-free antigen extract is a single and inexpensive source of antigens, and can be applied on the sporotrichosis serodiagnosis. © 2012 Blackwell Verlag GmbH.

  4. False Negative Cell-Free DNA Screening Result in a Newborn with Trisomy 13

    Directory of Open Access Journals (Sweden)

    Yang Cao

    2016-01-01

    Full Text Available Background. Noninvasive prenatal screening (NIPS is revolutionizing prenatal screening as a result of its increased sensitivity, specificity. NIPS analyzes cell-free fetal DNA (cffDNA circulating in maternal plasma to detect fetal chromosome abnormalities. However, cffDNA originates from apoptotic placental trophoblast; therefore cffDNA is not always representative of the fetus. Although the published data for NIPS testing states that the current technique ensures high sensitivity and specificity for aneuploidy detection, false positives are possible due to isolated placental mosaicism, vanishing twin or cotwin demise, and maternal chromosome abnormalities or malignancy. Results. We report a case of false negative cell-free DNA (cfDNA screening due to fetoplacental mosaicism. An infant male with negative cfDNA screening result was born with multiple congenital abnormalities. Postnatal chromosome and FISH studies on a blood specimen revealed trisomy 13 in 20/20 metaphases and 100% interphase nuclei, respectively. FISH analysis on tissues collected after delivery revealed extraembryonic mosaicism. Conclusions. Extraembryonic tissue mosaicism is likely responsible for the false negative cfDNA screening result. This case illustrates that a negative result does not rule out the possibility of a fetus affected with a trisomy, as cffDNA is derived from the placenta and therefore may not accurately represent the fetal genetic information.

  5. Durability of a low shrinkage TEGDMA/HEMA-free resin composite system in Class II restorations

    DEFF Research Database (Denmark)

    van Dijken, Jan WV; Pallesen, Ulla

    2017-01-01

    Objective: The objective of this randomized controlled prospective trial was to evaluate the durability of a low shrinkage and TEGDMA/HEMA-free resin composite system in posterior restorations in a 6-year follow up. Material and methods: 139 Class II restorations were placed in 67 patients...... with a mean age of 53 years (range 29-82). Each participant received at random two, as similar as possible, Class II restorations. In the first cavity of each pair the TEGDMA/HEMA-free resin composite system was placed with its 3-step etch-and-rinse adhesive (cmf-els). In the second cavity a 1-step HEMA...... for failure were fracture followed by recurrent caries. Most fractures and all caries lesions were found in high risk participants. Significance: The tested Class II resin composite restorations performed with the new TEGDMA/HEMA-free low shrinkage resin composite system showed good durability over six years....

  6. 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.

  7. Washing of Cloth Contaminated with Radionuclides Using a Detergent-free Laundry System

    International Nuclear Information System (INIS)

    Yim, Sung Paal

    2005-01-01

    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

  8. Fuel cell systems and production aspects

    Energy Technology Data Exchange (ETDEWEB)

    Blomen, L.J.M.J. [Blomenco, Rumpt (Netherlands)

    1995-02-01

    Attention is paid to some system integration and production aspects of fuel cell systems. After an introduction of the main performance differences of fuel cell systems with different types of fuel cells, and the causes for such differences, a few aspects of building up sub-systems towards large and small capacity fuel cell systems are being discussed. Subsequently, some production aspects are being highlighted: the concept of an automated plant and automated plant contracting, the trends towards greater simplicity m reactors, and a systematic evaluation of the differences in fuel processing steps required in systems with different types of fuel cells. In this section also the cost aspects of a total plant contracting project are being discussed. As a next step, some emphasis has been placed on system integration aspects, explaining that hydrogen purification does not always `destroy` system efficiency, and can be used in dispersed hydrogen generation concepts. Advanced system integration concepts include simplified flow sheet design, in which instead of special dedicated rotating equipment standard gas turbine rotary components are being employed. As an example, it is claimed that gas turbine - fuel cell combinations can be made with very high electrical efficiencies. 5 figs., 2 tabs.

  9. Water reactive hydrogen fuel cell power system

    Science.gov (United States)

    Wallace, Andrew P; Melack, John M; Lefenfeld, Michael

    2014-01-21

    A water reactive hydrogen fueled power system includes devices and methods to combine reactant fuel materials and aqueous solutions to generate hydrogen. The generated hydrogen is converted in a fuel cell to provide electricity. The water reactive hydrogen fueled power system includes a fuel cell, a water feed tray, and a fuel cartridge to generate power for portable power electronics. The removable fuel cartridge is encompassed by the water feed tray and fuel cell. The water feed tray is refillable with water by a user. The water is then transferred from the water feed tray into a fuel cartridge to generate hydrogen for the fuel cell which then produces power for the user.

  10. Sunitinib-induced hypothyroidism predicts progression-free survival in metastatic renal cell carcinoma patients.

    Science.gov (United States)

    Buda-Nowak, Anna; Kucharz, Jakub; Dumnicka, Paulina; Kuzniewski, Marek; Herman, Roman Maria; Zygulska, Aneta L; Kusnierz-Cabala, Beata

    2017-04-01

    Sunitinib is a tyrosine kinase inhibitor (TKI) used in treatment of metastatic renal cell carcinoma (mRCC), gastrointestinal stromal tumors and pancreatic neuroendocrine tumors. One of the most common side effects related to sunitinib is hypothyroidism. Recent trials suggest correlation between the incidence of hypothyroidism and treatment outcome in patients treated with TKI. This study evaluates whether development of hypothyroidism is a predictive marker of progression-free survival (PFS) in patients with mRCC treated with sunitinib. Twenty-seven patients diagnosed with clear cell mRCC, after nephrectomy and in 'good' or 'intermediate' MSKCC risk prognostic group, were included in the study. All patients received sunitinib as a first-line treatment on a standard schedule (initial dose 50 mg/day, 4 weeks on, 2 weeks off). The thyroid-stimulating hormone serum levels were obtained at the baseline and every 12 weeks of treatment. In statistic analyses, we used Kaplan-Meier method for assessment of progression-free survival; for comparison of survival, we used log-rank test. In our study, the incidence of hypothyroidism was 44%. The patients who had developed hypothyroidism had better median PFS to patients with normal thyroid function 28,3 months [95% (CI) 20.4-36.2 months] versus 9.8 months (6.4-13.1 months). In survival analysis, we perceive that thyroid dysfunction is a predictive factor of a progression-free survival (PFS). In the unified group of patients, the development of hypothyroidism during treatment with sunitinib is a positive marker for PFS. During that treatment, thyroid function should be evaluated regularly.

  11. Increased Levels of Cell-Free miR-517a and Decreased Levels of Cell-Free miR-518b in Maternal Plasma Samples From Placenta Previa Pregnancies at 32 Weeks of Gestation.

    Science.gov (United States)

    Hasegawa, Yuri; Miura, Kiyonori; Higashijima, Ai; Abe, Shuhei; Miura, Shoko; Yoshiura, Koh-ichiro; Masuzaki, Hideaki

    2015-12-01

    The aim of this study was to clarify the association between placenta previa and circulating levels of cell-free pregnancy-associated placenta-specific microRNAs (miRNAs) in maternal plasma. Twenty singleton pregnancies with placenta previa (placenta previa group) and 26 uncomplicated pregnancies (control group) were recruited. Blood sampling was performed at 32 weeks of gestation, and cesarean delivery in all cases of placenta previa was performed at a mean gestational age of 37 weeks. The maternal plasma concentrations of cell-free pregnancy-associated placenta-specific miRNAs (miR-517a and miR-518b) were measured by absolute quantitative real-time reverse transcription-polymerase chain reaction. Plasma concentrations of cell-free miR-517a were significantly higher in the placenta previa group than that in the control group (P = .011), while the plasma concentration of cell-free miR-518b was significantly lower in the placenta previa group than that in the control group (P = .004). Plasma concentrations of cell-free miR-517a in placenta previa were significantly higher in placenta previa with alert bleeding later group than those in placenta previa without alert bleeding group or control group (P = .030 or .047, respectively) and correlated with the volume of hemorrhage at delivery (R and P value: .512 and .025). Plasma concentrations of cell-free miR-517a and miR-518b at 32 weeks of gestation were altered in pregnant women with placenta previa, and the circulating level of cell-free miR-517a in placenta previa may be a predictive marker for the risks of alert bleeding later and massive hemorrhage at delivery. © The Author(s) 2015.

  12. 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.

  13. Scale-free flow of life: on the biology, economics, and physics of the cell

    Directory of Open Access Journals (Sweden)

    Kurakin Alexei

    2009-05-01

    Full Text Available Abstract The present work is intended to demonstrate that most of the paradoxes, controversies, and contradictions accumulated in molecular and cell biology over many years of research can be readily resolved if the cell and living systems in general are re-interpreted within an alternative paradigm of biological organization that is based on the concepts and empirical laws of nonequilibrium thermodynamics. In addition to resolving paradoxes and controversies, the proposed re-conceptualization of the cell and biological organization reveals hitherto unappreciated connections among many seemingly disparate phenomena and observations, and provides new and powerful insights into the universal principles governing the emergence and organizational dynamics of living systems on each and every scale of biological organizational hierarchy, from proteins and cells to economies and ecologies.

  14. Label-free multidimensional information acquisition from optogenetically engineered cells using a graphene transistor.

    Science.gov (United States)

    Li, Gongxin; Yang, Jia; Yang, Wenguang; Wang, Feifei; Wang, Yuechao; Wang, Wenxue; Liu, Lianqing

    2018-02-01

    The optogenetic technique, which allows the manipulation of cellular activity patterns in space and time by light, has transformed the field of neuroscience. However, acquiring multidimensional optogenetic information remains challenging despite the fact that several cellular information detection methods have been proposed. Herein, we present a new method to acquire label-free multidimensional information from optogenetically engineered cells using a graphene transistor. Using a graphene film to form a strong densely packed layer with cells, the cellular action potentials were characterized as light-activated transistor conductance signals, which quantified the multidimensional optogenetic information. Based on this approach, some important cellular optogenetic information, including electrophysiological state, cell concentration, expression levels of opsin and response to variable light intensity, were also precisely detected. Furthermore, the graphene transistor was also used to distinguish cells expressing different channelrhodopsin-2 variants. Our study offers a general detection method of multidimensional optogenetic information for extending the applications of the optogenetic technique and provides a novel sensor for the development of future biological prosthetic devices.

  15. Kinerja Sistem Komunikasi FSO (Free Space Optics Menggunakan Cell-site Diversity di Daerah Tropis

    Directory of Open Access Journals (Sweden)

    Octiana Widyarena

    2012-09-01

    Full Text Available Kebutuhan masyarakat akan adanya layanan komunikasi multimedia seperti video conference, high speed internet, video streaming, dan lain sebagainya, saat ini terus meningkat. Untuk memenuhi kebutuhan tersebut, perlu adanya suatu sistem komunikasi nirkabel dengan kecepatan tinggi. Salah satunya yaitu dengan menggunakan FSO (Free Space Optics. FSO merupakan sistem komunikasi yang memungkinkan memiliki koneksi layaknya serat optik, namun media transmisi yang digunakan yaitu melalui atmosfer. Penggunaan FSO di daerah tropis memiliki kendala yang cukup serius yaitu tingginya intensitas curah hujan yang dapat mempengaruhi kinerja dari FSO. Semakin tinggi intensitas curah hujan, maka nilai redaman hujan juga semakin besar. Untuk mengatasi dampak redaman hujan tersebut, maka digunakan teknik cell-site diversity dengan selection combining. Penerapan teknik cell-site diversity pada sistem komunikasi FSO menggunakan variasi panjang lintasan 0,5 km, 1 km, 1,5 km, dan 2 km serta variasi sudut antar link sebesar 45°, 90°, 135°, dan 180°. Hasil dari penerapan teknik cell-site diversity menunjukkan bahwa adanya peningkatan kualitas sinyal FSO, dalam hal ini yaitu nilai SNR. Peningkatan nilai SNR terbesar didapatkan pada panjang lintasan 2 km dengan sudut antar link 180° serta pada link availability 99,9 %. Untuk konfigurasi cell-site diversity terbaik didapatkan pada sudut antar link sebesar 90° dan 180°.

  16. Suppression of vapor cell temperature error for spin-exchange-relaxation-free magnetometer

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Jixi, E-mail: lujixi@buaa.edu.cn; Qian, Zheng; Fang, Jiancheng; Quan, Wei [School of Instrument Science and Opto-Electronics Engineering, Beihang University, Beijing 100191 (China)

    2015-08-15

    This paper presents a method to reduce the vapor cell temperature error of the spin-exchange-relaxation-free (SERF) magnetometer. The fluctuation of cell temperature can induce variations of the optical rotation angle, resulting in a scale factor error of the SERF magnetometer. In order to suppress this error, we employ the variation of the probe beam absorption to offset the variation of the optical rotation angle. The theoretical discussion of our method indicates that the scale factor error introduced by the fluctuation of the cell temperature could be suppressed by setting the optical depth close to one. In our experiment, we adjust the probe frequency to obtain various optical depths and then measure the variation of scale factor with respect to the corresponding cell temperature changes. Our experimental results show a good agreement with our theoretical analysis. Under our experimental condition, the error has been reduced significantly compared with those when the probe wavelength is adjusted to maximize the probe signal. The cost of this method is the reduction of the scale factor of the magnetometer. However, according to our analysis, it only has minor effect on the sensitivity under proper operating parameters.

  17. Divergent Label-free Cell Phenotypic Pharmacology of Ligands at the Overexpressed β2-Adrenergic Receptors

    Science.gov (United States)

    Ferrie, Ann M.; Sun, Haiyan; Zaytseva, Natalya; Fang, Ye

    2014-01-01

    We present subclone sensitive cell phenotypic pharmacology of ligands at the β2-adrenergic receptor (β2-AR) stably expressed in HEK-293 cells. The parental cell line was transfected with green fluorescent protein (GFP)-tagged β2-AR. Four stable subclones were established and used to profile a library of sixty-nine AR ligands. Dynamic mass redistribution (DMR) profiling resulted in a pharmacological activity map suggesting that HEK293 endogenously expresses functional Gi-coupled α2-AR and Gs-coupled β2-AR, and the label-free cell phenotypic activity of AR ligands are subclone dependent. Pathway deconvolution revealed that the DMR of epinephrine is originated mostly from the remodeling of actin microfilaments and adhesion complexes, to less extent from the microtubule networks and receptor trafficking, and certain agonists displayed different efficacy towards the cAMP-Epac pathway. We demonstrate that receptor signaling and ligand pharmacology is sensitive to the receptor expression level, and the organization of the receptor and its signaling circuitry.

  18. Materials towards carbon-free, emission-free and oil-free mobility: hydrogen fuel-cell vehicles--now and in the future.

    Science.gov (United States)

    Hirose, Katsuhiko

    2010-07-28

    In the past, material innovation has changed society through new material-induced technologies, adding a new value to society. In the present world, engineers and scientists are expected to invent new materials to solve the global problem of climate change. For the transport sector, the challenge for material engineers is to change the oil-based world into a sustainable world. After witnessing the recent high oil price and its adverse impact on the global economy, it is time to accelerate our efforts towards this change. Industries are tackling global energy issues such as oil and CO2, as well as local environmental problems, such as NO(x) and particulate matter. Hydrogen is the most promising candidate to provide carbon-free, emission-free and oil-free mobility. As such, engineers are working very hard to bring this technology into the real society. This paper describes recent progress of vehicle technologies, as well as hydrogen-storage technologies to extend the cruise range and ensure the easiness of refuelling and requesting material scientists to collaborate with industry to fight against global warming.

  19. Efficient Lead-Free Solar Cells Based on Hollow {en}MASnI3Perovskites.

    Science.gov (United States)

    Ke, Weijun; Stoumpos, Constantinos C; Spanopoulos, Ioannis; Mao, Lingling; Chen, Michelle; Wasielewski, Michael R; Kanatzidis, Mercouri G

    2017-10-18

    Tin-based perovskites have very comparable electronic properties to lead-based perovskites and are regarded as possible lower toxicity alternates for solar cell applications. However, the efficiency of tin-based perovskite solar cells is still low and they exhibit poor air stability. Here, we report lead-free tin-based solar cells with greatly enhanced performance and stability using so-called "hollow" ethylenediammonium and methylammonium tin iodide ({en}MASnI 3 ) perovskite as absorbers. Our results show that en can improve the film morphology and most importantly can serve as a new cation to be incorporated into the 3D MASnI 3 lattice. When the cation of en becomes part of the 3D structure, a high density of SnI 2 vacancies is created resulting in larger band gap, larger unit cell volume, lower trap-state density, and much longer carrier lifetime compared to classical MASnI 3 . The best-performing {en}MASnI 3 solar cell has achieved a high efficiency of 6.63% with an open circuit voltage of 428.67 mV, a short-circuit current density of 24.28 mA cm -2 , and a fill factor of 63.72%. Moreover, the {en}MASnI 3 device shows much better air stability than the neat MASnI 3 device. Comparable performance is also achieved for cesium tin iodide solar cells with en loading, demonstrating the broad scope of this approach.

  20. Methylcellulose cell culture as a new cytotoxicity test system for biomaterials

    OpenAIRE

    van Luyn, M.J.A.; van Wachem, P.B.; Nieuwenhuis, P.; Olde-Damink, L.; ten Hoopen, Hermina W.M.; Feijen, Jan

    1991-01-01

    The cytotoxicity of biomaterials can be testedin vitro using various culture systems. Liquid culture systems may detect cytotoxicity of a material either by culture of cells with extracts or with the material itself. In the latter instance, renewing the medium will remove possible released cytotoxic products. The agar-overlay test is a short term semi-solid culture system in which the possible cytotoxicity of biomaterials is identified only by the presence of cell free zones. The aim of this ...