WorldWideScience

Sample records for cell-free synthesizing systems

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

    International Nuclear Information System (INIS)

    Azrolan, N.; Coleman, P.S.

    1986-01-01

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

  2. 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

  3. 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

  4. 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.

  5. 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.

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

    Science.gov (United States)

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

    1978-09-01

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

  7. Reconstituted AIM2 inflammasome in cell-free system.

    Science.gov (United States)

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

    2015-11-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  9. 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...

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Science.gov (United States)

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

    2009-10-01

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

  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 ... control group. Thorough clinical examination stressing on the central nervous system, vascular, ... sis/necrosis of blood and tissue cells) and, second, active metabolic ..... alternative biomarkers should be tested. There was ...

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

    Science.gov (United States)

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

    2017-06-01

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

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

    OpenAIRE

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

    2009-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-12-09

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

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

    Directory of Open Access Journals (Sweden)

    Daniel eLewis

    2014-12-01

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

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

    International Nuclear Information System (INIS)

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

    2014-01-01

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

  18. 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.

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

    Halleran, Andrew D; Murray, Richard M

    2018-02-16

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

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  2. 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

  3. 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.

  4. 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.

  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. γ-irradiated ribosomes from Micrococcus radiodurans in a cell-free protein synthesizing system

    International Nuclear Information System (INIS)

    Suessmuth, R.; Widmann, A.

    1979-01-01

    γ-irradiation inactivation of isolated ribosomes of Micrococcus radiodurans was studied by examining poly U directed synthesis of polyphenylalanine. Ribosomes of M. radiodurans did not show significant γ-radiation sensitivity up to a dose of approx. 11.6 k Gy. Cells of M. radiodurans take up more magnesium than E. coli cells under the same conditions. The magnesium content of ribosomes of M. radiodurans was 18% higher than that of E.coli ribosomes. A possible relation between Mg 2+ -content and γ-resistance is discussed. (orig.) [de

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

    Science.gov (United States)

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

    2014-05-03

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

  9. Establishment of a Wheat Cell-Free Synthesized Protein Array Containing 250 Human and Mouse E3 Ubiquitin Ligases to Identify Novel Interaction between E3 Ligases and Substrate Proteins.

    Directory of Open Access Journals (Sweden)

    Hirotaka Takahashi

    Full Text Available Ubiquitination is a key post-translational modification in the regulation of numerous biological processes in eukaryotes. The primary roles of ubiquitination are thought to be the triggering of protein degradation and the regulation of signal transduction. During protein ubiquitination, substrate specificity is mainly determined by E3 ubiquitin ligase (E3. Although more than 600 genes in the human genome encode E3, the E3s of many target proteins remain unidentified owing to E3 diversity and the instability of ubiquitinated proteins in cell. We demonstrate herein a novel biochemical analysis for the identification of E3s targeting specific proteins. Using wheat cell-free protein synthesis system, a protein array containing 227 human and 23 mouse recombinant E3s was synthesized. To establish the high-throughput binding assay using AlphaScreen technology, we selected MDM2 and p53 as the model combination of E3 and its target protein. The AlphaScreen assay specifically detected the binding of p53 and MDM2 in a crude translation mixture. Then, a comprehensive binding assay using the E3 protein array was performed. Eleven of the E3s showed high binding activity, including four previously reported E3s (e.g., MDM2, MDM4, and WWP1 targeting p53. This result demonstrated the reliability of the assay. Another interactors, RNF6 and DZIP3-which there have been no report to bind p53-were found to ubiquitinate p53 in vitro. Further analysis showed that RNF6 decreased the amount of p53 in H1299 cells in E3 activity-dependent manner. These results suggest the possibility that the RNF6 ubiquitinates and degrades p53 in cells. The novel in vitro screening system established herein is a powerful tool for finding novel E3s of a target protein.

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

    Science.gov (United States)

    Genji, Takahisa; Nozawa, Akira; Tozawa, Yuzuru

    2010-10-01

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

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

    Science.gov (United States)

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

    2018-01-19

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

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

    Science.gov (United States)

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

    2015-05-01

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

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

    Science.gov (United States)

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

    1981-03-01

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

  14. Biochemical and biophysical characterization of cell-free synthesized Rift Valley fever virus nucleoprotein capsids enables in vitro screening to identify novel antivirals.

    Science.gov (United States)

    Broce, Sean; Hensley, Lisa; Sato, Tomoharu; Lehrer-Graiwer, Joshua; Essrich, Christian; Edwards, Katie J; Pajda, Jacqueline; Davis, Christopher J; Bhadresh, Rami; Hurt, Clarence R; Freeman, Beverly; Lingappa, Vishwanath R; Kelleher, Colm A; Karpuj, Marcela V

    2016-05-14

    Viral capsid assembly involves the oligomerization of the capsid nucleoprotein (NP), which is an essential step in viral replication and may represent a potential antiviral target. An in vitro transcription-translation reaction using a wheat germ (WG) extract in combination with a sandwich ELISA assay has recently been used to identify small molecules with antiviral activity against the rabies virus. Here, we examined the application of this system to viruses with capsids with a different structure, such as the Rift Valley fever virus (RVFV), the etiological agent of a severe emerging infectious disease. The biochemical and immunological characterization of the in vitro-generated RVFV NP assembly products enabled the distinction between intermediately and highly ordered capsid structures. This distinction was used to establish a screening method for the identification of potential antiviral drugs for RVFV countermeasures. These results indicated that this unique analytical system, which combines nucleoprotein oligomerization with the specific immune recognition of a highly ordered capsid structure, can be extended to various viral families and used both to study the early stages of NP assembly and to assist in the identification of potential antiviral drugs in a cost-efficient manner. Reviewed by Jeffry Skolnick and Noah Isakov. For the full reviews please go to the Reviewers' comments section.

  15. 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.

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

    Science.gov (United States)

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

    2018-01-04

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

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

    Directory of Open Access Journals (Sweden)

    Hsiu-Mei Chiang

    2011-07-01

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

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

    Directory of Open Access Journals (Sweden)

    Noireaux Vincent

    2010-06-01

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

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

    Science.gov (United States)

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

    2014-05-20

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

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

    Science.gov (United States)

    Cosgrove, J W; Brown, I R

    1984-05-01

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

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

    Directory of Open Access Journals (Sweden)

    Lena Thoring

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

  2. 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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2017-12-01

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

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

    Science.gov (United States)

    He, Mingyue

    2008-01-01

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

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

    Science.gov (United States)

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

    2010-05-01

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

  7. Tail-extension following the termination codon is critical for release of the nascent chain from membrane-bound ribosomes in a reticulocyte lysate cell-free system.

    Science.gov (United States)

    Takahara, Michiyo; Sakaue, Haruka; Onishi, Yukiko; Yamagishi, Marifu; Kida, Yuichiro; Sakaguchi, Masao

    2013-01-11

    Nascent chain release from membrane-bound ribosomes by the termination codon was investigated using a cell-free translation system from rabbit supplemented with rough microsomal membrane vesicles. Chain release was extremely slow when mRNA ended with only the termination codon. Tail extension after the termination codon enhanced the release of the nascent chain. Release reached plateau levels with tail extension of 10 bases. This requirement was observed with all termination codons: TAA, TGA and TAG. Rapid release was also achieved by puromycin even in the absence of the extension. Efficient translation termination cannot be achieved in the presence of only a termination codon on the mRNA. Tail extension might be required for correct positioning of the termination codon in the ribosome and/or efficient recognition by release factors. Copyright © 2012. Published by Elsevier Inc.

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

    Science.gov (United States)

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

    2015-03-01

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

  9. Studies on cell-free metabolism: ethanol production by a yeast glycolytic system reconstituted from purified enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Welch, P; Scopes, R K

    1985-07-01

    A reconstituted glycolytic system has been established from individually purified enzymes to simulate the conversion of glucose to ethanol plus CO/sub 2/ by yeast. Sustained and extensive conversion occurred provided that input of glucose matched the rate of ATP degradation appropriately. ATPase activity could be replaced by arsenate, which uncoupled ATP synthesis from glycolysis. The mode of uncoupling was investigated, and it was concluded that the artificial intermediate, 1-arseno-3-phosphoglycerate, has a half-life of no more than a few milliseconds. Arsenate at 4 mM concentration could simulate the equivalent of 10 ..mu..mol/ml min. of ATPase activity. The reconstituted enzyme system was capable of totally degrading one M (18% w/v) glucose in 8 hours giving 9% (w/v) ethanol. The levels of metabolites during metabolism were measured to detect rate-limiting steps. The successful operation of the reconstituted enzyme system demonstrates that it is possible to carry out complex chemical transformations with multiple enzyme systems in vitro. 36 references.

  10. 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.

  11. 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.

  12. 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.

  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. Arraying proteins by cell-free synthesis.

    Science.gov (United States)

    He, Mingyue; Wang, Ming-Wei

    2007-10-01

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

  15. 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.

  16. Is Ghrelin Synthesized in the Central Nervous System?

    Science.gov (United States)

    Cabral, Agustina; López Soto, Eduardo J; Epelbaum, Jacques; Perelló, Mario

    2017-03-15

    Ghrelin is an octanoylated peptide that acts via its specific receptor, the growth hormone secretagogue receptor type 1a (GHSR-1a), and regulates a vast variety of physiological functions. It is well established that ghrelin is predominantly synthesized by a distinct population of endocrine cells located within the gastric oxyntic mucosa. In addition, some studies have reported that ghrelin could also be synthesized in some brain regions, such as the hypothalamus. However, evidences of neuronal production of ghrelin have been inconsistent and, as a consequence, it is still as a matter of debate if ghrelin can be centrally produced. Here, we provide a comprehensive review and discussion of the data supporting, or not, the notion that the mammalian central nervous system can synthetize ghrelin. We conclude that no irrefutable and reproducible evidence exists supporting the notion that ghrelin is synthetized, at physiologically relevant levels, in the central nervous system of adult mammals.

  17. Is Ghrelin Synthesized in the Central Nervous System?

    Directory of Open Access Journals (Sweden)

    Agustina Cabral

    2017-03-01

    Full Text Available Ghrelin is an octanoylated peptide that acts via its specific receptor, the growth hormone secretagogue receptor type 1a (GHSR-1a, and regulates a vast variety of physiological functions. It is well established that ghrelin is predominantly synthesized by a distinct population of endocrine cells located within the gastric oxyntic mucosa. In addition, some studies have reported that ghrelin could also be synthesized in some brain regions, such as the hypothalamus. However, evidences of neuronal production of ghrelin have been inconsistent and, as a consequence, it is still as a matter of debate if ghrelin can be centrally produced. Here, we provide a comprehensive review and discussion of the data supporting, or not, the notion that the mammalian central nervous system can synthetize ghrelin. We conclude that no irrefutable and reproducible evidence exists supporting the notion that ghrelin is synthetized, at physiologically relevant levels, in the central nervous system of adult mammals.

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

    Directory of Open Access Journals (Sweden)

    Takayoshi Matsuda

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

  19. Development of an Intelligent System to Synthesize Petrophysical Well Logs

    Directory of Open Access Journals (Sweden)

    Morteza Nouri Taleghani

    2013-07-01

    Full Text Available Porosity is one of the fundamental petrophysical properties that should be evaluated for hydrocarbon bearing reservoirs. It is a vital factor in precise understanding of reservoir quality in a hydrocarbon field. Log data are exceedingly crucial information in petroleum industries, for many of hydrocarbon parameters are obtained by virtue of petrophysical data. There are three main petrophysical logging tools for the determination of porosity, namely neutron, density, and sonic well logs. Porosity can be determined by the use of each of these tools; however, a precise analysis requires a complete set of these tools. Log sets are commonly either incomplete or unreliable for many reasons (i.e. incomplete logging, measurement errors, and loss of data owing to unsuitable data storage. To overcome this drawback, in this study several intelligent systems such as fuzzy logic (FL, neural network (NN, and support vector machine are used to predict synthesized petrophysical logs including neutron, density, and sonic. To accomplish this, the petrophysical well logs data were collected from a real reservoir in one of Iran southwest oil fields. The corresponding correlation was obtained through the comparison of synthesized log values with real log values. The results showed that all intelligent systems were capable of synthesizing petrophysical well logs, but SVM had better accuracy and could be used as the most reliable method compared to the other techniques.

  20. Nonhomologous DNA End Joining in Cell-Free Extracts

    Directory of Open Access Journals (Sweden)

    Sheetal Sharma

    2010-01-01

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

  1. 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.

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

  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. 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.

  6. Synthesizing International Understanding of Changes in the Arctic Hydrological System

    Science.gov (United States)

    Pundsack, J. W.; Vorosmarty, C. J.; Hinzman, L. D.

    2009-12-01

    There are several notable gaps in our current level of understanding of Arctic hydrological systems. At the same time, rapidly emerging data sets, technologies, and modeling resources provide us with an unprecedented opportunity to move substantially forward. The Arctic Community-Wide Hydrological Analysis and Monitoring Program (Arctic-CHAMP), funded by NSF/ARCSS, was established to initiate a major effort to improve our current monitoring of water cycle variables, and to foster collaboration with the many relevant U.S. and international arctic research initiatives. These projects, funded under ARCSS through the ‘Freshwater Integration (FWI) study’, links CHAMP, the Arctic/Subarctic Ocean Fluxes (ASOF) Programme, and SEARCH. As part of the overall synthesis and integration efforts of the NSF-ARCSS Freshwater Integration (FWI) study, the program carried-out a major International Synthesis Capstone Workshop in Fall 2009 as an International Polar Year (IPY) affiliated meeting. The workshop, "Synthesizing International Understanding of Changes in the Arctic Hydrological System,” was held 30 September to 4 October 2009 in Stockholm at the Beijer Auditorium of the Royal Swedish Academy. The workshop was sponsored by the NSF-ARCSS Arctic-CHAMP Science Management Office (City College of New York / Univ. of New Hampshire), the International Study of Arctic Change (ISAC), and the International Arctic Research Center (IARC; Univ. of Alaska Fairbanks). The overarching goals of the meeting were to stage a post-IPY lessons-learned workshop with co-equal numbers of FWI, IPY, and ICARP-II researchers, using insights from recent scientific findings, data, and strategies to afford synthesis. The workshop aimed to: (1) take stock of recent advances in our understanding of changes in the Arctic hydrological system; (2) identify key remaining research gaps / unanswered questions; and (3) gather insight on where to focus future research efforts/initiatives (nationally and

  7. Cell-Free, De Nova Synthesis of Poliovirus

    Science.gov (United States)

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

    1991-12-01

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

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

    Science.gov (United States)

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

    2017-11-01

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

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

    Science.gov (United States)

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

    2017-06-15

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

  10. A frequency tracking synthesizer for beam diagnostic systems

    International Nuclear Information System (INIS)

    Peterson, D.; Marriner, J.

    1991-01-01

    In low and medium energy synchrotrons the beam revolution frequency changes by a large factor during the acceleration process. High production rates require that these machines cycle rapidly. In attempting to diagnose instabilities which develop during the acceleration process it is useful to be able to select some frequency segment between revolution harmonics for viewing. Most types of test equipment operating in the frequency domain, such as spectrum analyzers and network analyzers, are not suited to making direct measurements on such rapidly sweeping signals. Ideally, one would want to set the frequency frame of reference to the spot in the accelerating revolution harmonic domain where the measurements are to be made. A scheme using a direct digital synthesizer (DDS) was developed to provide this moving reference frame. This paper describes a synthesizer scheme combining digital and analog synthesizer techniques to allow tracking of signals during acceleration. Virtually any ratio of synthesizer to beam revolution frequency may be generated by this scheme. Details of hardware and measurement results are presented

  11. Observing System Simulations for ASCENDS: Synthesizing Science Measurement Requirements (Invited)

    Science.gov (United States)

    Kawa, S. R.; Baker, D. F.; Schuh, A. E.; Crowell, S.; Rayner, P. J.; Hammerling, D.; Michalak, A. M.; Wang, J. S.; Eluszkiewicz, J.; Ott, L.; Zaccheo, T.; Abshire, J. B.; Browell, E. V.; Moore, B.; Crisp, D.

    2013-12-01

    The measurement of atmospheric CO2 from space using active (lidar) sensing techniques has several potentially significant advantages in comparison to current and planned passive CO2 instruments. Application of this new technology aims to advance CO2 measurement capability and carbon cycle science into the next decade. The NASA Active Sensing of Carbon Emissions, Nights, Days, and Seasons (ASCENDS) mission has been recommended by the US National Academy of Sciences Decadal Survey for the next generation of space-based CO2 observing systems. ASCENDS is currently planned for launch in 2022. Several possible lidar instrument approaches have been demonstrated in airborne campaigns and the results indicate that such sensors are quite feasible. Studies are now underway to evaluate performance requirements for space mission implementation. Satellite CO2 observations must be highly precise and unbiased in order to accurately infer global carbon source/sink fluxes. Measurement demands are likely to further increase in the wake of GOSAT, OCO-2, and enhanced ground-based in situ and remote sensing CO2 data. The objective of our work is to quantitatively and consistently evaluate the measurement capabilities and requirements for ASCENDS in the context of advancing our knowledge of carbon flux distributions and their dependence on underlying physical processes. Considerations include requirements for precision, relative accuracy, spatial/temporal coverage and resolution, vertical information content, interferences, and possibly the tradeoffs among these parameters, while at the same time framing a mission that can be implemented within a constrained budget. Here, we attempt to synthesize the results of observing system simulation studies, commissioned by the ASCENDS Science Requirements Definition Team, into a coherent set of mission performance guidelines. A variety of forward and inverse model frameworks are employed to reduce the potential dependence of the results on model

  12. Stimulation of poliovirus RNA synthesis and virus maturation in a HeLa cell-free in vitro translation-RNA replication system by viral protein 3CDpro

    Directory of Open Access Journals (Sweden)

    Wimmer Eckard

    2005-11-01

    Full Text Available Abstract Poliovirus protein 3CDpro possesses both proteinase and RNA binding activities, which are located in the 3Cpro domain of the protein. The RNA polymerase (3Dpol domain of 3CDpro modulates these activities of the protein. We have recently shown that the level of 3CDpro in HeLa cell-free in vitro translation-RNA replication reactions is suboptimal for efficient virus production. However, the addition of either 3CDpro mRNA or of purified 3CDpro protein to in vitro reactions, programmed with viral RNA, results in a 100-fold increase in virus yield. Mutational analyses of 3CDpro indicated that RNA binding by the 3Cpro domain and the integrity of interface I in the 3Dpol domain of the protein are both required for function. The aim of these studies was to determine the exact step or steps at which 3CDpro enhances virus yield and to determine the mechanism by which this occurs. Our results suggest that the addition of extra 3CDpro to in vitro translation RNA-replication reactions results in a mild enhancement of both minus and plus strand RNA synthesis. By examining the viral particles formed in the in vitro reactions on sucrose gradients we determined that 3CDpro has only a slight stimulating effect on the synthesis of capsid precursors but it strikingly enhances the maturation of virus particles. Both the stimulation of RNA synthesis and the maturation of the virus particles are dependent on the presence of an intact RNA binding site within the 3Cpro domain of 3CDpro. In addition, the integrity of interface I in the 3Dpol domain of 3CDpro is required for efficient production of mature virus. Surprisingly, plus strand RNA synthesis and virus production in in vitro reactions, programmed with full-length transcript RNA, are not enhanced by the addition of extra 3CDpro. Our results indicate that the stimulation of RNA synthesis and virus maturation by 3CDpro in vitro is dependent on the presence of a VPg-linked RNA template.

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

    Science.gov (United States)

    Wuu, Jessica J; Swartz, James R

    2008-05-01

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

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

    Directory of Open Access Journals (Sweden)

    Tomasz Misztal

    2011-09-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1980-01-01

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

  16. A Synthesized Framework for Formal Verification of Computing Systems

    Directory of Open Access Journals (Sweden)

    Nikola Bogunovic

    2003-12-01

    Full Text Available Design process of computing systems gradually evolved to a level that encompasses formal verification techniques. However, the integration of formal verification techniques into a methodical design procedure has many inherent miscomprehensions and problems. The paper explicates the discrepancy between the real system implementation and the abstracted model that is actually used in the formal verification procedure. Particular attention is paid to the seamless integration of all phases of the verification procedure that encompasses definition of the specification language and denotation and execution of conformance relation between the abstracted model and its intended behavior. The concealed obstacles are exposed, computationally expensive steps identified and possible improvements proposed.

  17. 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 ...

  18. 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.

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

    Science.gov (United States)

    Terada, Takaho; Yokoyama, Shigeyuki

    2015-01-01

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

  20. 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.

  1. Optimization of film synthesized rare earth transition metal permanent magnet systems

    International Nuclear Information System (INIS)

    Cadieu, F.J.

    1990-01-01

    This report reviews work on the optimization of film synthesized rare earth transition metal permanent magnet systems. Topics include: high coercivity in Sm-Fe-Ti-V, Sm-Fe-V, and two element systems; ThMn 12 type pseudobinary SmFe 12 - X T X ; and sputter process control for the synthesis of precisely textured RE-TM magnetic films. (JL)

  2. Fibronectin-synthesizing activity of free and membrane-bound polyribosomes from human embryonic fibroblasts and chick embryos

    International Nuclear Information System (INIS)

    Belkin, V.M.; Volodarskaya, S.M.

    1986-01-01

    The fibronectin-synthesizing activity of membrane-bound and free polyribosomes in a cell-free system was studied using immunochemical methods. It was found that fibronectin biosynthesis on membrane-bound polyribosomes from human embryonic fibroblasts accounts for 4.9% and those from 10-day-old chick embryos for 1.1% of the total amount of newly synthesized proteins, whereas on free polyribosomes it is 1.0 and 0.3%, respectively. Fibronectin monomers with a molecular weight of 220,000 were found only in the material of the cell-free system containing heavy fractions of membrane-bound polyribosomes newly synthesized in the presence of spermidine. Thus, it was shown that fibronectin is synthesized primarily on membrane-bound polyribosomes

  3. Translation of satellite tobacco necrosis virus RNA modified by (not equal to)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene is inhibited in a wheat germ cell-free system

    International Nuclear Information System (INIS)

    Haas, R.; Pulkrabek, P.; Takanami, Y.; Grunberger, D.

    1983-01-01

    It has been shown that (not equal to)-r-7-,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) modification of rabbit globin mRNA results in inhibition of translational initiation. In order to explore the possibility that modification of the 5' cap structure was responsible for this inhibition, the naturally non-capped mRNA from satellite tobacco necrosis virus (STNV) was reacted with BPDE and translated in a wheat germ cell-free system. The extent of modification was 1.3 and 2.9 BPDE residues/molecule. High performance liquid chromatography of the modified nucleosides from enzymatically hydrolyzed STNV RNA revealed that greater than 90% of the nucleoside adducts were substituted at the exocyclic amino group of guanosine. The translational ability of the lower and higher modified STNV, measured by incorporation of [ 14 C]amino acids into acid-precipitable polypeptides is inhibited by 55% and 63%, respectively. Polyacrylamide gel electrophoretic analyses of the translation products indicate that predominantly full-length coat proteins are synthesized but with the carcinogen-modified STNV the amount is reduced. On the other hand, 80S initiation complex formation is not inhibited as measured by binding of the BPDE-modified STNV to ribosomes and followed by glycerol gradient centrifugation. Under these conditions, aurintricarboxylic acid completely inhibits 80S initiation complex formation in the presence of either modified or native STNV. These results suggest that inhibition of in vitro translation of BPDE-modified STNV, in contrast to that of globin mRNA, is not at the level of initiation complex formation but possibly by premature termination of growing polypeptides

  4. Recent progress in solution plasma-synthesized-carbon-supported catalysts for energy conversion systems

    Science.gov (United States)

    Lun Li, Oi; Lee, Hoonseung; Ishizaki, Takahiro

    2018-01-01

    Carbon-based materials have been widely utilized as the electrode materials in energy conversion and storage technologies, such as fuel cells and metal-air batteries. In these systems, the oxygen reduction reaction is an important step that determines the overall performance. A novel synthesis route, named the solution plasma process, has been recently utilized to synthesize various types of metal-based and heteroatom-doped carbon catalysts. In this review, we summarize cutting-edge technologies involving the synthesis and modeling of carbon-supported catalysts synthesized via solution plasma process, followed by current progress on the electrocatalytic performance of these catalysts. This review provides the fundamental and state-of-the-art performance of solution-plasma-synthesized electrode materials, as well as the remaining scientific and technological challenges for this process.

  5. Enhancement of a radiation safety system through the use of a microprocessor-controlled speech synthesizer

    International Nuclear Information System (INIS)

    Keefe, D.J.; McDowell, W.P.

    1980-01-01

    A speech synthesizer is being used to differentiate eight separate safety alarms on a high energy accelerator at Argonne National Laboratory. A single board microcomputer monitors eight signals from an existing radiation safety logic circuit. The microcomputer is programmed to output the proper code at the proper time and sequence to a speech synthesizer which supplies the audio input to a local public address system. This eliminates the requirement for eight different alarm tones and the personnel training required to differentiate among them. A twenty-word vocabulary was found adequate to supply the necessary safety announcements. The article describes the techniques used to interface the speech synthesizer into the existing safety logic circuit

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

    Science.gov (United States)

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

    2018-01-01

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

  7. 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.

    Science.gov (United States)

    Pais, Pilar; Villar, Agustí; Rull, Santiago

    2016-01-01

    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. 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. 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. 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. 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 bioactivity of SPSE corresponds favorably to that reported for the hexane extract used in a large number of positive BPH clinical trials, as well as to finasteride

  8. 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

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    Joseph A. Wayman

    2015-03-01

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

  11. A general digital computer procedure for synthesizing linear automatic control systems

    International Nuclear Information System (INIS)

    Cummins, J.D.

    1961-10-01

    The fundamental concepts required for synthesizing a linear automatic control system are considered. A generalized procedure for synthesizing automatic control systems is demonstrated. This procedure has been programmed for the Ferranti Mercury and the IBM 7090 computers. Details of the programmes are given. The procedure uses the linearized set of equations which describe the plant to be controlled as the starting point. Subsequent computations determine the transfer functions between any desired variables. The programmes also compute the root and phase loci for any linear (and some non-linear) configurations in the complex plane, the open loop and closed loop frequency responses of a system, the residues of a function of the complex variable 's' and the time response corresponding to these residues. With these general programmes available the design of 'one point' automatic control systems becomes a routine scientific procedure. Also dynamic assessments of plant may be carried out. Certain classes of multipoint automatic control problems may also be solved with these procedures. Autonomous systems, invariant systems and orthogonal systems may also be studied. (author)

  12. Systems including catalysts in porous zeolite materials within a reactor for use in synthesizing hydrocarbons

    Science.gov (United States)

    Rolllins, Harry W [Idaho Falls, ID; Petkovic, Lucia M [Idaho Falls, ID; Ginosar, Daniel M [Idaho Falls, ID

    2012-07-24

    Catalytic structures include a catalytic material disposed within a zeolite material. The catalytic material may be capable of catalyzing a formation of methanol from carbon monoxide and/or carbon dioxide, and the zeolite material may be capable of catalyzing a formation of hydrocarbon molecules from methanol. The catalytic material may include copper and zinc oxide. The zeolite material may include a first plurality of pores substantially defined by a crystal structure of the zeolite material and a second plurality of pores dispersed throughout the zeolite material. Systems for synthesizing hydrocarbon molecules also include catalytic structures. Methods for synthesizing hydrocarbon molecules include contacting hydrogen and at least one of carbon monoxide and carbon dioxide with such catalytic structures. Catalytic structures are fabricated by forming a zeolite material at least partially around a template structure, removing the template structure, and introducing a catalytic material into the zeolite material.

  13. EXAFS Phase Retrieval Solution Tracking for Complex Multi-Component System: Synthesized Topological Inverse Computation

    International Nuclear Information System (INIS)

    Lee, Jay Min; Yang, Dong-Seok; Bunker, Grant B

    2013-01-01

    Using the FEFF kernel A(k,r), we describe the inverse computation from χ(k)-data to g(r)-solution in terms of a singularity regularization method based on complete Bayesian statistics process. In this work, we topologically decompose the system-matched invariant projection operators into two distinct types, (A + AA + A) and (AA + AA + ), and achieved Synthesized Topological Inversion Computation (STIC), by employing a 12-operator-closed-loop emulator of the symplectic transformation. This leads to a numerically self-consistent solution as the optimal near-singular regularization parameters are sought, dramatically suppressing instability problems connected with finite precision arithmetic in ill-posed systems. By statistically correlating a pair of measured data, it was feasible to compute an optimal EXAFS phase retrieval solution expressed in terms of the complex-valued χ(k), and this approach was successfully used to determine the optimal g(r) for a complex multi-component system.

  14. Polyaniline/silver nanocomposites synthesized via UV-Vis-Assisted aniline polymerization with a reversed micellar microemulsion system

    NARCIS (Netherlands)

    Li, Z.; Li, Y.; Lin, W.; Zheng, F.; Laven, J.

    Polyaniline (PANI)/silver (Ag) nanocomposites were successfully synthesized within a sodium dodecyl sulfate reverse micro-emulsion system and characterized by Fourier transform infrared spectroscopy, X-ray diffraction, ultraviolet spectrometry, thermogravimetric analysis, scanning electron

  15. Performance of active feedforward control systems in non-ideal, synthesized diffuse sound fields.

    Science.gov (United States)

    Misol, Malte; Bloch, Christian; Monner, Hans Peter; Sinapius, Michael

    2014-04-01

    The acoustic performance of passive or active panel structures is usually tested in sound transmission loss facilities. A reverberant sending room, equipped with one or a number of independent sound sources, is used to generate a diffuse sound field excitation which acts as a disturbance source on the structure under investigation. The spatial correlation and coherence of such a synthesized non-ideal diffuse-sound-field excitation, however, might deviate significantly from the ideal case. This has consequences for the operation of an active feedforward control system which heavily relies on the acquisition of coherent disturbance source information. This work, therefore, evaluates the spatial correlation and coherence of ideal and non-ideal diffuse sound fields and considers the implications on the performance of a feedforward control system. The system under consideration is an aircraft-typical double panel system, equipped with an active sidewall panel (lining), which is realized in a transmission loss facility. Experimental results for different numbers of sound sources in the reverberation room are compared to simulation results of a comparable generic double panel system excited by an ideal diffuse sound field. It is shown that the number of statistically independent noise sources acting on the primary structure of the double panel system depends not only on the type of diffuse sound field but also on the sample lengths of the processed signals. The experimental results show that the number of reference sensors required for a defined control performance exhibits an inverse relationship to control filter length.

  16. Synthesizing modeling of power generation and power limits in energy systems

    International Nuclear Information System (INIS)

    Sieniutycz, Stanislaw

    2015-01-01

    Applying the common mathematical procedure of thermodynamic optimization the paper offers a synthesizing or generalizing modeling of power production in various energy generators, such as thermal, solar and electrochemical engines (fuel cells). Static and dynamical power systems are investigated. Dynamical models take into account the gradual downgrading of a resource, caused by power delivery. Analytical modeling includes conversion efficiencies expressed in terms of driving fluxes. Products of efficiencies and driving fluxes determine the power yield and power maxima. While optimization of static systems requires using of differential calculus and Lagrange multipliers, dynamic optimization involves variational calculus and dynamic programming. In reacting mixtures balances of mass and energy serve to derive power yield in terms of an active part of chemical affinity. Power maximization approach is also applied to fuel cells treated as flow engines driven by heat flux and fluxes of chemical reagents. The results of power maxima provide limiting indicators for thermal, solar and SOFC generators. They are more exact than classical reversible limits of energy transformation. - Highlights: • Systematic evaluation of power limits by optimization. • Common thermodynamic methodology for engine systems. • Original, in-depth study of power maxima. • Inclusion of fuel cells to a class of thermodynamic power systems

  17. Writing syntheses for managers: Lessons from the Rainbow Series and Fire Effects Information System

    Science.gov (United States)

    Jane Kapler Smith; Kristin L. Zouhar; Janet Fryer

    2009-01-01

    Scientific knowledge is essential for sound wildland management, but this knowledge is a complex, ever-expanding resource. Managers often request syntheses or reviews of available knowledge, and scientists have responded with an increasing number of syntheses for managers. Unfortunately, little guidance is available for this kind of writing. While most scientists have...

  18. Kinetics and mechanism of oxygen reduction reaction at CoPd system synthesized on XC72

    International Nuclear Information System (INIS)

    Tarasevich, M.R.; Chalykh, A.E.; Bogdanovskaya, V.A.; Kuznetsova, L.N.; Kapustina, N.A.; Efremov, B.N.; Ehrenburg, M.R.; Reznikova, L.A.

    2006-01-01

    Studies are presented of the kinetics and mechanism of oxygen electroreduction reaction on CoPd catalysts synthesized on carbon black XC72. As shown both in model conditions and in the tests within the cathodes of hydrogen-oxygen fuel cells with proton conducting electrolyte, CoPd/C system features a higher activity, as compared to Co/C. The highest activity in the oxygen reduction reaction is demonstrated by the catalysts with the Pd:Co atomic ratio being 7:3 and 4:1. The structural studies (XPS and XRD, and also the data of CO desorption measurements) evidence the CoPd alloy formation, which is reflected in the negative shift of the bonding energy maximum as compared to Pd/C and in the appearance of the additional CO desorption maximums on the voltammograms. It is found by means of structural research that CoPd alloy is formed in the course of the catalyst synthesis which features a higher catalytic activity of the binary systems. Besides, CoPd/C catalyst is more stable in respect to corrosion than Pd supported on carbon black. The measurements on the rotating disc electrode and rotating ring-disc electrode evidence that CoPd/C system provides the predominant oxygen reduction to water in the practically important range of potentials (E > 0.7 V). The proximity of kinetic parameters of the oxygen reduction reaction on CoPd/C and Pt/C catalysts points to the similar reaction mechanism. The slow step of the reaction is the addition of the first electron to the adsorbed and previously protonated O 2 molecule. The assumptions are offered about the reasons causing the higher activity and selectivity of the binary catalyst towards oxygen reduction to water, as compared to Co/C. The studies of the most active catalysts within the fuel cell cathodes are performed

  19. Cellulose esters synthesized using a tetrabutylammonium acetate and dimethylsulfoxide solvent system

    Science.gov (United States)

    Yu, Yongqi; Miao, Jiaojiao; Jiang, Zeming; Sun, Haibo; Zhang, Liping

    2016-07-01

    Cellulose acetate (CA) and cellulose acetate propionate (CAP) were homogeneously synthesized in a novel tetrabutylammonium acetate/dimethyl sulfoxide (DMSO) solvent system, without any catalyst, at temperatures below 70 °C. The molecular structures of the cellulose esters (CEs) and distributions of the substituents in the anhydroglucose repeating units were determined using 13C cross-polarization magic angle spinning nuclear magnetic resonance spectroscopy, and the degree of substitution (DS) values were determined using 1H nuclear magnetic resonance spectroscopy. The structures of the CEs, regenerated cellulose (RC), and pulp were determined using Fourier transform infrared spectroscopy. The thermal properties of the products were determined using thermogravimetric analysis. The temperatures of initial decomposition of the CEs were up to 40 °C higher than those of the RC and pulp. All the CEs were highly soluble in DMSO, but were insoluble in acetone. CAs with DS values less than 2.6 swelled or were poorly dissolved in CHCl3, but those with DS values above 2.9 dissolved rapidly. CAPs with DS values above 2.6 had good solubilities in ethyl acetate.

  20. tRNA-mediated labelling of proteins with biotin. A nonradioactive method for the detection of cell-free translation products.

    Science.gov (United States)

    Kurzchalia, T V; Wiedmann, M; Breter, H; Zimmermann, W; Bauschke, E; Rapoport, T A

    1988-03-15

    We have developed a new method for the rapid and sensitive detection of cell-free translation products. Biotinylated lysine is incorporated into newly synthesized proteins by means of lysyl-tRNA that is modified in the epsilon-position. After electrophoresis in a dodecyl sulfate gel and blotting onto nitrocellulose, the translation products can be identified by probing with streptavidin and biotinylated alkaline phosphatase, followed by incubation with a chromogenic enzyme substrate. The non-radioactive labelling by biotin approaches in its sensitivity that obtained by radioactive amino acids. The products are absolutely stable and can be rapidly identified. The new method has been tested with different mRNAs in the cell-free translation systems of wheat germ and reticulocytes. Neither the interaction of secretory proteins with the signal recognition particle nor the in vitro translocation across the endoplasmic reticulum membrane or core glycosylation of nascent polypeptides are prevented by the incorporation of biotinylated lysine residues. The results indicate that both the ribosome and the endoplasmic reticulum membrane permit the passage of polypeptides carrying bulky groups attached to the amino acids (by atomic models it was estimated that the size of the side chain of lysine changes from approximately equal to 0.8 nm to approximately equal to 2 nm after modification.

  1. Extracellular biosynthesis of silver nanoparticles using the cell-free filtrate of nematophagous fungus Duddingtonia flagrans

    Directory of Open Access Journals (Sweden)

    Costa Silva LP

    2017-08-01

    Full Text Available Laryssa Pinheiro Costa Silva,1 Jairo Pinto Oliveira,2 Wanderson Juvencio Keijok,2 André Romero da Silva,3 Anderson Rocha Aguiar,1 Marco Cesar Cunegundes Guimarães,2 Carolina Magri Ferraz,1 Jackson Victor Araújo,4 Fernando Luiz Tobias,5 Fábio Ribeiro Braga1 1Department of Parasitology, University Vila Velha, Vila Velha, Espírito Santo, Brazil; 2Morphology Department, Federal University of Espírito Santo, Vitória, Espírito Santo, Brazil; 3Federal Institute of Education, Science and Technology of Espírito Santo, Aracruz, Espírito Santo, Brazil; 4Department of Veterinary Medicine, Federal University of Viçosa, Viçosa, Minas Gerais, Brazil; 5Department of Microbiology, University Vila Velha, Vila Velha, Espírito Santo, Brazil Abstract: The biosynthesis of metallic nanoparticles (NPs using biological systems such as fungi has evolved to become an important area of nanobiotechnology. Herein, we report for the first time the extracellular synthesis of highly stable silver NPs (AgNPs using the nematophagous fungus Duddingtonia flagrans (AC001. The fungal cell-free filtrate was analyzed by the Bradford method and 3,5-dinitrosalicylic acid assay and used to synthesize the AgNPs in the presence of a 1 mM AgNO3 solution. They have been characterized by UV–Vis spectroscopy, X-ray diffraction, transmission electron microscopy, dynamic light scattering, Zeta potential measurements, Fourier-transform infrared, and Raman spectroscopes. UV–Vis spectroscopy confirmed bioreduction, while X-ray diffractometry established the crystalline nature of the AgNPs. Dynamic light scattering and transmission electron microscopy images showed approximately 11, 38 nm monodisperse and quasispherical AgNPs. Zeta potential analysis was able to show a considerable stability of AgNPs. The N–H stretches in Fourier-transform infrared spectroscopy indicate the presence of protein molecules. The Raman bands suggest that chitinase was involved in the growth and

  2. 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.

  3. A fast-hopping 3-band CMOS frequency synthesizer for MB-OFDM UWB system

    International Nuclear Information System (INIS)

    Zheng Yongzheng; Xia Lingli; Li Weinan; Huang Yumei; Hong Zhiliang

    2009-01-01

    A fast-hopping 3-band (mode 1) multi-band orthogonal frequency division multiplexing ultra-wideband frequency synthesizer is presented. This synthesizer uses two phase-locked loops for generating steady frequencies and one quadrature single-sideband mixer for frequency shifting and quadrature frequency generation. The generated carriers can hop among 3432 MHz, 3960 MHz, and 4488 MHz. Implemented in a 0.13 μm CMOS process, this fully integrated synthesizer consumes 27 mA current from a 1.2 V supply. Measurement shows that the out-of-band spurious tones are below -50 dBc, while the in-band spurious tones are below -34 dBc. The measured hopping time is below 2 ns. The core die area is 1.0 x 1.8 mm 2 .

  4. A fast-hopping 3-band CMOS frequency synthesizer for MB-OFDM UWB system

    Energy Technology Data Exchange (ETDEWEB)

    Zheng Yongzheng; Xia Lingli; Li Weinan; Huang Yumei; Hong Zhiliang, E-mail: yumeihuang@fudan.edu.c [State Key Laboratory of ASIC and System, Fudan University, Shanghai 201203 (China)

    2009-09-15

    A fast-hopping 3-band (mode 1) multi-band orthogonal frequency division multiplexing ultra-wideband frequency synthesizer is presented. This synthesizer uses two phase-locked loops for generating steady frequencies and one quadrature single-sideband mixer for frequency shifting and quadrature frequency generation. The generated carriers can hop among 3432 MHz, 3960 MHz, and 4488 MHz. Implemented in a 0.13 {mu}m CMOS process, this fully integrated synthesizer consumes 27 mA current from a 1.2 V supply. Measurement shows that the out-of-band spurious tones are below -50 dBc, while the in-band spurious tones are below -34 dBc. The measured hopping time is below 2 ns. The core die area is 1.0 x 1.8 mm{sup 2}.

  5. 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.

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

    Science.gov (United States)

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

    2010-01-01

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

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    Lihong Jiang

    2018-06-01

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

  9. Cell-Free Expression and In Situ Immobilization of Parasite Proteins from Clonorchis sinensis for Rapid Identification of Antigenic Candidates.

    Directory of Open Access Journals (Sweden)

    Christy Catherine

    Full Text Available Progress towards genetic sequencing of human parasites has provided the groundwork for a post-genomic approach to develop novel antigens for the diagnosis and treatment of parasite infections. To fully utilize the genomic data, however, high-throughput methodologies are required for functional analysis of the proteins encoded in the genomic sequences. In this study, we investigated cell-free expression and in situ immobilization of parasite proteins as a novel platform for the discovery of antigenic proteins. PCR-amplified parasite DNA was immobilized on microbeads that were also functionalized to capture synthesized proteins. When the microbeads were incubated in a reaction mixture for cell-free synthesis, proteins expressed from the microbead-immobilized DNA were instantly immobilized on the same microbeads, providing a physical linkage between the genetic information and encoded proteins. This approach of in situ expression and isolation enables streamlined recovery and analysis of cell-free synthesized proteins and also allows facile identification of the genes coding antigenic proteins through direct PCR of the microbead-bound DNA.

  10. Rare azido-bridged manganese(II) systems: syntheses, crystal structures, and magnetic properties.

    Science.gov (United States)

    Ghosh, A K; Ghoshal, D; Zangrando, E; Ribas, J; Ray Chaudhuri, N

    2005-03-21

    Two new polymeric azido-bridged manganese complexes of formulas [Mn(N3)2 (bpee)]n (1) and {[Mn(N3)(dpyo)Cl(H2O)2](H2O)}n (2) [bpee, trans-1,2-bis(4-pyridyl)ethylene; dpyo, 4,4'-dipyridyl N,N'-dioxide] have been synthesized and characterized by single-crystal X-ray diffraction analysis and low-temperature magnetic study. Both the complexes 1 and 2 crystallize in the triclinic system, space group P1, with a = 8.877(3) A, b = 11.036(3) A, c = 11.584(4) A, alpha = 72.62(2) degrees, beta = 71.06(2) degrees, gamma = 87.98(3) degrees, and Z = 1 and a = 7.060(3) A, b = 10.345(3) A, c = 11.697(4) A, alpha = 106.86(2) degrees, beta = 113.33(2) degrees, gamma = 96.39(3) degrees, and Z = 2, respectively. Complex 1 exhibits a 2D structure of [-Mn(N3)2-]n chains, connected by bpee ligands, whose pyridine rings undergo pi-pi and C-H...pi interactions. This facilitates the rare arrangement of doubly bridged azide ligands with one end-on and two end-to-end (EO-EE-EE) sequence. Complex 2 is a neutral 1D polymer built up by [Mn(N3)(dpyo)Cl(H2O)2] units and lattice water molecules. The metals are connected by single EE azide ligands, which are arranged in a cis position to the Mn(II) center. The 1D zipped chains are linked by H-bonds involving lattice water molecules and show pi-pi stacking of dpyo pyridine rings to form a supramolecular 2D layered structure. The magnetic studies were performed in 2-300 K temperature range, and the data were fitted by considering an alternating chain of exchange interactions with S = 5/2 (considered as classical spin) with the spin Hamiltonians H = -Ji sigma(S(3i)S(3i+1) + S(3i+1)S(3i+2)) - J2 sigmaS(3i-1)S(3i) and H = -Ji sigmaS(2i)S(2i+1) - J2 sigmaS(2i+1)S(2i+2) for complexes 1 and 2, respectively. Complex 2 exhibits small antiferromagnetic coupling between the metal centers, whereas 1 exhibits a new case of topological ferromagnetism, which is very unusual.

  11. 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.

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

    Science.gov (United States)

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

    2008-10-14

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

  13. Integrating cell-free biosyntheses of heme prosthetic group and apoenzyme for the synthesis of functional P450 monooxygenase.

    Science.gov (United States)

    Kwon, Yong-Chan; Oh, In-Seok; Lee, Nahum; Lee, Kyung-Ho; Yoon, Yeo Joon; Lee, Eun Yeol; Kim, Byung-Gee; Kim, Dong-Myung

    2013-04-01

    Harnessing the isolated protein synthesis machinery, cell-free protein synthesis reproduces the cellular process of decoding genetic information in artificially controlled environments. More often than not, however, generation of functional proteins requires more than simple translation of genetic sequences. For instance, many of the industrially important enzymes require non-protein prosthetic groups for biological activity. Herein, we report the complete cell-free biogenesis of a heme prosthetic group and its integration with concurrent apoenzyme synthesis for the production of functional P450 monooxygenase. Step reactions required for the syntheses of apoenzyme and the prosthetic group have been designed so that these two separate pathways take place in the same reaction mixture, being insulated from each other. Combined pathways for the synthesis of functional P450 monooxygenase were then further integrated with in situ assay reactions to enable real-time measurement of enzymatic activity during its synthesis. Copyright © 2012 Wiley Periodicals, Inc.

  14. 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.

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

    Science.gov (United States)

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

    2018-04-01

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

  16. The cell-free integration of a polytopic mitochondrial membrane protein into liposomes occurs cotranslationally and in a lipid-dependent manner.

    Directory of Open Access Journals (Sweden)

    Ashley R Long

    Full Text Available The ADP/ATP Carrier (AAC is the most abundant transporter of the mitochondrial inner membrane. The central role that this transporter plays in cellular energy production highlights the importance of understanding its structure, function, and the basis of its pathologies. As a means of preparing proteoliposomes for the study of membrane proteins, several groups have explored the use of cell-free translation systems to facilitate membrane protein integration directly into preformed unilamellar vesicles without the use of surfactants. Using AAC as a model, we report for the first time the detergent-free reconstitution of a mitochondrial inner membrane protein into liposomes using a wheat germ-based in vitro translation system. Using a host of independent approaches, we demonstrate the efficient integration of AAC into vesicles with an inner membrane-mimetic lipid composition and, more importantly, that the integrated AAC is functionally active in transport. By adding liposomes at different stages of the translation reaction, we show that this direct integration is obligatorily cotranslational, and by synthesizing stable ribosome-bound nascent chain intermediates, we show that the nascent AAC polypeptide interacts with lipid vesicles while ribosome-bound. Finally, we show that the presence of the phospholipid cardiolipin in the liposomes specifically enhances AAC translation rate as well as the efficiency of vesicle association and integration. In light of these results, the possible mechanisms of liposome-assisted membrane protein integration during cell-free translation are discussed with respect to the mode of integration and the role of specific lipids.

  17. Preparation of a Bis-GMA-Free Dental Resin System with Synthesized Fluorinated Dimethacrylate Monomers

    Directory of Open Access Journals (Sweden)

    Shuzhen Luo

    2016-12-01

    Full Text Available With the aim of reducing human exposure to Bisphenol A (BPA derivatives in dentistry, a fluorinated dimethacrylate monomer was synthesized to replace 2,2-bis[4-(2-hydroxy-3-methacryloy-loxypropyl-phenyl]propane (Bis-GMA as the base monomer of dental resin. After mixing with reactive diluent triethyleneglycol dimethacrylate (TEGDMA, fluorinated dimethacrylate (FDMA/TEGDMA was prepared and compared with Bis-GMA/TEGDMA in physicochemical properties, such as double bond conversion (DC, volumetric shrinkage (VS, water sorption (WS and solubility (WSL, flexural strength (FS and modulus (FM. The results showed that, when compared with Bis-GMA based resin, FDMA-based resin had several advantages, such as higher DC, lower VS, lower WS, and higher FS after water immersion. All of these revealed that FDMA had potential to be used as a substitute for Bis-GMA. Of course, many more studies, such as biocompatibility testing, should be undertaken to prove whether FDMA could be applied in clinic.

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

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    Nitzan Krinsky

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ana Ferreras

    2002-04-01

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

  1. Morphological classification of mesoporous silicas synthesized in a binary water-ether solvent system

    NARCIS (Netherlands)

    Cai, Qiang; Geng, Yi; Zhao, Xiang; Cui, Kai; Sun, Qianyao; Chen, Xihua; Feng, Qingling; Li, Hengde; Vrieling, Engel G.

    2008-01-01

    Using diethyl ether as a co-solvent, a non-stable interface of biphasic oil-water system (the so-called oil-water two-phase (OWTP) system) was employed in the preparation of mesostructured silicas with diversified particle morphologies. By adjusting the molar ratios of H2O:C2H5OC2H5:NH3 center dot

  2. Synthesizing Configurable Biochemical Implementation of Linear Systems from Their Transfer Function Specifications.

    Directory of Open Access Journals (Sweden)

    Tai-Yin Chiu

    Full Text Available The ability to engineer synthetic systems in the biochemical context is constantly being improved and has a profound societal impact. Linear system design is one of the most pervasive methods applied in control tasks, and its biochemical realization has been proposed by Oishi and Klavins and advanced further in recent years. However, several technical issues remain unsolved. Specifically, the design process is not fully automated from specification at the transfer function level, systems once designed often lack dynamic adaptivity to environmental changes, matching rate constants of reactions is not always possible, and implementation may be approximative and greatly deviate from the specifications. Building upon the work of Oishi and Klavins, this paper overcomes these issues by introducing a design flow that transforms a transfer-function specification of a linear system into a set of chemical reactions, whose input-output response precisely conforms to the specification. This system is implementable using the DNA strand displacement technique. The underlying configurability is embedded into primitive components and template modules, and thus the entire system is adaptive. Simulation of DNA strand displacement implementation confirmed the feasibility and superiority of the proposed synthesis flow.

  3. Completion of proteomic data sets by Kd measurement using cell-free synthesis of site-specifically labeled proteins.

    Directory of Open Access Journals (Sweden)

    Paul Majkut

    Full Text Available The characterization of phosphotyrosine mediated protein-protein interactions is vital for the interpretation of downstream pathways of transmembrane signaling processes. Currently however, there is a gap between the initial identification and characterization of cellular binding events by proteomic methods and the in vitro generation of quantitative binding information in the form of equilibrium rate constants (Kd values. In this work we present a systematic, accelerated and simplified approach to fill this gap: using cell-free protein synthesis with site-specific labeling for pull-down and microscale thermophoresis (MST we were able to validate interactions and to establish a binding hierarchy based on Kd values as a completion of existing proteomic data sets. As a model system we analyzed SH2-mediated interactions of the human T-cell phosphoprotein ADAP. Putative SH2 domain-containing binding partners were synthesized from a cDNA library using Expression-PCR with site-specific biotinylation in order to analyze their interaction with fluorescently labeled and in vitro phosphorylated ADAP by pull-down. On the basis of the pull-down results, selected SH2's were subjected to MST to determine Kd values. In particular, we could identify an unexpectedly strong binding of ADAP to the previously found binding partner Rasa1 of about 100 nM, while no evidence of interaction was found for the also predicted SH2D1A. Moreover, Kd values between ADAP and its known binding partners SLP-76 and Fyn were determined. Next to expanding data on ADAP suggesting promising candidates for further analysis in vivo, this work marks the first Kd values for phosphotyrosine/SH2 interactions on a phosphoprotein level.

  4. Microprocessor controlled system for automatic and semi-automatic syntheses of radiopharmaceuticals

    International Nuclear Information System (INIS)

    Ruth, T.J.; Adam, M.J.; Morris, D.; Jivan, S.

    1986-01-01

    A computer based system has been constructed to control the automatic synthesis of 2-deoxy-2-( 18 F)fluoro-D-glucose and is also being used in the development of an automatic synthesis of L-6-( 18 F)fluorodopa. (author)

  5. Reviews and syntheses: Systematic Earth observations for use in terrestrial carbon cycle data assimilation systems

    Science.gov (United States)

    Scholze, Marko; Buchwitz, Michael; Dorigo, Wouter; Guanter, Luis; Quegan, Shaun

    2017-07-01

    The global carbon cycle is an important component of the Earth system and it interacts with the hydrology, energy and nutrient cycles as well as ecosystem dynamics. A better understanding of the global carbon cycle is required for improved projections of climate change including corresponding changes in water and food resources and for the verification of measures to reduce anthropogenic greenhouse gas emissions. An improved understanding of the carbon cycle can be achieved by data assimilation systems, which integrate observations relevant to the carbon cycle into coupled carbon, water, energy and nutrient models. Hence, the ingredients for such systems are a carbon cycle model, an algorithm for the assimilation and systematic and well error-characterised observations relevant to the carbon cycle. Relevant observations for assimilation include various in situ measurements in the atmosphere (e.g. concentrations of CO2 and other gases) and on land (e.g. fluxes of carbon water and energy, carbon stocks) as well as remote sensing observations (e.g. atmospheric composition, vegetation and surface properties).We briefly review the different existing data assimilation techniques and contrast them to model benchmarking and evaluation efforts (which also rely on observations). A common requirement for all assimilation techniques is a full description of the observational data properties. Uncertainty estimates of the observations are as important as the observations themselves because they similarly determine the outcome of such assimilation systems. Hence, this article reviews the requirements of data assimilation systems on observations and provides a non-exhaustive overview of current observations and their uncertainties for use in terrestrial carbon cycle data assimilation. We report on progress since the review of model-data synthesis in terrestrial carbon observations by Raupach et al.(2005), emphasising the rapid advance in relevant space-based observations.

  6. 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

  7. Solving Boundary Value Problem for a Nonlinear Stationary Controllable System with Synthesizing Control

    Directory of Open Access Journals (Sweden)

    Alexander N. Kvitko

    2017-01-01

    Full Text Available An algorithm for constructing a control function that transfers a wide class of stationary nonlinear systems of ordinary differential equations from an initial state to a final state under certain control restrictions is proposed. The algorithm is designed to be convenient for numerical implementation. A constructive criterion of the desired transfer possibility is presented. The problem of an interorbital flight is considered as a test example and it is simulated numerically with the presented method.

  8. Arctic sea-ice syntheses: Charting across scope, scale, and knowledge systems

    Science.gov (United States)

    Druckenmiller, M. L.; Perovich, D. K.; Francis, J. A.

    2017-12-01

    Arctic sea ice supports and intersects a multitude of societal benefit areas, including regulating regional and global climates, structuring marine food webs, providing for traditional food provisioning by indigenous peoples, and constraining marine shipping and access. At the same time, sea ice is one of the most rapidly changing elements of the Arctic environment and serves as a source of key physical indicators for monitoring Arctic change. Before the present scientific interest in Arctic sea ice for climate research, it has long been, and remains, a focus of applied research for industry and national security. For generations, the icy coastal seas of the North have also provided a basis for the sharing of local and indigenous knowledge between Arctic residents and researchers, including anthropologists, biologists, and geoscientists. This presentation will summarize an ongoing review of existing synthesis studies of Arctic sea ice. We will chart efforts to achieve system-level understanding across geography, temporal scales, and the ecosystem services that Arctic sea ice supports. In doing so, we aim to illuminate the role of interdisciplinary science, together with local and indigenous experts, in advancing knowledge of the roles of sea ice in the Arctic system and beyond, reveal the historical and scientific evolution of sea-ice research, and assess current gaps in system-scale understanding.

  9. Synthesizing Marketing, Community Engagement, and Systems Science Approaches for Advancing Translational Research.

    Science.gov (United States)

    Kneipp, Shawn M; Leeman, Jennifer; McCall, Pamela; Hassmiller-Lich, Kristen; Bobashev, Georgiy; Schwartz, Todd A; Gilmore, Robert; Riggan, Scott; Gil, Benjamin

    2015-01-01

    The adoption and implementation of evidence-based interventions (EBIs) are the goals of translational research; however, potential end-users' perceptions of an EBI value have contributed to low rates of adoption. In this article, we describe our application of emerging dissemination and implementation science theoretical perspectives, community engagement, and systems science principles to develop a novel EBI dissemination approach. Using consumer-driven, graphics-rich simulation, the approach demonstrates predicted implementation effects on health and employment outcomes for socioeconomically disadvantaged women at the local level and is designed to increase adoption interest of county program managers accountable for improving these outcomes in their communities.

  10. Solid solution and amorphous phase in Ti–Nb–Ta–Mn systems synthesized by mechanical alloying

    Energy Technology Data Exchange (ETDEWEB)

    Aguilar, C., E-mail: claudio.aguilar@usm.cl [Departamento de Ingeniería Metalúrgica y Materiales, Universidad Técnica Federico Santa María, Av. España 1680, Valparaíso (Chile); Guzman, P. [Departamento de Ingeniería Metalúrgica y Materiales, Universidad Técnica Federico Santa María, Av. España 1680, Valparaíso (Chile); Lascano, S. [Departamento de Ingeniería Mecánica, Universidad Técnica Federico Santa María, Av. España 1680, Valparaíso (Chile); Parra, C. [Departamento de Física, Universidad Técnica Federico Santa María, Av. España 1680, Valparaíso (Chile); Bejar, L. [Instituto de Investigaciones Metalúrgicas, Universidad Michoacana de San Nicolás de Hidalgo, Ciudad Universitaria, Morelia C.P. 58000, Michoacán (Mexico); Medina, A. [Facultad de Ingeniería Mecánica, Universidad Michoacana de San Nicolás de Hidalgo, Ciudad Universitaria, C.P. 58000, Michoacán (Mexico); Guzman, D. [Departamento de Metalurgia, Universidad de Atacama, Av. España 485, Copiapó (Chile)

    2016-06-15

    This work discusses the formation of Ti–30Nb–13Ta–xMn (x: 2, 4 and 6 wt%) solid solution by mechanical alloying using a shaker mill. A solid solution was formed after 15 h of milling and an amorphous phase was formed after 30 h of milling, according to X-ray diffraction results. Disappearance of strongest X-ray diffraction peaks of Nb, Ta and Mn indicated the formation of solid solution, while, X-ray diffraction patterns of powders milled for 30 h showed an amorphous hump with crystalline peaks in the angular range of 35–45° in 2θ. TEM image analysis showed the presence of nanocrystalline intermetallic compounds embedded in an amorphous matrix. Mn{sub 2}Ti, MnTi and NbTi{sub 4} intermetallic compounds were detected and revealed crystallites with size ranging from 3 to 20 nm. The Gibbs free energy for the formation of solid solution and amorphous phase of three ternary systems (Ti–Nb–Ta, Ti–Nb–Mn and Ti–Ta–Mn) was calculated using extended Miedema's model. Experimental and thermodynamic data confirmed that solid solution was first formed in the alloy with 6wt% Mn followed by the formation of an amorphous phase as milling time increases. The presence of Mn promoted the formation of amorphous phase because the atomic radius difference between Mn with Ti, Nb and Ta. - Highlights: • Thermodynamics analysis of extension of solid solution of the Ti–Nb–Ta–Mn system. • Formation of amorphous phase and intermetallic compounds were observed. • Nanocrystalline intermetallic compounds were formed with the sizes between 3 and 20 nm.

  11. Quasi-experimental study designs series-paper 11: supporting the production and use of health systems research syntheses that draw on quasi-experimental study designs.

    Science.gov (United States)

    Lavis, John N; Bärnighausen, Till; El-Jardali, Fadi

    2017-09-01

    To describe the infrastructure available to support the production of policy-relevant health systems research syntheses, particularly those incorporating quasi-experimental evidence, and the tools available to support the use of these syntheses. Literature review. The general challenges associated with the available infrastructure include their sporadic nature or limited coverage of issues and countries, whereas the specific ones related to policy-relevant syntheses of quasi-experimental evidence include the lack of mechanism to register synthesis titles and scoping review protocols, the limited number of groups preparing user-friendly summaries, and the difficulty of finding quasi-experimental studies for inclusion in rapid syntheses and research syntheses more generally. Although some new tools have emerged in recent years, such as guidance workbooks and citizen briefs and panels, challenges related to using available tools to support the use of policy-relevant syntheses of quasi-experimental evidence arise from such studies potentially being harder for policymakers and stakeholders to commission and understand. Policymakers, stakeholders, and researchers need to expand the coverage and institutionalize the use of the available infrastructure and tools to support the use of health system research syntheses containing quasi-experimental evidence. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2004-11-15

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

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

    Science.gov (United States)

    Mizuuchi, Kiyoshi; Vecchiarelli, Anthony G.

    2018-05-01

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

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

    International Nuclear Information System (INIS)

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

    2004-01-01

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

  15. Final Technical Report - Use of Systems Biology Approaches to Develop Advanced Biofuel-Synthesizing Cyanobacterial Strains

    Energy Technology Data Exchange (ETDEWEB)

    Pakrasi, Himadri [Washington Univ., St. Louis, MO (United States)

    2016-09-01

    The overall objective of this project was to use a systems biology approach to evaluate the potentials of a number of cyanobacterial strains for photobiological production of advanced biofuels and/or their chemical precursors. Cyanobacteria are oxygen evolving photosynthetic prokaryotes. Among them, certain unicellular species such as Cyanothece can also fix N2, a process that is exquisitely sensitive to oxygen. To accommodate such incompatible processes in a single cell, Cyanothece produces oxygen during the day, and creates an O2-limited intracellular environment during the night to perform O2-sensitive processes such as N2-fixation. Thus, Cyanothece cells are natural bioreactors for the storage of captured solar energy with subsequent utilization at a different time during a diurnal cycle. Our studies include the identification of a novel, fast-growing, mixotrophic, transformable cyanobacterium. This strain has been sequenced and will be made available to the community. In addition, we have developed genome-scale models for a family of cyanobacteria to assess their metabolic repertoire. Furthermore, we developed a method for rapid construction of metabolic models using multiple annotation sources and a metabolic model of a related organism. This method will allow rapid annotation and screening of potential phenotypes based on the newly available genome sequences of many organisms.

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

    Science.gov (United States)

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

    2017-07-25

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

  17. 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.

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

    International Nuclear Information System (INIS)

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

    1997-01-01

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

  19. 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)

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

    Science.gov (United States)

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

    2009-05-01

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

  1. Syntheses of super-hard boron-rich solids in the B-C-N-O system

    Science.gov (United States)

    Hubert, Herve Pierre

    Alpha-rhombohedral (alpha-rh.) B-rich materials belonging to the B-C-N-O system were prepared using high-pressure, high-temperature techniques. The samples were synthesized using a multianvil device and characterized by powder X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and parallel electron energy-loss spectroscopy (PEELS). In the B-O system, the formation of BsbxO materials produced from mixtures of B and Bsb2Osb3 between 1 to 10 GPa and 1000 to 1800sp°C was investigated. Graphitic and diamond-like Bsb2O, reported in previous studies, were not detected. The refractory boron suboxide, nominally Bsb6O, which has the alpha-rh. B structure, is the dominant suboxide in the P and T range of our investigation. High-pressure techniques were used successfully to synthesize boron suboxide of improved purity and crystallinity, and less oxygen-deficient (i.e., closer to the nominal Bsb6O composition) in comparison to room-pressure syntheses. Quantitative analyses indicate compositions of Bsb6Osb{0.95} and Bsb6Osb{0.77} for high-pressure and room-pressure samples, respectively. The first preparation, between 4 to 5.5 GPa, of Bsb6O in which the preferred form of the material is as macroscopic near-perfect regular icosahedra (to 30 mum in diameter) is reported. The Bsb6O icosahedra are similar to the multiply-twinned particles observed in some cubic materials. However, a major difference is that Bsb6O has a rhombohedral structure that closely fits the geometrical requirements for obtaining icosahedral twins. The Bsb6O grains are neither 3D-periodic nor quasicrystalline. Their formation can be described as a Mackay packing of icosahedral Bsb{12} units and provides an alternative to crystal formation by propagation of translational symmetry. Icosahedral twins ranging from 20 nm to 30 mum in diameter, as well as micron-sized euhedral crystals (to 40 mum) were prepared. The structural similarity of compounds with the alpha

  2. 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.

  3. 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.

  4. Cell-Free DNA in Metastatic Colorectal Cancer: A Systematic Review and Meta-Analysis.

    Science.gov (United States)

    Spindler, Karen-Lise G; Boysen, Anders K; Pallisgård, Niels; Johansen, Julia S; Tabernero, Josep; Sørensen, Morten M; Jensen, Benny V; Hansen, Torben F; Sefrioui, David; Andersen, Rikke F; Brandslund, Ivan; Jakobsen, Anders

    2017-09-01

    systemic therapy. Small fragments of circulating cell-free DNA (cfDNA) can be measured in a simple blood sample. This report presents the first meta-analysis of the prognostic value of total cfDNA measurement in patients with metastatic colorectal cancer. Data from 1,076 patients confirmed that patients with the lowest pre-treatment levels of cfDNA had a significantly higher chance of longer survival than those with higher levels. Cell-free DNA analysis can also be used for detection of tumor-specific mutations, and hold potential as a valuable tool in colorectal cancer treatment. © AlphaMed Press 2017.

  5. Optimization and Implementation of Scaling-Free CORDIC-Based Direct Digital Frequency Synthesizer for Body Care Area Network Systems

    Directory of Open Access Journals (Sweden)

    Ying-Shen Juang

    2012-01-01

    Full Text Available Coordinate rotation digital computer (CORDIC is an efficient algorithm for computations of trigonometric functions. Scaling-free-CORDIC is one of the famous CORDIC implementations with advantages of speed and area. In this paper, a novel direct digital frequency synthesizer (DDFS based on scaling-free CORDIC is presented. The proposed multiplier-less architecture with small ROM and pipeline data path has advantages of high data rate, high precision, high performance, and less hardware cost. The design procedure with performance and hardware analysis for optimization has also been given. It is verified by Matlab simulations and then implemented with field programmable gate array (FPGA by Verilog. The spurious-free dynamic range (SFDR is over 86.85 dBc, and the signal-to-noise ratio (SNR is more than 81.12 dB. The scaling-free CORDIC-based architecture is suitable for VLSI implementations for the DDFS applications in terms of hardware cost, power consumption, SNR, and SFDR. The proposed DDFS is very suitable for medical instruments and body care area network systems.

  6. 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.

  7. 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.

  8. 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

  9. 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

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

    Science.gov (United States)

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

    2013-09-01

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

  11. A comparative study of field-emission from different one dimensional carbon nanostructures synthesized via thermal CVD system

    International Nuclear Information System (INIS)

    Jha, A.; Banerjee, D.; Chattopadhyay, K.K.

    2011-01-01

    Different one dimensional (1D) carbon nanostructures, such as carbon nanonoodles (CNNs), carbon nanospikes (CNSs) and carbon nanotubes (CNTs) have been synthesized via thermal chemical vapour deposition (TCVD) technique. The different 1D morphologies were synthesized by varying the substrate material and the deposition conditions. The as-prepared samples were characterized by X-ray diffraction (XRD), field emission scanning electron microscope (FESEM) and transmission electron microscope (TEM). FESEM and TEM images showed that the diameters of the CNNs and CNTs were ∼40 nm while the diameters of the CNSs were around 100 nm. Field emission studies of the as-prepared samples showed that CNSs to be a better field emitter than CNNs, whereas CNTs are the best among the three producing large emission current. The variation of field emission properties with inter-electrode distance has been studied in detail. Also the time dependent field emission studies of all the nanostructures have been carried out.

  12. Silica-coated quantum dots fluorescent spheres synthesized using a quaternary 'water-in-oil' microemulsion system

    International Nuclear Information System (INIS)

    Chu Maoquan; Sun Ye; Xu Shi

    2008-01-01

    Nanoscale and microscale silica spheres embedded with multiple CdSe quantum dots (QDs, having average diameters of about 2.4 and 5.0 nm, respectively.) were synthesized by using a quaternary 'water-in-oil' microemulsion. Comparing the uncoated QDs, the quantum yields (QYs) of the silica-coated QD spheres were enhanced when the QD cores were synthesized using mercaptoacetic acid (MA) as a stabilizer, while the QYs were dramatically decreased when the cores were synthesized using citric acid (CA) as a stabilizer. The enhanced QYs could be further improved by heating the silica-coated QDs in aqueous solution. Although the QYs of the silica-coated QDs were not high, these spheres emitted bright fluorescence. The silica shells contained numerous micropores (∼0.58-0.91 nm), and small amounts of toxic ions (such as Cd 2+ ) could be released from the silica spheres. However, the release rate of toxic ions from the silica spheres was significantly reduced compared with that of the uncoated QDs

  13. Lipid-protein nanodiscs for cell-free production of integral membrane proteins in a soluble and folded state: comparison with detergent micelles, bicelles and liposomes.

    Science.gov (United States)

    Lyukmanova, E N; Shenkarev, Z O; Khabibullina, N F; Kopeina, G S; Shulepko, M A; Paramonov, A S; Mineev, K S; Tikhonov, R V; Shingarova, L N; Petrovskaya, L E; Dolgikh, D A; Arseniev, A S; Kirpichnikov, M P

    2012-03-01

    Production of integral membrane proteins (IMPs) in a folded state is a key prerequisite for their functional and structural studies. In cell-free (CF) expression systems membrane mimicking components could be added to the reaction mixture that promotes IMP production in a soluble form. Here lipid-protein nanodiscs (LPNs) of different lipid compositions (DMPC, DMPG, POPC, POPC/DOPG) have been compared with classical membrane mimicking media such as detergent micelles, lipid/detergent bicelles and liposomes by their ability to support CF synthesis of IMPs in a folded and soluble state. Three model membrane proteins of different topology were used: homodimeric transmembrane (TM) domain of human receptor tyrosine kinase ErbB3 (TM-ErbB3, 1TM); voltage-sensing domain of K(+) channel KvAP (VSD, 4TM); and bacteriorhodopsin from Exiguobacterium sibiricum (ESR, 7TM). Structural and/or functional properties of the synthesized proteins were analyzed. LPNs significantly enhanced synthesis of the IMPs in a soluble form regardless of the lipid composition. A partial disintegration of LPNs composed of unsaturated lipids was observed upon co-translational IMP incorporation. Contrary to detergents the nanodiscs resulted in the synthesis of ~80% active ESR and promoted correct folding of the TM-ErbB3. None of the tested membrane mimetics supported CF synthesis of correctly folded VSD, and the protocol of the domain refolding was developed. The use of LPNs appears to be the most promising approach to CF production of IMPs in a folded state. NMR analysis of (15)N-Ile-TM-ErbB3 co-translationally incorporated into LPNs shows the great prospects of this membrane mimetics for structural studies of IMPs produced by CF systems. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. Analysis of signal transduction in cell-free extracts and rafts of Xenopus eggs.

    Science.gov (United States)

    Tokmakov, Alexander A; Iwasaki, Tetsushi; Sato, Ken-Ichi; Fukami, Yasuo

    2010-05-01

    Intracellular signaling during egg activation/fertilization has been extensively studied using intact eggs, which can be manipulated by microinjection of different mRNAs, proteins, or chemical drugs. Furthermore, egg extracts, which retain high CSF activity (CSF-arrested extracts), were developed for studying fertilization/activation signal transduction, which have significant advantages as a model system. The addition of calcium to CSF-arrested extracts initiates a plethora of signaling events that take place during egg activation. Hence, the signaling downstream of calcium mobilization has been successfully studied in the egg extracts. Moreover, despite disruption of membrane-associated signaling compartments and ordered compartmentalization during extract preparation, CSF-arrested extracts can be successfully used to study early signaling events, which occur upstream of calcium release during egg activation/fertilization. In combination with the CSF-arrested extracts, activated egg rafts can reproduce some events of egg activation, including PLCgamma activation, IP3 production, transient calcium release, MAPK inactivation, and meiotic exit. This becomes possible due to complementation of the sperm-induced egg activation signaling machinery present in the rafts with the components of signal transduction system localized in the extracts. Herein, we describe protocols for studying molecular mechanisms of egg fertilization/activation using cell-free extracts and membrane rafts prepared from metaphase-arrested Xenopus eggs.

  15. 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.

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

    Science.gov (United States)

    Baltar, Federico

    2018-01-01

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

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

    Science.gov (United States)

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

    2018-01-02

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

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

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

    Science.gov (United States)

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

    2017-10-25

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

  20. 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.

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

    Science.gov (United States)

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

    2015-09-11

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

  2. Effect of hydrogen on the growth and morphology of single wall carbon nanotubes synthesized on a Fe-Mo/MgO catalytic system

    Energy Technology Data Exchange (ETDEWEB)

    Biris, Alexandru R. [National Institute for Research and Development of Isotopic and Molecular Technologies, Cluj Napoca, RO-3400 (Romania)], E-mail: biris@oc1.itim-cj.ro; Li Zhongrui; Dervishi, Enkeleda [Applied Science Department, University of Arkansas at Little Rock, 2801 S. University Ave, Little Rock, AR 72204 (United States); Nanotechnology Center, University of Arkansas at Little Rock, 2801 S. University Ave, Little Rock, AR 72204 (United States); Lupu, Dan [National Institute for Research and Development of Isotopic and Molecular Technologies, Cluj Napoca, RO-3400 (Romania); Xu Yang; Saini, Viney [Applied Science Department, University of Arkansas at Little Rock, 2801 S. University Ave, Little Rock, AR 72204 (United States); Nanotechnology Center, University of Arkansas at Little Rock, 2801 S. University Ave, Little Rock, AR 72204 (United States); Watanabe, Fumiya [Nanotechnology Center, University of Arkansas at Little Rock, 2801 S. University Ave, Little Rock, AR 72204 (United States); Biris, Alexandru S. [Applied Science Department, University of Arkansas at Little Rock, 2801 S. University Ave, Little Rock, AR 72204 (United States); Nanotechnology Center, University of Arkansas at Little Rock, 2801 S. University Ave, Little Rock, AR 72204 (United States)], E-mail: asbiris@ualr.edu

    2008-04-21

    Single wall carbon nanotubes were synthesized from thermal pyrolysis of methane on a Fe-Mo/MgO catalyst by radio frequency catalytic chemical vapor deposition (RF-CVD) using argon as a carrier gas. Controlled amounts of hydrogen (H{sub 2}/CH{sub 4}=0-1 v/v) were introduced in separate experiments along with the carbon source. The properties and morphology of the synthesized single wall carbon nanotubes were monitored by transmission electron microscopy, Raman scattering, and thermogravimetric analysis. The nanotubes with the highest crystallinity were obtained with H{sub 2}/CH{sub 4}=0.6. By monitoring the Radial Breathing Modes present in the Raman spectra of the single-wall carbon nanotube samples, the variation of the structural and morphological properties of the carbon nanotubes with the flow level of hydrogen, reflect changes of the catalyst systems induced by the presence of hydrogen.

  3. Catalytic reduction of 4-nitrophenol using gold nanoparticles biosynthesized by cell-free extracts of Aspergillus sp. WL-Au

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Wenli; Qu, Yuanyuan, E-mail: qyy@dlut.edu.cn; Pei, Xiaofang; Li, Shuzhen; You, Shengnan; Wang, Jingwei; Zhang, Zhaojing; Zhou, Jiti

    2017-01-05

    Highlights: • A green process for AuNPs synthesis was achieved by fungus Aspergillus. • Uniform spherical AuNPs with well dispersity and stability were biosynthesized. • The biogenic AuNPs possessed remarkable catalytic activities for 4-NP reduction. - Abstract: A facile one-pot eco-friendly process for synthesis of gold nanoparticles (AuNPs) with high catalytic activity was achieved using cell-free extracts of Aspergillus sp. WL-Au as reducing, capping and stabilizing agents. The surface plasmon resonance band of UV–vis spectrum at 532 nm confirmed the presence of AuNPs. Transmission electron microscopy images showed that quite uniform spherical AuNPs were synthesized and the average size of nanoparticles increased from 4 nm to 29 nm with reaction time. X-ray diffraction analysis verified the formation of nano-crystalline gold particles. Fourier transform infrared spectra showed the presence of functional groups on the surface of biosynthesized AuNPs, such as O−H, N−H, C=O, C−H, C−OH and C−O−C groups, which increased the stability of AuNPs. The biogenic AuNPs could serve as a highly efficient catalyst for 4-nitrophenol reduction. The reaction rate constant was linearly correlated with the concentration of AuNPs, which increased from 0.59 min{sup −1} to 1.51 min{sup −1} with the amount of AuNPs increasing form 1.46 × 10{sup −6} to 17.47 × 10{sup −6} mmol. Moreover, the as-synthesized AuNPs exhibited a remarkable normalized catalytic activity (4.04 × 10{sup 5} min{sup −1} mol{sup −1}), which was much higher than that observed for AuNPs synthesized by other biological and conventional chemical methods.

  4. Catalytic reduction of 4-nitrophenol using gold nanoparticles biosynthesized by cell-free extracts of Aspergillus sp. WL-Au

    International Nuclear Information System (INIS)

    Shen, Wenli; Qu, Yuanyuan; Pei, Xiaofang; Li, Shuzhen; You, Shengnan; Wang, Jingwei; Zhang, Zhaojing; Zhou, Jiti

    2017-01-01

    Highlights: • A green process for AuNPs synthesis was achieved by fungus Aspergillus. • Uniform spherical AuNPs with well dispersity and stability were biosynthesized. • The biogenic AuNPs possessed remarkable catalytic activities for 4-NP reduction. - Abstract: A facile one-pot eco-friendly process for synthesis of gold nanoparticles (AuNPs) with high catalytic activity was achieved using cell-free extracts of Aspergillus sp. WL-Au as reducing, capping and stabilizing agents. The surface plasmon resonance band of UV–vis spectrum at 532 nm confirmed the presence of AuNPs. Transmission electron microscopy images showed that quite uniform spherical AuNPs were synthesized and the average size of nanoparticles increased from 4 nm to 29 nm with reaction time. X-ray diffraction analysis verified the formation of nano-crystalline gold particles. Fourier transform infrared spectra showed the presence of functional groups on the surface of biosynthesized AuNPs, such as O−H, N−H, C=O, C−H, C−OH and C−O−C groups, which increased the stability of AuNPs. The biogenic AuNPs could serve as a highly efficient catalyst for 4-nitrophenol reduction. The reaction rate constant was linearly correlated with the concentration of AuNPs, which increased from 0.59 min −1 to 1.51 min −1 with the amount of AuNPs increasing form 1.46 × 10 −6 to 17.47 × 10 −6 mmol. Moreover, the as-synthesized AuNPs exhibited a remarkable normalized catalytic activity (4.04 × 10 5 min −1 mol −1 ), which was much higher than that observed for AuNPs synthesized by other biological and conventional chemical methods.

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

    OpenAIRE

    Roberta Troia; Massimo Giunti; Stefano Calipa; Robert Goggs

    2018-01-01

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

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

    Science.gov (United States)

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

    2017-05-01

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

  7. Reaction Mechanisms and Structural and Physicochemical Properties of Caffeic Acid Grafted Chitosan Synthesized in Ascorbic Acid and Hydroxyl Peroxide Redox System.

    Science.gov (United States)

    Liu, Jun; Pu, Huimin; Chen, Chong; Liu, Yunpeng; Bai, Ruyu; Kan, Juan; Jin, Changhai

    2018-01-10

    The ascorbic acid (AA) and hydroxyl peroxide (H 2 O 2 ) redox pair induced free radical grafting reaction is a promising approach to conjugate phenolic groups with chitosan (CS). In order to reveal the exact mechanisms of the AA/H 2 O 2 redox pair induced grafting reaction, free radicals generated in the AA/H 2 O 2 redox system were compared with hydroxyl radical ( • OH) produced in the Fe 2+ /H 2 O 2 redox system. Moreover, the structural and physicochemical properties of caffeic acid grafted CS (CA-g-CS) synthesized in these two redox systems were compared. Results showed that only ascorbate radical (Asc •- ) was produced in the AA/H 2 O 2 system. The reaction between Asc •- and CS produced novel carbon-centered radicals, whereas no new free radicals were detected when • OH reacted with CS. Thin layer chromatography, UV-vis, Fourier transform infrared, and nuclear magnetic resonance spectroscopic analyses all confirmed that CA was successfully grafted onto CS through Asc •- . However, CA could be hardly grafted onto CS via • OH. CA-g-CS synthesized through Asc •- exhibited lower thermal stability and crystallinity than the reaction product obtained through • OH. For the first time, our results demonstrated that the synthesis of CA-g-CS in the AA/H 2 O 2 redox system was mediated by Asc •- rather than • OH.

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

    Science.gov (United States)

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

    2017-11-01

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

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

    Science.gov (United States)

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

    2017-05-01

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

  10. A fractional-N frequency synthesizer-based multi-standard I/Q carrier generation system in 0.13 μm CMOS

    International Nuclear Information System (INIS)

    Lou Wenfeng; Geng Zhiqing; Feng Peng; Wu Nanjian

    2011-01-01

    This paper proposes a sigma-delta fractional-N frequency synthesizer-based multi-standard I/Q carrier generation system. With reasonable frequency planning, the system can be used in multi-standard wireless communication applications (GSM, WCDMA, GPRS, TD-SCDMA, WLAN (802.11a/b/g)). The implementation is achieved by a 0.13 μm RF CMOS process. The measured results demonstrate that three quadrature VCOs (QVCO) continuously cover the frequency from 3.1 to 6.1 GHz (65.2%), and through the successive divide-by-2 prescalers to achieve the frequency from 0.75 to 6.1 GHz continuously. The chip was fully integrated with the exception of an off-chip filter. The entire chip area is only 3.78 mm 2 , and the system consumes a 21.7 mA - 1.2 V supply without output buffers. The lock-in time of the PLL frequency synthesizer is less than 4 μs over the entire frequency range with a direct frequency presetting technique and the auxiliary non-volatile memory (NVM) can store the digital configuration signal of the system, including presetting signals to avoid the calibration process case by case. (semiconductor integrated circuits)

  11. A fractional-N frequency synthesizer-based multi-standard I/Q carrier generation system in 0.13 {mu}m CMOS

    Energy Technology Data Exchange (ETDEWEB)

    Lou Wenfeng; Geng Zhiqing; Feng Peng; Wu Nanjian, E-mail: nanjian@semi.ac.cn [Institute of Semiconductors, Chinese Academy of Sciences, Beijing 100083 (China)

    2011-06-15

    This paper proposes a sigma-delta fractional-N frequency synthesizer-based multi-standard I/Q carrier generation system. With reasonable frequency planning, the system can be used in multi-standard wireless communication applications (GSM, WCDMA, GPRS, TD-SCDMA, WLAN (802.11a/b/g)). The implementation is achieved by a 0.13 {mu}m RF CMOS process. The measured results demonstrate that three quadrature VCOs (QVCO) continuously cover the frequency from 3.1 to 6.1 GHz (65.2%), and through the successive divide-by-2 prescalers to achieve the frequency from 0.75 to 6.1 GHz continuously. The chip was fully integrated with the exception of an off-chip filter. The entire chip area is only 3.78 mm{sup 2}, and the system consumes a 21.7 mA - 1.2 V supply without output buffers. The lock-in time of the PLL frequency synthesizer is less than 4 {mu}s over the entire frequency range with a direct frequency presetting technique and the auxiliary non-volatile memory (NVM) can store the digital configuration signal of the system, including presetting signals to avoid the calibration process case by case. (semiconductor integrated circuits)

  12. Magnetic and structural properties of Cu0.85Fe0.15O system synthesized by co-precipitation

    International Nuclear Information System (INIS)

    Colorado, H. D.; Pérez Alcázar, G. A.

    2011-01-01

    Cu 0.94 Fe 0.06 O and Cu 0.85 Fe 0.15 O samples were synthesized by using the co-precipitation chemical method. Starting from aqueous solutions of copper nitrate, CuO (NO 3 ) 2 3H 2 O, iron nitrate, Fe (NO 3 ) 3 9H 2 O and sodium hydroxide as precipitating agent, NaOH. The precipitate of three samples for Cu 0.94 Fe 0.06 O and five for Cu 0.85 Fe 0.15 O of fine powder were calcined for 5 h at different temperatures. The obtained X rays diffraction patterns refined by the Rietveld method show the CuO characteristic pattern, showing that the Fe atoms enter to replace Cu atoms. Furthermore, it was obtained that the crystallite size decreases with calcination temperatures for Cu 0.94 Fe 0.06 O. The transmission Mössbauer spectroscopy showed that the samples present a disordered paramagnetic behavior due to the big value of the half-width of line of the quadrupolar splitting. Vibrating sample magnetometry confirms the paramagnetic character. The XRD results indicate that the material is nanostructured, due that the crystallite sizes are of the order of 10 nm for Cu 0.94 Fe 0.06 O and 40 nm for Cu 0.85 Fe 0.15 O.

  13. 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.

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

    NARCIS (Netherlands)

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

    2014-01-01

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

  15. 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

  16. Cell-free DNA in healthy individuals, noncancerous disease and strong prognostic value in colorectal cancer

    DEFF Research Database (Denmark)

    Spindler, Karen-Lise Garm; Appelt, Ane L; Pallisgaard, Niels

    2014-01-01

    The purpose was to investigate total cell-free DNA (cfDNA) in colorectal cancer (CRC) patients during treatment with second-line chemotherapy and in healthy controls and patients with different comorbidities. Patient treated with second-line irinotecan for metastatic CRC (n = 100), a cohort...

  17. 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.

  18. Plasma cell-free mitochondrial DNA declines in response to prolonged moderate aerobic exercise.

    Science.gov (United States)

    Shockett, Penny E; Khanal, Januka; Sitaula, Alina; Oglesby, Christopher; Meachum, William A; Castracane, V Daniel; Kraemer, Robert R

    2016-01-01

    Increased plasma cell-free mitochondrial DNA (cf-mDNA), a damage-associated molecular pattern (DAMP) produced by cellular injury, contributes to neutrophil activation/inflammation in trauma patients and arises in cancer and autoimmunity. To further understand relationships between cf-mDNA released by tissue injury, inflammation, and health benefits of exercise, we examined cf-mDNA response to prolonged moderate aerobic exercise. Seven healthy moderately trained young men (age = 22.4 ± 1.2) completed a treadmill exercise trial for 90 min at 60% VO2 max and a resting control trial. Blood was sampled immediately prior to exercise (0 min = baseline), during (+18, +54 min), immediately after (+90 min), and after recovery (R40). Plasma was analyzed for cf-mDNA, IL-6, and lactate. A significant difference in cf-mDNA response was observed between exercise and control trials, with cf-mDNA levels reduced during exercise at +54 and +90 (with or without plasma volume shift correction). Declines in cf-mDNA were accompanied by increased lactate and followed by an increase in IL-6, suggesting a temporal association with muscle stress and inflammatory processes. Our novel finding of cf-mDNA decline with prolonged moderate treadmill exercise provides evidence for increased clearance from or reduced release of cf-mDNA into the blood with prolonged exercise. These studies contrast with previous investigations involving exhaustive short-term treadmill exercise, in which no change in cf-mDNA levels were reported, and contribute to our understanding of differences between exercise- and trauma-induced inflammation. We propose that transient declines in cf-mDNA may induce health benefits, by reducing systemic inflammation. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  19. 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.

  20. 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.

  1. Fe-N-C electrocatalysts for oxygen reduction reaction synthesized by using aniline salt and Fe3+/H2O2 catalytic system

    KAUST Repository

    Bukola, Saheed; Merzougui, Belabbes A.; Akinpelu, Akeem; Laoui, Tahar; Hedhili, Mohamed N.; Swain, Greg M.; Shao, Minhua

    2014-01-01

    Non-precious metal (NPM) catalysts are synthesized by polymerizing aniline salt using an aqueous Fe3+/H2O2 coupled catalytic system on a carbon matrix with a porous creating agent. The sulfur containing compunds such as ammonium peroxydisulfate, are eliminated in this method resulting in a much simpler process. The catalysts' porous structures are enhanced with ammonium carbonate as a sacrificial material that yields voids when decomposed during the heat treatment at 900 °C in N2 atmosphere. Two catalysts Fe-N-C/Vu and Fe-N-C/KB (Vu = Vulcan and KB = Ketjen black) were synthesized and characterized. Their oxygen reduction reaction (ORR) activities were investigated using a rotating ring-disk electrode (RRDE) in both 0.1 M KOH and 0.1 M HClO4. The catalysts show improved ORR activities close to that of Pt-based catalysts, low H2O2 formation and also demonstrated a remarkable tolerance towards methanol oxidation.

  2. Fe-N-C electrocatalysts for oxygen reduction reaction synthesized by using aniline salt and Fe3+/H2O2 catalytic system

    KAUST Repository

    Bukola, Saheed

    2014-11-01

    Non-precious metal (NPM) catalysts are synthesized by polymerizing aniline salt using an aqueous Fe3+/H2O2 coupled catalytic system on a carbon matrix with a porous creating agent. The sulfur containing compunds such as ammonium peroxydisulfate, are eliminated in this method resulting in a much simpler process. The catalysts\\' porous structures are enhanced with ammonium carbonate as a sacrificial material that yields voids when decomposed during the heat treatment at 900 °C in N2 atmosphere. Two catalysts Fe-N-C/Vu and Fe-N-C/KB (Vu = Vulcan and KB = Ketjen black) were synthesized and characterized. Their oxygen reduction reaction (ORR) activities were investigated using a rotating ring-disk electrode (RRDE) in both 0.1 M KOH and 0.1 M HClO4. The catalysts show improved ORR activities close to that of Pt-based catalysts, low H2O2 formation and also demonstrated a remarkable tolerance towards methanol oxidation.

  3. A novel prosodic-information synthesizer based on recurrent fuzzy neural network for the Chinese TTS system.

    Science.gov (United States)

    Lin, Chin-Teng; Wu, Rui-Cheng; Chang, Jyh-Yeong; Liang, Sheng-Fu

    2004-02-01

    In this paper, a new technique for the Chinese text-to-speech (TTS) system is proposed. Our major effort focuses on the prosodic information generation. New methodologies for constructing fuzzy rules in a prosodic model simulating human's pronouncing rules are developed. The proposed Recurrent Fuzzy Neural Network (RFNN) is a multilayer recurrent neural network (RNN) which integrates a Self-cOnstructing Neural Fuzzy Inference Network (SONFIN) into a recurrent connectionist structure. The RFNN can be functionally divided into two parts. The first part adopts the SONFIN as a prosodic model to explore the relationship between high-level linguistic features and prosodic information based on fuzzy inference rules. As compared to conventional neural networks, the SONFIN can always construct itself with an economic network size in high learning speed. The second part employs a five-layer network to generate all prosodic parameters by directly using the prosodic fuzzy rules inferred from the first part as well as other important features of syllables. The TTS system combined with the proposed method can behave not only sandhi rules but also the other prosodic phenomena existing in the traditional TTS systems. Moreover, the proposed scheme can even find out some new rules about prosodic phrase structure. The performance of the proposed RFNN-based prosodic model is verified by imbedding it into a Chinese TTS system with a Chinese monosyllable database based on the time-domain pitch synchronous overlap add (TD-PSOLA) method. Our experimental results show that the proposed RFNN can generate proper prosodic parameters including pitch means, pitch shapes, maximum energy levels, syllable duration, and pause duration. Some synthetic sounds are online available for demonstration.

  4. Transport, Thermal, and Magnetic Properties of YbNi3X9 (X = Al, Ga): A Newly Synthesized Yb-Based Kondo Lattice System

    Science.gov (United States)

    Yamashita, Tetsuro; Miyazaki, Ryoichi; Aoki, Yuji; Ohara, Shigeo

    2012-03-01

    We have succeeded in synthesizing a new Yb-based Kondo lattice system, YbNi3X9 (X = Al, Ga). Our study reveals that YbNi3Al9 shows typical features of a heavy-fermion antiferromagnet with a Néel temperature of TN = 3.4 K. All of the properties reflect a competition between the Kondo effect and the crystalline electric field (CEF) effect. The moderate heavy-fermion state leads to an enhanced Sommerfeld coefficient of 100 mJ/(mol\\cdotK2), even if ordered antiferromagnetically. On the other hand, the isostructural gallide YbNi3Ga9 is an intermediate-valence system with a Kondo temperature of TK = 570 K. A large hybridization scale can overcome the CEF splitting energy, and a moderately heavy Fermi-liquid ground state with high local moment degeneracy should form at low temperatures. Note that the quality of single-crystalline YbNi3X9 is extremely high compared with those of other Yb-based Kondo lattice compounds. We conclude that YbNi3X9 is a suitable system for investigating the electronic structure of Yb-based Kondo lattice systems from a heavy-fermion system with an antiferromagnetically ordered ground state to an intermediate-valence system.

  5. Positive effects of cell-free porous PLGA implants and early loading exercise on hyaline cartilage regeneration in rabbits.

    Science.gov (United States)

    Chang, Nai-Jen; Lin, Chih-Chan; Shie, Ming-You; Yeh, Ming-Long; Li, Chien-Feng; Liang, Peir-In; Lee, Kuan-Wei; Shen, Pei-Hsun; Chu, Chih-Jou

    2015-12-01

    The regeneration of hyaline cartilage remains clinically challenging. Here, we evaluated the therapeutic effects of using cell-free porous poly(lactic-co-glycolic acid) (PLGA) graft implants (PGIs) along with early loading exercise to repair a full-thickness osteochondral defect. Rabbits were randomly allocated to a treadmill exercise (TRE) group or a sedentary (SED) group and were prepared as either a PGI model or an empty defect (ED) model. TRE was performed as a short-term loading exercise; SED was physical inactivity in a free cage. The knees were evaluated at 6 and 12 weeks after surgery. At the end of testing, none of the knees developed synovitis, formed osteophytes, or became infected. Macroscopically, the PGI-TRE group regenerated a smooth articular surface, with transparent new hyaline-like tissue soundly integrated with the neighboring cartilage, but the other groups remained distinct at the margins with fibrous or opaque tissues. In a micro-CT analysis, the synthesized bone volume/tissue volume (BV/TV) was significantly higher in the PGI-TRE group, which also had integrating architecture in the regeneration site. The thickness of the trabecular (subchondral) bone was improved in all groups from 6 to 12 weeks. Histologically, remarkable differences in the cartilage regeneration were visible. At week 6, compared with SED groups, the TRE groups manifested modest inflammatory cells with pro-inflammatory cytokines (i.e., TNF-α and IL-6), improved collagen alignment and higher glycosaminoglycan (GAG) content, particularly in the PGI-TRE group. At week 12, the PGI-TRE group had the best regeneration outcomes, showing the formation of hyaline-like cartilage, the development of columnar rounded chondrocytes that expressed enriched levels of collagen type II and GAG, and functionalized trabecular bone with osteocytes. In summary, the combination of implanting cell-free PLGA and performing an early loading exercise can significantly promote the full

  6. System Development from Organic Solvents to Ionic Liquids for Synthesiz-ing Ascorbyl Esters with Conjugated Linoleic Acids

    DEFF Research Database (Denmark)

    Yang, Zhiyong; Schultz, Lise; Guo, Zheng

    2012-01-01

    . Results show that only Novozym® 435 turned out to be a useful enzymatic preparation for the production of ascorbyl-CLA ester. The optimum reaction conditions in the or-ganic solvent system were 4 h at 55°C and at a molar ratio of 5 (CLA/ascorbic acid). The esterification reaction was trans......-ferred to an ionic liquid system for the purpose of improving solubility of the polar substrate and avoiding the application of organic solvents. From screening experiments, it was evident that only methyltrioctylammonium triflouroacetate (tO-MA·TFA) could provide a proper reaction environment for production...... of ascorbyl-CLA ester when using Novozym® 435 as biocatalyst. It was possible to significantly increase the productivity (150 g/l) through the increase of ascorbic acid sol-ubility in ionic liquids by super saturation together with the increase of reaction temperature to 70°C, far beyond than that in organic...

  7. 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.

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

    Science.gov (United States)

    2015-03-03

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

  9. 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.

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

    Science.gov (United States)

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

    2015-01-01

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

  11. Incorporation of [1-C14] Isopentenyl Pyrophosphate into Carotenoids and Homo carotenoids using a Cell-free Preparation of Micrococcus Luteus

    International Nuclear Information System (INIS)

    Al-Wandawi, H.

    1998-01-01

    The early steps up to the formation of acyclic unsaturated carotenes (e.g.,phytoene to lycopene) are presumed to be common to the biosynthesis of all carotenoids with 40 or more carbon atoms, nevertheless, no direct evidence so far available to confirm this for homo carotenoids (c 45 and c 50 carotenoids). In the present study, an active cell-free preparation was obtained from diphenylamine-inhibited cells of Micrococcus Iuteus and found to be capable to incorporate radioactivity from Isopentenyl pyrophosphate (labelled with C-14)into carotenoids and homo carotenoids, providing for the first time a direct evidence which suggests that both carotenoids and homo carotenoids are sharing the same biological origin. Furthermore, the technique developed in this study may be considered as a valuable method for preparation of biological-active labelled compounds which may have some advantages over conventional chemical syntheses methods

  12. Synthesizing diverse stakeholder needs for a drought early warning information system in the Apalachicola-Chattahoochee-Flint River Basin

    Science.gov (United States)

    Darby, L. S.; Mcnutt, C. A.; Ingram, K.; Knox, P.; Martinez, C. J.; Zierden, D.; Pulwarty, R. S.; Verdin, J. P.

    2011-12-01

    From fall 2009 to fall 2010 the National Integrated Drought Information System (NIDIS) Program Office coordinated several stakeholder meetings in the Apalachicola-Chattahoochee-Flint (ACF) River Basin, which extends from Georgia into Alabama and Florida. The purpose of the meetings was to ascertain which products and services are needed by basin stakeholders for drought early warning. Drought vulnerabilities across the basin are quite diverse - from changes in salinity that harm oyster bed productivity in Apalachicola Bay, to the health of crops in the agricultural fields of the Flint River basin, to municipal water supply issues for the city of Atlanta and smaller communities along the tributaries. These, and many other vulnerabilities, exist against a backdrop of decades-long water allocation litigation among the three states. The benefits of these stakeholder meetings went beyond information gathering by serving as opportunities for communication across state lines among people with differing needs and perspectives regarding water management decisions in the basin. The meetings also provided a good opportunity for stakeholders from all three states to share lessons learned from various management perspectives during the drought that affected the basin from 2006 to 2009. Common issues and needs identified from all regions of the basin include: (1) Education and Communication - People across the basin agree that education and communication regarding drought needs improvement (e.g., definition of drought, sector-specific impacts); (2) Improved interactions with the US Army Corps of Engineers (e.g., increased data sharing and opportunities for communication between the Corps and other stakeholders); (3) Data - easier access to real-time calibrated and quality-controlled data; (4) ACF Basin-wide webinars and climate outlooks; (5) Drought Index - Can a basin-wide drought index be established?; (6) Resolve perceived discrepancies regarding groundwater (How much

  13. 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.

  14. Reviews and syntheses: guiding the evolution of the observing system for the carbon cycle through quantitative network design

    Science.gov (United States)

    Kaminski, Thomas; Rayner, Peter Julian

    2017-10-01

    Various observational data streams have been shown to provide valuable constraints on the state and evolution of the global carbon cycle. These observations have the potential to reduce uncertainties in past, current, and predicted natural and anthropogenic surface fluxes. In particular such observations provide independent information for verification of actions as requested by the Paris Agreement. It is, however, difficult to decide which variables to sample, and how, where, and when to sample them, in order to achieve an optimal use of the observational capabilities. Quantitative network design (QND) assesses the impact of a given set of existing or hypothetical observations in a modelling framework. QND has been used to optimise in situ networks and assess the benefit to be expected from planned space missions. This paper describes recent progress and highlights aspects that are not yet sufficiently addressed. It demonstrates the advantage of an integrated QND system that can simultaneously evaluate a multitude of observational data streams and assess their complementarity and redundancy.

  15. Reviews and syntheses: Anthropogenic perturbations to carbon fluxes in Asian river systems – concepts, emerging trends, and research challenges

    Directory of Open Access Journals (Sweden)

    J.-H. Park

    2018-05-01

    Full Text Available Human activities are drastically altering water and material flows in river systems across Asia. These anthropogenic perturbations have rarely been linked to the carbon (C fluxes of Asian rivers that may account for up to 40–50 % of the global fluxes. This review aims to provide a conceptual framework for assessing the human impacts on Asian river C fluxes, along with an update on anthropogenic alterations of riverine C fluxes. Drawing on case studies conducted in three selected rivers (the Ganges, Mekong, and Yellow River and other major Asian rivers, the review focuses on the impacts of river impoundment and pollution on CO2 outgassing from the rivers draining South, Southeast, and East Asian regions that account for the largest fraction of river discharge and C exports from Asia and Oceania. A critical examination of major conceptual models of riverine processes against observed trends suggests that to better understand altered metabolisms and C fluxes in anthropogenic land-water-scapes, or riverine landscapes modified by human activities, the traditional view of the river continuum should be complemented with concepts addressing spatial and temporal discontinuities created by human activities, such as river impoundment and pollution. Recent booms in dam construction on many large Asian rivers pose a host of environmental problems, including increased retention of sediment and associated C. A small number of studies that measured greenhouse gas (GHG emissions in dammed Asian rivers have reported contrasting impoundment effects: decreased GHG emissions from eutrophic reservoirs with enhanced primary production vs. increased emissions from the flooded vegetation and soils in the early years following dam construction or from the impounded reaches and downstream estuaries during the monsoon period. These contrasting results suggest that the rates of metabolic processes in the impounded and downstream reaches can vary greatly longitudinally

  16. Reviews and syntheses: Anthropogenic perturbations to carbon fluxes in Asian river systems - concepts, emerging trends, and research challenges

    Science.gov (United States)

    Park, Ji-Hyung; Nayna, Omme K.; Begum, Most S.; Chea, Eliyan; Hartmann, Jens; Keil, Richard G.; Kumar, Sanjeev; Lu, Xixi; Ran, Lishan; Richey, Jeffrey E.; Sarma, Vedula V. S. S.; Tareq, Shafi M.; Xuan, Do Thi; Yu, Ruihong

    2018-05-01

    Human activities are drastically altering water and material flows in river systems across Asia. These anthropogenic perturbations have rarely been linked to the carbon (C) fluxes of Asian rivers that may account for up to 40-50 % of the global fluxes. This review aims to provide a conceptual framework for assessing the human impacts on Asian river C fluxes, along with an update on anthropogenic alterations of riverine C fluxes. Drawing on case studies conducted in three selected rivers (the Ganges, Mekong, and Yellow River) and other major Asian rivers, the review focuses on the impacts of river impoundment and pollution on CO2 outgassing from the rivers draining South, Southeast, and East Asian regions that account for the largest fraction of river discharge and C exports from Asia and Oceania. A critical examination of major conceptual models of riverine processes against observed trends suggests that to better understand altered metabolisms and C fluxes in anthropogenic land-water-scapes, or riverine landscapes modified by human activities, the traditional view of the river continuum should be complemented with concepts addressing spatial and temporal discontinuities created by human activities, such as river impoundment and pollution. Recent booms in dam construction on many large Asian rivers pose a host of environmental problems, including increased retention of sediment and associated C. A small number of studies that measured greenhouse gas (GHG) emissions in dammed Asian rivers have reported contrasting impoundment effects: decreased GHG emissions from eutrophic reservoirs with enhanced primary production vs. increased emissions from the flooded vegetation and soils in the early years following dam construction or from the impounded reaches and downstream estuaries during the monsoon period. These contrasting results suggest that the rates of metabolic processes in the impounded and downstream reaches can vary greatly longitudinally over time as a

  17. Interdiffusion within model TiN/Cu and TiTaN/Cu systems synthesized by combinatorial thin film deposition

    International Nuclear Information System (INIS)

    Mühlbacher, M.

    2015-01-01

    Continued device miniaturization in microelectronics calls for a fundamental understanding of diffusion processes and damage mechanisms in the Cu metallization/TiN barrier layer system. Thus, the starting point of the present study is a combined experimental and theoretical examination of lattice diffusion in ideal single-crystal TiN/Cu stacks grown on MgO(001) by unbalanced DC magnetron sputter deposition. After a 12 h annealing treatment at 1000 °C, a uniform Cu diffusion layer of 7-12 nm is observed by scanning transmission electron microscopy and atom probe tomography (APT). Density-functional theory calculations predict a stoichiometry-dependent atomic diffusion mechanism of Cu in bulk TiN, with Cu diffusing on the N sublattice for the experimental N/Ti ratio of 0.92. These findings are extended to a comparison of grain boundary diffusion of Cu in dense polycrystalline TiN sputter-deposited on Si at 700 °C and underdense polycrystalline TiN grown on Si without external substrate heating. While the Cu diffusion path along dense TiN grain boundaries can be restricted to approximately 30 nm after a 1 h annealing treatment at 900 °C as visualized by 3D APT reconstructions, it already exceeds 500 nm after annealing at 700 °C in the underdense low-temperature TiN barrier. In this case, the formation of the Cu3Si phase, which characteristically grows along the close-packed directions in Si, is identified as the main damage mechanism leading to complete barrier failure. To meet the low-temperature processing needs of semiconductor industry and at the same time exploit the improved performance of dense polycrystalline barrier layers, deposition of TiTaN barriers on Si is demonstrated by a reactive hybrid high-power impulse/DC magnetron sputtering process, where barrier densification is achieved by pulsed irradiation of the growth surface with only a few at.% of energetic Ta ions without external substrate heating. These barrier layers delay the onset of Cu grain

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

    Science.gov (United States)

    Chen, Angela; Contreras, Lydia M; Keitz, Benjamin K

    2017-09-15

    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 organisms

  19. PULSE SYNTHESIZING GENERATOR

    Science.gov (United States)

    Kerns, Q.A.

    1963-08-01

    >An electronlc circuit for synthesizing electrical current pulses having very fast rise times includes several sinewave generators tuned to progressively higher harmonic frequencies with signal amplitudes and phases selectable according to the Fourier series of the waveform that is to be synthesized. Phase control is provided by periodically triggering the generators at precisely controlled times. The outputs of the generators are combined in a coaxial transmission line. Any frequency-dependent delays that occur in the transmission line can be readily compensated for so that the desired signal wave shape is obtained at the output of the line. (AEC)

  20. 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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1983-12-01

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

  2. Souper: A Synthesizing Superoptimizer

    OpenAIRE

    Sasnauskas, Raimondas; Chen, Yang; Collingbourne, Peter; Ketema, Jeroen; Lup, Gratian; Taneja, Jubi; Regehr, John

    2017-01-01

    If we can automatically derive compiler optimizations, we might be able to sidestep some of the substantial engineering challenges involved in creating and maintaining a high-quality compiler. We developed Souper, a synthesizing superoptimizer, to see how far these ideas might be pushed in the context of LLVM. Along the way, we discovered that Souper's intermediate representation was sufficiently similar to the one in Microsoft Visual C++ that we applied Souper to that compiler as well. Shipp...

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

    Science.gov (United States)

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

    2017-07-26

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

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

    Science.gov (United States)

    Raik, Shalini; Kumar, Ajay; Bhattacharyya, Shalmoli

    2018-03-01

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

  5. 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.

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

    Science.gov (United States)

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

    2008-12-01

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

  7. Formation of Lignans(-)-Secoisolariciresinol and (-)-Matairesinol with Forsythia intermedia Cell-Free Extracts

    Science.gov (United States)

    Umezawa, Toshiaki; Davin, Laurence B.; Lewis, Norman G.

    1991-01-01

    In vivo labeling experiments of Forsythia intermedia plant tissue with [8-(C-14)]- and [9,9-(2)H2,OC(2)H3]coniferyl alcohols revealed that the lignans, (-)-secoisolariciresinol and (-)-matairesinol, were derived from two coniferyl alcohol molecules; no evidence for the formation of the corresponding (+)-enantiomers was found. Administration of (+/-)-[Ar-(H-3)] secoisolariciresinols to excised shoots of F.intermedia resulted in a significant conversion into (-)-matairesinol; again, the (+)-antipode was not detected. Experiments using cell-free extracts of F.intermedia confirmed and extended these findings. In the presence of NAD(P)H and H2O2, the cell-free extracts catalyzed the formation of (-)- secoisolariciresinol, with either [8-(C-14)]- or [9,9-(2)H2,OC(2)H3]coniferyl alcohols as substrates. The (+)- enantiomer was not formed. Finally, when either (-)-[Ar-(H-3)] or (+/-)-[Ar-(H-2)]secoisolariciresinols were used as substrates, in the presence of NAD(P), only (-)- and not (+)-matairesinol formation occurred. The other antipode, (+)-secoisolariciresinol, did not serve as a substrate for the formation of either (+)- or (-)-matairesinol. Thus, in F.intermedia, the formation of the lignan, (-)-secoisolariciresinol, occurs under strict stereochemical control, in a reaction or reactions requiring NAD(P)H and H2O2 as cofactors. This stereoselectivity is retained in the subsequent conversion into (-)-matairesinol, since (+)-secoisolariciresinol is not a substrate. These are the first two enzymes to be discovered in lignan formation.

  8. Widely tunable THz synthesizer

    Science.gov (United States)

    Hindle, F.; Mouret, G.; Eliet, S.; Guinet, M.; Cuisset, A.; Bocquet, R.; Yasui, T.; Rovera, D.

    2011-09-01

    The generation of cw-THz radiation by photomixing is particularly suited to the high resolution spectroscopy of gases; nevertheless, until recently, it has suffered from a lack of frequency metrology. Frequency combs are a powerful tool that can transfer microwave frequency standards to optical frequencies and a single comb has permitted accurate (10-8) THz frequency synthesis with a limited tuning range. A THz synthesizer composed of three extended cavity laser diodes phase locked to a frequency comb has been constructed and its utility for high resolution gas phase spectroscopy demonstrated. The third laser diode allows a larger tuning range of up to 300 MHz to be achieved without the need for large frequency excursions, while the frequency comb provides a versatile link to be established from any traceable microwave frequency standard. The use of a single frequency comb as a reference for all of the cw-lasers eliminates the dependency of synthesized frequency on the carrier envelope offset frequency. This greatly simplifies the frequency comb stabilization requirements and leads to a reduced instrument complexity.

  9. SYNTH: A spectrum synthesizer

    International Nuclear Information System (INIS)

    Hensley, W.K.; McKinnon, A.D.; Miley, H.S.; Panisko, M.E.; Savard, R.M.

    1994-07-01

    A computer code has been written at the Pacific Northwest Laboratory (PNL) to synthesize the results of typical gamma-ray spectroscopy experiments. The code, dubbed SYNTH, allows a use r to specify physical characteristics of a gamma-ray source, the quantity of the nuclides producing the radiation, the source-to-detector distance and the type and thickness of absorbers, the size and composition of the detector (Ge or NaI), and the electronic set up used to gather the data. In the process of specifying the parameters needed to synthesize a spectrum, several interesting intermediate results are produced, including a photopeak transmission function vs energy, a detector efficiency curve, and a weighted list of gamma and x rays produced from a set of nuclides. All of these intermediate results are available for graphical inspection and for printing. SYNTH runs on personal computers. It is menu driven and can be customized to user specifications. SYNTH contains robust support for coaxial germanium detectors and some support for sodium iodide detectors. SYNTH is not a finished product. A number of additional developments are planned. However, the existing code has been compared carefully to spectra obtained from National Institute for Standards and Technology (NIST) certified standards with very favorable results. Examples of the use of SYNTH and several spectral results will be presented

  10. SYNTH: A spectrum synthesizer

    International Nuclear Information System (INIS)

    Hensley, W.K.; McKinnon, A.D.; Miley, H.S.; Panisko, M.E.; Savard, R.M.

    1993-10-01

    A computer code has been written at the Pacific Northwest Laboratory (PNL) to synthesize the results of typical gamma ray spectroscopy experiments. The code, dubbed SYNTH, allows a user to specify physical characteristics of a gamma ray source, the quantity of the nuclides producing the radiation, the source-to-detector distance and the presence of absorbers, the type and size of the detector, and the electronic set up used to gather the data. In the process of specifying the parameters needed to synthesize a spectrum, several interesting intermediate results are produced, including a photopeak transmission function versus energy, a detector efficiency curve, and a weighted list of gamma and x rays produced from a set of nuclides. All of these intermediate results are available for graphical inspection and for printing. SYNTH runs on personal computers. It is menu driven and can be customized to user specifications. SYNTH contains robust support for coaxial germanium detectors and some support for sodium iodide detectors. SYNTH is not a finished product. A number of additional developments are planned. However, the existing code has been compared carefully to spectra obtained from National Institute for Standards and Technology (NIST) certified standards with very favorable results. Examples of the use of SYNTH and several spectral results are presented

  11. U.S. Geological Survey core science systems strategy: characterizing, synthesizing, and understanding the critical zone through a modular science framework

    Science.gov (United States)

    Bristol, R. Sky; Euliss, Ned H.; Booth, Nathaniel L.; Burkardt, Nina; Diffendorfer, Jay E.; Gesch, Dean B.; McCallum, Brian E.; Miller, David M.; Morman, Suzette A.; Poore, Barbara S.; Signell, Richard P.; Viger, Roland J.

    2013-01-01

    collaborations that transcend all USGS missions. The framework is designed to improve the efficiency of scientific work within USGS by establishing a means to preserve and recall data for future applications, organizing existing scientific knowledge and data to facilitate new use of older information, and establishing a future workflow that naturally integrates new data, applications, and other science products to make interdisciplinary research easier and more efficient. Given the increasing need for integrated data and interdisciplinary approaches to solve modern problems, leadership by the Core Science Systems mission will facilitate problem solving by all USGS missions in ways not formerly possible. The report lays out a strategy to achieve this vision through three goals with accompanying objectives and actions. The first goal builds on and enhances the strengths of the Core Science Systems mission in characterizing and understanding the Earth system from the geologic framework to the topographic characteristics of the land surface and biodiversity across the Nation. The second goal enhances and develops new strengths in computer and information science to make it easier for USGS scientists to discover data and models, share and publish results, and discover connections between scientific information and knowledge. The third goal brings additional focus to research and development methods to address complex issues affecting society that require integration of knowledge and new methods for synthesizing scientific information. Collectively, the report lays out a strategy to create a seamless connection between all USGS activities to accelerate and make USGS science more efficient by fully integrating disciplinary expertise within a new and evolving science paradigm for a changing world in the 21st century.

  12. Optimised 'on demand' protein arraying from DNA by cell free expression with the 'DNA to Protein Array' (DAPA) technology.

    Science.gov (United States)

    Schmidt, Ronny; Cook, Elizabeth A; Kastelic, Damjana; Taussig, Michael J; Stoevesandt, Oda

    2013-08-02

    We have previously described a protein arraying process based on cell free expression from DNA template arrays (DNA Array to Protein Array, DAPA). Here, we have investigated the influence of different array support coatings (Ni-NTA, Epoxy, 3D-Epoxy and Polyethylene glycol methacrylate (PEGMA)). Their optimal combination yields an increased amount of detected protein and an optimised spot morphology on the resulting protein array compared to the previously published protocol. The specificity of protein capture was improved using a tag-specific capture antibody on a protein repellent surface coating. The conditions for protein expression were optimised to yield the maximum amount of protein or the best detection results using specific monoclonal antibodies or a scaffold binder against the expressed targets. The optimised DAPA system was able to increase by threefold the expression of a representative model protein while conserving recognition by a specific antibody. The amount of expressed protein in DAPA was comparable to those of classically spotted protein arrays. Reaction conditions can be tailored to suit the application of interest. DAPA represents a cost effective, easy and convenient way of producing protein arrays on demand. The reported work is expected to facilitate the application of DAPA for personalized medicine and screening purposes. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. A pilot study on the use of cerebrospinal fluid cell-free DNA in intramedullary spinal ependymoma.

    Science.gov (United States)

    Connolly, Ian David; Li, Yingmei; Pan, Wenying; Johnson, Eli; You, Linya; Vogel, Hannes; Ratliff, John; Hayden Gephart, Melanie

    2017-10-01

    Cerebrospinal fluid (CSF) represents a promising source of cell-free DNA (cfDNA) for tumors of the central nervous system. A CSF-based liquid biopsy may obviate the need for riskier tissue biopsies and serve as a means for monitoring tumor recurrence or response to therapy. Spinal ependymomas most commonly occur in adults, and aggressive resection must be delicately balanced with the risk of injury to adjacent normal tissue. In patients with subtotal resection, recurrence commonly occurs. A CSF-based liquid biopsy matched to the patient's spinal ependymoma mutation profile has potential to be more sensitive then surveillance MRI, but the utility has not been well characterized for tumors of the spinal cord. In this study, we collected matched blood, tumor, and CSF samples from three adult patients with WHO grade II intramedullary spinal ependymoma. We performed whole exome sequencing on matched tumor and normal DNA to design Droplet Digital™ PCR (ddPCR) probes for tumor and wild-type mutations. We then interrogated CSF samples for tumor-derived cfDNA by performing ddPCR on extracted cfDNA. Tumor cfDNA was not reliably detected in the CSF of our cohort. Anatomic sequestration and low grade of intramedullary spinal cord tumors likely limits the role of CSF liquid biopsy.

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

    Science.gov (United States)

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

    2017-11-01

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

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

    Science.gov (United States)

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

    2010-04-01

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

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

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  17. 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......-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.......AIM: Treatment of multiresistant epithelial ovarian cancer (EOC) is palliative and patients who have become resistant after multiple lines of chemotherapy often have an unmet need for further and less toxic treatment. Anti-angiogenic therapy has attracted considerable attention in the treatment...

  18. Diagnostic and Prognostic MicroRNA Biomarkers for Prostate Cancer in Cell-free Urine

    DEFF Research Database (Denmark)

    Fredsøe, Jacob Christian; Rasmussen, Anne Karin; Thomsen, Anni Rønfeldt

    2017-01-01

    Background: Widespread use of prostate-specific antigen (PSA) testing for prostate cancer (PC) detection has led to extensive overdiagnosis and overtreatment. Urine-based microRNA (miRNA) biomarkers could be useful in PC diagnosis and prognosis. Objective: To train and validate urine-based micro......RNA (miRNA) biomarkers that may assist in PC diagnosis and prognosis. Design, setting, and participants: We profiled the expression levels of 92 miRNAs via reverse transcriptase–poymerase chain reaction in cell-free urine samples from 29 patients with benign prostatic hyperplasia (BPH) and 215 patients...... could help in primary diagnosis of PC and guide treatment decisions. Further validation studies are warranted. Patient summary: Using two large patient cohorts, we searched for novel prostate cancer biomarkers in urine. We found two new sets of microRNA biomarkers in urine that could accurately predict...

  19. 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

    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 decrease varying......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...... from 1-17 percentage points. This was due to a significant increase in the concentration of cfDNA (p physical activity. CONCLUSION: When planning the timing of noninvasive...

  20. Analysis, Design and Synthesis Methods for Guidance and Control Systems (Les Methodes d’Analyse de Conception et de Synthese Pour les Systemes de Guidage et de Pilotage)

    Science.gov (United States)

    1990-06-01

    factor anomalies and shifts can also ,. from changes In the gain medium, which ,akes a significant contribution to the optica length of the cavity...felt that this application may be more appropriate for a mature fibra optic gyro system, exhibitin!, a more acceptable cost-performance ratio. A

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

    Science.gov (United States)

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

    2015-06-17

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

  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

    with antibodies to FANCI, FANCD2, DNA pol e, FANCA , FANCM. We first wanted to test whether Approach, which is inhibited in BRCA1-depleted egg...novel proteins whose binding to chromatin depends on BRCA1: clone the gene , express the protein, raise antibodies, immunodeplete the protein from egg... Gene Regulation and Genomics Seminar Series, UT Southwestern 2016 Keynote Speaker Gordon Research Seminar on Mutagenesis, Ventura, CA 2016 Invited

  4. Systemic and Pulmonary Hypertension After Resuscitation with Cell-Free Hemoglobin

    Science.gov (United States)

    1992-07-01

    Endotoxin was measured using a kinetic turbidimetric modification of the Limulus amoebocyte lysate assay (9). The rabbit pyrogen test was performed...renal failure. This injury occurred in half of the animals that were resuscitated with HPLC purified, pyrogen -negative, low-residual- phosphate HbAo...oxide: an endogenous modulator of leukocyte adhesion. Proc. Nat. Acad. Sci. USA 88:4651-4655, 1991. 17. Moncada, S., K. M. J. Palmer, E. A. Higgs. Nitric

  5. Gibberellin biosynthesis in cell-free extracts from developing Cucurbita maxima embryos and the identification of new endogenous gibberellins.

    Science.gov (United States)

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

    1993-03-01

    Gibberellin (GA) biosynthetic pathways from GA12-aldehyde, GA12 and GA53 were investigated in cell-free systems from developing embryos of Cucurbita maxima L. Gibberellin A12-aldehyde and GA12 were converted to GA25, putative 12α-hydroxyGA25, GA13 and GA39 as main products. Minor products were GA4, GA34 and, when GA12 was the substrate, putative 12α-hydroxyGA12. The intermediates GA15 and GA24 accumulated at low protein concentrations. The influence of various factors on GA12 metabolism was examined. At low 2-oxoglutarate and ascorbate concentrations, or at acid pH, 3β-hydroxylated products predominated, whereas with increasing 2-oxoglutarate and ascorbate concentrations, or at neutral pH, the yield of 12α-hydroxylated GAs increased. Gibberellin A53 was metabolised mainly to the C20-GAs GA44, GA19, GA17, GA23 and GA28, with the C19-GAs GA20, GA1 and GA8 as minor products. Only C19-GAs were 2β-hydroxylated, which is a main characteristic of the embryo systems. In addition to GA13, GA25, GA39, GA43, GA49, GA58, GA74, 12α-hydroxyGA25 and GA39 3-isovalerate, which were known previously from embryos of C. maxima, GA1, GA4, GA17, GA28, GA37, GA38, GA48, GA85, 12α-hydroxyGA37 and putative 12α-hydroxyGA43 were identified as endogenous components by full-scan capillary gas chromatography-mass spectrometry and Kovats retention indices. Evidence for putative 2β-hydroxyGA28 and GA23 was also obtained but it was less conclusive because of contamination.

  6. The role of total cell-free DNA in predicting outcomes among trauma patients in the intensive care unit

    DEFF Research Database (Denmark)

    Gögenur, Mikail; Burcharth, Jakob; Gögenur, Ismail

    2017-01-01

    searched Pubmed, Embase, Scopus and the Cochrane Central Register for Controlled Trials and reference lists of relevant articles for studies that assessed the prognostic value of cell-free DNA detection in trauma patients in the intensive care unit. Outcomes of interest included survival, posttraumatic...

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

    DEFF Research Database (Denmark)

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

    2016-01-01

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

  8. Preanalytical blood sample workup for cell-free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics

    NARCIS (Netherlands)

    van Ginkel, Joost H.; van den Broek, Daan A.; van Kuik, Joyce; Linders, Dorothé; de Weger, Roel; Willems, Stefan M.; Huibers, Manon M.H.

    2017-01-01

    In current molecular cancer diagnostics, using blood samples of cancer patients for the detection of genetic alterations in plasma (cell-free) circulating tumor DNA (ctDNA) is an emerging practice. Since ctDNA levels in blood are low, highly sensitive Droplet Digital PCR (ddPCR) can be used for

  9. Bioreduction of trivalent aurum to nano-crystalline gold particles by active and inactive cells and cell-free extract of Aspergillus oryzae var. viridis

    International Nuclear Information System (INIS)

    Binupriya, A.R.; Sathishkumar, M.; Vijayaraghavan, K.; Yun, S.-I.

    2010-01-01

    Bioreduction efficacy of both active (AB) and inactive (IB) cells/biomass of Aspergillus oryzae var. viridis and their respective cell-free extracts (ACE and ICE) to convert trivalent aurum to gold nanoparticles were tested in the present study. Strong plasmon resonance of gold nanoparticles was observed between 540 and 560 nm in the samples obtained from AB, IB, ACE and ICE. Transmission electron microscopy (TEM), field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray (EDX) and X-ray diffraction (XRD) were performed to examine the formation of gold nanoparticles. Comparing all four forms of A. oryzae var. viridis, ICE showed high gold nanoparticle productivity. The nanoparticles formed were quite uniform in shape and ranged in size from 10 to 60 nm. In addition some triangle, pentagon and hexagon-shaped nanoplates with size range of 30-400 nm were also synthesized especially at lower pH. Organics from the inactive cells are believed to be responsible for reduction of trivalent aurum to nano-sized gold particles. Organic content of the ICE was found to be double the amount of ACE. High productivity of gold nanoparticles by metabolic-independent process opens up an interesting area of nanoparticle synthesis using waste fungal biomass from industries.

  10. Bioreduction of trivalent aurum to nano-crystalline gold particles by active and inactive cells and cell-free extract of Aspergillus oryzae var. viridis

    Energy Technology Data Exchange (ETDEWEB)

    Binupriya, A.R. [Department of Food Science and Technology, College of Agriculture and Life Science, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Sathishkumar, M., E-mail: cvemuthu@nus.edu.sg [Singapore-Delft Water Alliance, National University of Singapore, 2 Engineering Drive 2, Singapore 117577 (Singapore); Vijayaraghavan, K. [Singapore-Delft Water Alliance, National University of Singapore, 2 Engineering Drive 2, Singapore 117577 (Singapore); Yun, S.-I., E-mail: siyun@chonbuk.ac.kr [Department of Food Science and Technology, College of Agriculture and Life Science, Chonbuk National University, Jeonju 561-756 (Korea, Republic of)

    2010-05-15

    Bioreduction efficacy of both active (AB) and inactive (IB) cells/biomass of Aspergillus oryzae var. viridis and their respective cell-free extracts (ACE and ICE) to convert trivalent aurum to gold nanoparticles were tested in the present study. Strong plasmon resonance of gold nanoparticles was observed between 540 and 560 nm in the samples obtained from AB, IB, ACE and ICE. Transmission electron microscopy (TEM), field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray (EDX) and X-ray diffraction (XRD) were performed to examine the formation of gold nanoparticles. Comparing all four forms of A. oryzae var. viridis, ICE showed high gold nanoparticle productivity. The nanoparticles formed were quite uniform in shape and ranged in size from 10 to 60 nm. In addition some triangle, pentagon and hexagon-shaped nanoplates with size range of 30-400 nm were also synthesized especially at lower pH. Organics from the inactive cells are believed to be responsible for reduction of trivalent aurum to nano-sized gold particles. Organic content of the ICE was found to be double the amount of ACE. High productivity of gold nanoparticles by metabolic-independent process opens up an interesting area of nanoparticle synthesis using waste fungal biomass from industries.

  11. Circulating cell-free DNA and circulating tumor cells, the "liquid biopsies" in ovarian cancer.

    Science.gov (United States)

    Cheng, Xianliang; Zhang, Lei; Chen, Yajuan; Qing, Chen

    2017-11-13

    Limited understanding of ovarian cancer (OC) genome portrait has hindered the therapeutic advances. The serial monitoring of tumor genotypes is becoming increasingly attainable with circulating cell-free DNA (cf-DNA) and circulating tumor cells (CTCs) emerging as "liquid biopsies". They represent non-invasive biomarkers and are viable, as they can be isolated from human plasma, serum and other body fluids. Molecular characterization of circulating tumor DNA (ct-DNA) and CTCs offer unique potentials to better understand the biology of metastasis and resistance to therapies. The liquid biopsies may also give innovative insights into the process of rapid and accurate identification, resistant genetic alterations and a real time monitoring of treatment responses. In addition, liquid biopsies are shedding light on elucidating signal pathways involved in invasiveness and metastasis competence; but the detection and molecular characterization of ct-DNA and CTCs are still challenging, since they are rare, and the amount of available samples are very limited. This review will focus on the clinical potential of ct-DNA and CTCs in both the early and advanced diagnosis, prognosis, and in the identification of resistance mutations in OC.

  12. Cell-free oxygen carriers: scientific foundations, clinical development, and new directions.

    Science.gov (United States)

    Winslow, Robert M

    2008-10-01

    The most significant hurdle to the development of a safe and effective hemoglobin-based oxygen carrier ("blood substitute") is generally thought to be its propensity to cause vasoconstriction in the microcirculation and hypertension. Two theories for this effect are currently being studied: in one, scavenging NO by hemoglobin reduces vasorelaxation; in the other, cell-free hemoglobin oversupplies O2 (a known vasoconstrictor) to vascular walls by facilitated diffusion. While both mechanisms might lead to reduction of local NO concentration, the important distinction between the two is that if the NO scavenging theory is correct, it greatly diminishes the prospects to develop any solution based on free hemoglobin. However, if the O2-oversupply theory is correct, modifications to the hemoglobin molecule can be envisioned that can prevent oversupply and reduce toxicity. This review summarizes the development of Hemospan, a novel modification of human hemoglobin whose design is based on the O2-oversupply theory. Because of its low P50 and increased molecular size, the release of O2 in resistance vessels (arterioles) by Hemospan is restricted, and vasoconstriction is greatly reduced.

  13. Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors.

    Science.gov (United States)

    Adalsteinsson, Viktor A; Ha, Gavin; Freeman, Samuel S; Choudhury, Atish D; Stover, Daniel G; Parsons, Heather A; Gydush, Gregory; Reed, Sarah C; Rotem, Denisse; Rhoades, Justin; Loginov, Denis; Livitz, Dimitri; Rosebrock, Daniel; Leshchiner, Ignaty; Kim, Jaegil; Stewart, Chip; Rosenberg, Mara; Francis, Joshua M; Zhang, Cheng-Zhong; Cohen, Ofir; Oh, Coyin; Ding, Huiming; Polak, Paz; Lloyd, Max; Mahmud, Sairah; Helvie, Karla; Merrill, Margaret S; Santiago, Rebecca A; O'Connor, Edward P; Jeong, Seong H; Leeson, Rachel; Barry, Rachel M; Kramkowski, Joseph F; Zhang, Zhenwei; Polacek, Laura; Lohr, Jens G; Schleicher, Molly; Lipscomb, Emily; Saltzman, Andrea; Oliver, Nelly M; Marini, Lori; Waks, Adrienne G; Harshman, Lauren C; Tolaney, Sara M; Van Allen, Eliezer M; Winer, Eric P; Lin, Nancy U; Nakabayashi, Mari; Taplin, Mary-Ellen; Johannessen, Cory M; Garraway, Levi A; Golub, Todd R; Boehm, Jesse S; Wagle, Nikhil; Getz, Gad; Love, J Christopher; Meyerson, Matthew

    2017-11-06

    Whole-exome sequencing of cell-free DNA (cfDNA) could enable comprehensive profiling of tumors from blood but the genome-wide concordance between cfDNA and tumor biopsies is uncertain. Here we report ichorCNA, software that quantifies tumor content in cfDNA from 0.1× coverage whole-genome sequencing data without prior knowledge of tumor mutations. We apply ichorCNA to 1439 blood samples from 520 patients with metastatic prostate or breast cancers. In the earliest tested sample for each patient, 34% of patients have ≥10% tumor-derived cfDNA, sufficient for standard coverage whole-exome sequencing. Using whole-exome sequencing, we validate the concordance of clonal somatic mutations (88%), copy number alterations (80%), mutational signatures, and neoantigens between cfDNA and matched tumor biopsies from 41 patients with ≥10% cfDNA tumor content. In summary, we provide methods to identify patients eligible for comprehensive cfDNA profiling, revealing its applicability to many patients, and demonstrate high concordance of cfDNA and metastatic tumor whole-exome sequencing.

  14. A reproducible and scalable procedure for preparing bacterial extracts for cell-free protein synthesis.

    Science.gov (United States)

    Katsura, Kazushige; Matsuda, Takayoshi; Tomabechi, Yuri; Yonemochi, Mayumi; Hanada, Kazuharu; Ohsawa, Noboru; Sakamoto, Kensaku; Takemoto, Chie; Shirouzu, Mikako

    2017-11-01

    Cell-free protein synthesis is a useful method for preparing proteins for functional or structural analyses. However, batch-to-batch variability with regard to protein synthesis activity remains a problem for large-scale production of cell extract in the laboratory. To address this issue, we have developed a novel procedure for large-scale preparation of bacterial cell extract with high protein synthesis activity. The developed procedure comprises cell cultivation using a fermentor, harvesting and washing of cells by tangential flow filtration, cell disruption with high-pressure homogenizer and continuous diafiltration. By optimizing and combining these methods, ∼100 ml of the cell extract was prepared from 150 g of Escherichia coli cells. The protein synthesis activities, defined as the yield of protein per unit of absorbance at 260 nm of the cell extract, were shown to be reproducible, and the average activity of several batches was twice that obtained using a previously reported method. In addition, combinatorial use of the high-pressure homogenizer and diafiltration increased the scalability, indicating that the cell concentration at disruption varies from 0.04 to 1 g/ml. Furthermore, addition of Gam protein and examinations of the N-terminal sequence rendered the extract prepared here useful for rapid screening with linear DNA templates. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  15. Effects of polymorphisms in ovine and caprine prion protein alleles on cell-free conversion

    Directory of Open Access Journals (Sweden)

    Eiden Martin

    2011-02-01

    Full Text Available Abstract In sheep polymorphisms of the prion gene (PRNP at the codons 136, 154 and 171 strongly influence the susceptibility to scrapie and bovine spongiform encephalopathy (BSE infections. In goats a number of other gene polymorphisms were found which are suspected to trigger similar effects. However, no strong correlation between polymorphisms and TSE susceptibility in goats has yet been obtained from epidemiological studies and only a low number of experimental challenge data are available at present. We have therefore studied the potential impact of these polymorphisms in vitro by cell-free conversion assays using mouse scrapie strain Me7. Mouse scrapie brain derived PrPSc served as seeds and eleven recombinant single mutation variants of sheep and goat PrPC as conversion targets. With this approach it was possible to assign reduced conversion efficiencies to specific polymorphisms, which are associated to low frequency in scrapie-affected goats or found only in healthy animals. Moreover, we could demonstrate a dominant-negative inhibition of prion polymorphisms associated with high susceptibility by alleles linked to low susceptibility in vitro.

  16. Antimicrobial Activity of Cell Free Supernatant of Irradiated Lactic Acid Bacteria Isolates

    International Nuclear Information System (INIS)

    Abdelaleem, M.A.; AL-Hagar, O.E.Aa.

    2015-01-01

    Attempts were made to isolate bio preservatives using food wastes with no value and low cost. Whey is the raw material achieved that value. Whey and many other food wastes are used in our study to isolate Lactic acid bacteria (LAB). Cell free supernatants (CFS) of isolates are used to evaluate their antimicrobial activity against indicator pathogenic bacterial strains. CFS-9 isolate from whey has the highest inhibitory activity compared to all other isolates. The inhibitory activity of CFS-9, Nisin (400 IU / ml) and the standard Lactococcus Lactis Subsp. Lactis ATCC 11454 (Lacto) were determined. Furthermore, isolate-9 and Lacto strains were exposed to irradiation at different doses. The inhibition zones of; control isolate-9 (non-irradiated) showed the highest values against all indicator strains, CFS of irradiated Lacto at dose 250 Gy was the highest value against Bacillus cereus and Escherichia coli compared to other irradiation treatments, CFS of irradiated Lacto at dose 100 Gy was the highest value against Staph aureus, while the inhibition zone was in the highest value in CFS of irradiated Lacto at dose 500 Gy against Salmonella typhimurium. Nisin (400 IU / ml) was significantly higher than all CFS of irradiated isolate-9 while, the inhibition zones of all CFS-Lacto (irradiated and nonirradiated) are better and higher than nisin-400

  17. Urinary Cell-Free DNA Quantification as Non-Invasive Biomarker in Patients with Bladder Cancer.

    Science.gov (United States)

    Brisuda, Antonin; Pazourkova, Eva; Soukup, Viktor; Horinek, Ales; Hrbáček, Jan; Capoun, Otakar; Svobodova, Iveta; Pospisilova, Sarka; Korabecna, Marie; Mares, Jaroslav; Hanuš, Tomáš; Babjuk, Marek

    2016-01-01

    Concentration of urinary cell-free DNA (ucfDNA) belongs to potential bladder cancer markers, but the reported results are inconsistent due to the use of various non-standardised methodologies. The aim of the study was to standardise the methodology for ucfDNA quantification as a potential non-invasive tumour biomarker. In total, 66 patients and 34 controls were enrolled into the study. Volumes of each urine portion (V) were recorded and ucfDNA concentrations (c) were measured using real-time PCR. Total amounts (TA) of ucfDNA were calculated and compared between patients and controls. Diagnostic accuracy of the TA of ucfDNA was determined. The calculation of TA of ucfDNA in the second urine portion was the most appropriate approach to ucfDNA quantification, as there was logarithmic dependence between the volume and the concentration of a urine portion (p = 0.0001). Using this methodology, we were able to discriminate between bladder cancer patients and subjects without bladder tumours (p = 0.0002) with area under the ROC curve of 0.725. Positive and negative predictive value of the test was 90 and 45%, respectively. Quantification of ucf DNA according to our modified method could provide a potential non-invasive biomarker for diagnosis of patients with bladder cancer. © 2015 S. Karger AG, Basel.

  18. Quantification of transplant-derived circulating cell-free DNA in absence of a donor genotype.

    Science.gov (United States)

    Sharon, Eilon; Shi, Hao; Kharbanda, Sandhya; Koh, Winston; Martin, Lance R; Khush, Kiran K; Valantine, Hannah; Pritchard, Jonathan K; De Vlaminck, Iwijn

    2017-08-01

    Quantification of cell-free DNA (cfDNA) in circulating blood derived from a transplanted organ is a powerful approach to monitoring post-transplant injury. Genome transplant dynamics (GTD) quantifies donor-derived cfDNA (dd-cfDNA) by taking advantage of single-nucleotide polymorphisms (SNPs) distributed across the genome to discriminate donor and recipient DNA molecules. In its current implementation, GTD requires genotyping of both the transplant recipient and donor. However, in practice, donor genotype information is often unavailable. Here, we address this issue by developing an algorithm that estimates dd-cfDNA levels in the absence of a donor genotype. Our algorithm predicts heart and lung allograft rejection with an accuracy that is similar to conventional GTD. We furthermore refined the algorithm to handle closely related recipients and donors, a scenario that is common in bone marrow and kidney transplantation. We show that it is possible to estimate dd-cfDNA in bone marrow transplant patients that are unrelated or that are siblings of the donors, using a hidden Markov model (HMM) of identity-by-descent (IBD) states along the genome. Last, we demonstrate that comparing dd-cfDNA to the proportion of donor DNA in white blood cells can differentiate between relapse and the onset of graft-versus-host disease (GVHD). These methods alleviate some of the barriers to the implementation of GTD, which will further widen its clinical application.

  19. Cell-free nucleic acids as noninvasive biomarkers for colorectal cancer detection

    KAUST Repository

    Mansour, Hicham

    2014-08-27

    Cell-free nucleic acids (CFNA) have been reported by several authors in blood, stool, and urine of patients with colorectal cancer (CRC). These genetic biomarkers can be an indication of neoplastic colorectal epithelial cells, and can thus potentially be used as noninvasive tests for the detection of the disease in CRC patients and monitor their staging, without the need to use heavier and invasive tools. In a number of test-trials, these genetic tests have shown the advantage of non-invasiveness, making them well accepted by most of the patients, without major side effects. They have also shown a promising sensitivity and specificity in the detection of malignant and premalignant neoplasms. Moreover, costs for performing such tests are very low. Several studies reported and confirmed the proof of the principle for these genetic tests for screening, diagnosis, and prognosis; the main challenge of translating this approach from research to clinical laboratory is the validation from large and long-term randomized trials to prove sustainable high sensitivity and specificity. In this paper, we present a review on the noninvasive genetics biomarkers for CRC detection described in the literature and the challenges that can be encountered for validation processes.

  20. Cell-free nucleic acids as noninvasive biomarkers for colorectal cancer detection

    KAUST Repository

    Mansour, Hicham

    2014-01-01

    Cell-free nucleic acids (CFNA) have been reported by several authors in blood, stool, and urine of patients with colorectal cancer (CRC). These genetic biomarkers can be an indication of neoplastic colorectal epithelial cells, and can thus potentially be used as noninvasive tests for the detection of the disease in CRC patients and monitor their staging, without the need to use heavier and invasive tools. In a number of test-trials, these genetic tests have shown the advantage of non-invasiveness, making them well accepted by most of the patients, without major side effects. They have also shown a promising sensitivity and specificity in the detection of malignant and premalignant neoplasms. Moreover, costs for performing such tests are very low. Several studies reported and confirmed the proof of the principle for these genetic tests for screening, diagnosis, and prognosis; the main challenge of translating this approach from research to clinical laboratory is the validation from large and long-term randomized trials to prove sustainable high sensitivity and specificity. In this paper, we present a review on the noninvasive genetics biomarkers for CRC detection described in the literature and the challenges that can be encountered for validation processes.

  1. Green Synthesis and Antimicrobial Activities of Silver Nanoparticles using Cell Free-Extracts of Enterococcus species

    Directory of Open Access Journals (Sweden)

    Iyabo C. OLADIPO

    2017-06-01

    Full Text Available Cell-free extracts of six strains of Enterococcus species obtained from fermented foods were used for the green synthesis of silver nanoparticles (AgNPs, which was characterized by UV-Vis spectroscopy, Fourier-transform infrared spectroscopy (FTIR and transmission electron microscopy (TEM. The biosynthesized AgNPs were dark brown in colour having surface plasmon resonance in the range of 420-442 nm. The spherical shaped AgNPs had sizes of 4-55 nm, whose formations were facilitated by proteins as indicated by the presence of peaks 1,635-1,637 and 3,275-3,313 cm-1 in the FTIR spectra. The energy dispersive x-ray (EDX showed prominent presence of silver in the AgNPs colloidal solution, while the selected area electron diffraction was typified by the face-centred crystalline nature of silver. The particles inhibited the growth of multi-drug resistant clinical isolates of Escherichia coli, Klebsiella pneumoniae and Proteus vulgaris, and also potentiated the activities of ampicillin, ciprofloxacin and cefuroxime in the AgNPs-antibiotic synergy studies. In addition, the prospective relevance of the particles as nanopreservative in paints was demonstrated with the inhibition of growth of Staphylococcus aureus, Pseudomonas aeruginosa, Aspergillus niger and A. flavus in AgNPs-paint admixture. This report further demonstrates the green synthesis of AgNPs by strains of Enterococcus species.

  2. Quantification of transplant-derived circulating cell-free DNA in absence of a donor genotype.

    Directory of Open Access Journals (Sweden)

    Eilon Sharon

    2017-08-01

    Full Text Available Quantification of cell-free DNA (cfDNA in circulating blood derived from a transplanted organ is a powerful approach to monitoring post-transplant injury. Genome transplant dynamics (GTD quantifies donor-derived cfDNA (dd-cfDNA by taking advantage of single-nucleotide polymorphisms (SNPs distributed across the genome to discriminate donor and recipient DNA molecules. In its current implementation, GTD requires genotyping of both the transplant recipient and donor. However, in practice, donor genotype information is often unavailable. Here, we address this issue by developing an algorithm that estimates dd-cfDNA levels in the absence of a donor genotype. Our algorithm predicts heart and lung allograft rejection with an accuracy that is similar to conventional GTD. We furthermore refined the algorithm to handle closely related recipients and donors, a scenario that is common in bone marrow and kidney transplantation. We show that it is possible to estimate dd-cfDNA in bone marrow transplant patients that are unrelated or that are siblings of the donors, using a hidden Markov model (HMM of identity-by-descent (IBD states along the genome. Last, we demonstrate that comparing dd-cfDNA to the proportion of donor DNA in white blood cells can differentiate between relapse and the onset of graft-versus-host disease (GVHD. These methods alleviate some of the barriers to the implementation of GTD, which will further widen its clinical application.

  3. New insights into the microvascular mechanisms of drag reducing polymers: effect on the cell-free layer.

    Science.gov (United States)

    Brands, Judith; Kliner, Dustin; Lipowsky, Herbert H; Kameneva, Marina V; Villanueva, Flordeliza S; Pacella, John J

    2013-01-01

    Drag-reducing polymers (DRPs) significantly increase blood flow, tissue perfusion, and tissue oxygenation in various animal models. In rectangular channel microfluidic systems, DRPs were found to significantly reduce the near-wall cell-free layer (CFL) as well as modify traffic of red blood cells (RBC) into microchannel branches. In the current study we further investigated the mechanism by which DRP enhances microvascular perfusion. We studied the effect of various concentrations of DRP on RBC distribution in more relevant round microchannels and the effect of DRP on CFL in the rat cremaster muscle in vivo. In round microchannels hematocrit was measured in parent and daughter branch at baseline and after addition of DRP. At DRP concentrations of 5 and 10 ppm, the plasma skimming effect in the daughter branch was eliminated, as parent and daughter branch hematocrit were equivalent, compared to a significantly lowered hematocrit in the daughter branch without DRPs. In anesthetized rats (N=11) CFL was measured in the cremaster muscle tissue in arterioles with a diameter of 32.6 ± 1.7 µm. In the control group (saline, N=6) there was a significant increase in CFL in time compared to corresponding baseline. Addition of DRP at 1 ppm (N=5) reduced CFL significantly compared to corresponding baseline and the control group. After DRP administration the CFL reduced to about 85% of baseline at 5, 15, 25 and 35 minutes after DRP infusion was complete. These in vivo and in vitro findings demonstrate that DRPs induce a reduction in CFL width and plasma skimming in the microvasculature. This may lead to an increase of RBC flux into the capillary bed, and thus explain previous observations of a DRP mediated enhancement of capillary perfusion.

  4. An Economic Analysis of Cell-Free DNA Non-Invasive Prenatal Testing in the US General Pregnancy Population.

    Directory of Open Access Journals (Sweden)

    Peter Benn

    Full Text Available Analyze the economic value of replacing conventional fetal aneuploidy screening approaches with non-invasive prenatal testing (NIPT in the general pregnancy population.Using decision-analysis modeling, we compared conventional screening to NIPT with cell-free DNA (cfDNA analysis in the annual US pregnancy population. Sensitivity and specificity for fetal aneuploidies, trisomy 21, trisomy 18, trisomy 13, and monosomy X, were estimated using published data and modeling of both first- and second trimester screening. Costs were assigned for each prenatal test component and for an affected birth. The overall cost to the healthcare system considered screening costs, the number of aneuploid cases detected, invasive procedures performed, procedure-related euploid losses, and affected pregnancies averted. Sensitivity analyses evaluated the effect of variation in parameters. Costs were reported in 2014 US Dollars.Replacing conventional screening with NIPT would reduce healthcare costs if it can be provided for $744 or less in the general pregnancy population. The most influential variables were timing of screening entry, screening costs, and pregnancy termination rates. Of the 13,176 affected pregnancies undergoing screening, NIPT detected 96.5% (12,717/13,176 of cases, compared with 85.9% (11,314/13,176 by conventional approaches. NIPT reduced invasive procedures by 60.0%, with NIPT and conventional methods resulting in 24,596 and 61,430 invasive procedures, respectively. The number of procedure-related euploid fetal losses was reduced by 73.5% (194/264 in the general screening population.Based on our analysis, universal application of NIPT would increase fetal aneuploidy detection rates and can be economically justified. Offering this testing to all pregnant women is associated with substantial prenatal healthcare benefits.

  5. A Comparative Study for Detection of EGFR Mutations in Plasma Cell-Free DNA in Korean Clinical Diagnostic Laboratories

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    Yoonjung Kim

    2018-01-01

    Full Text Available Liquid biopsies to genotype the epidermal growth factor receptor (EGFR for targeted therapy have been implemented in clinical decision-making in the field of lung cancer, but harmonization of detection methods is still scarce among clinical laboratories. We performed a pilot external quality assurance (EQA scheme to harmonize circulating tumor DNA testing among laboratories. For EQA, we created materials containing different levels of spiked cell-free DNA (cfDNA in normal plasma. The limit of detection (LOD of the cobas® EGFR Mutation Test v2 (Roche Molecular Systems was also evaluated. From November 2016 to June 2017, seven clinical diagnostic laboratories participated in the EQA program. The majority (98.94% of results obtained using the cobas assay and next-generation sequencing (NGS were acceptable. Quantitative results from the cobas assay were positively correlated with allele frequencies derived from digital droplet PCR measurements and showed good reproducibility among laboratories. The LOD of the cobas assay was 5~27 copies/mL for p.E746_A750del (exon 19 deletion, 35~70 copies/mL for p.L858R, 18~36 copies/mL for p.T790M, and 15~31 copies/mL for p.A767_V769dup (exon 20 insertion. Deep sequencing of materials (>100,000X depth of coverage resulted in detection of low-level targets present at frequencies of 0.06~0.13%. Our results indicate that the cobas assay is a reliable and rapid method for detecting EGFR mutations in plasma cfDNA. Careful interpretation is particularly important for p.T790M detection in the setting of relapse. Individual laboratories should optimize NGS performance to maximize clinical utility.

  6. High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients.

    Science.gov (United States)

    Kukita, Yoji; Matoba, Ryo; Uchida, Junji; Hamakawa, Takuya; Doki, Yuichiro; Imamura, Fumio; Kato, Kikuya

    2015-08-01

    Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  7. The development of [18F]FDG synthesizer

    International Nuclear Information System (INIS)

    Hu, M. G.; Kim, S. W.; Lee, J. Y.; Yang, S. D.; Jun, G. S.

    2003-01-01

    The automatic system for [ 18 F]FDG production using for the diagnosis of cancer has been developed. This automation system was consisted of a synthesizer module, a PLC based controller and a PMU for graphic user interface. By this system, the radiochemical purity was over 98%, the production yield was over 30% after synthesize and elapsed time was 35 minute

  8. Influence of aluminum addition in the framework of MCM-41 mesoporous molecular sieve synthesized by non-hydrothermal method in an alkali-free system

    Energy Technology Data Exchange (ETDEWEB)

    La-Salvia, Nathália; Lovón-Quintana, Juan José; Lovón, Adriana Siviero Pagani; Valença, Gustavo Paim, E-mail: nathalialasalvia@gmail.com [Laboratório para o Estudo de Processos de Adsorção e Catálise - LEPAC, Faculdade de Engenharia Química, Universidade Estadual de Campinas - UNICAMP, Campinas, SP (Brazil)

    2017-11-15

    Purely siliceous MCM-41 and Al-containing MCM-41 (Al-MCM-41) mesoporous materials were synthesized by non-hydrothermal method in alkali-free ions medium at room temperature and short reaction times. Under these synthesis conditions, it was also investigated the influence of Al incorporation in the crystal structure of MCM-41. The solids were characterized by ICP-OES, AAS, N{sub 2} adsorption at 77 K, XRD, TEM, NH3 -TPD, {sup 27}Al and {sup 29}Si-MAS-NMR, FT-IR and TGA. The resulting mesoporous materials showed a well-defined hexagonally ordered pore geometry maintaining a uniform and unimodal pore size distribution with high specific surface areas (1000-1400 m{sup 2} g{sup -1}). The Al{sup +3} ions were introduced successfully in the structure of the purely siliceous MCM-41 expanding the unit cell parameter and forming four-coordinated Al species, and in a less extent, forming six-coordinated Al species. In addition, the surface acidity of the MCM-41 increased with Al loading. Contrary, the presence of Al in the MCM-41 mesoporous structure resulted in a decrease of the crystallinity and specific surface area possibly due to the presence of Al species in highly distorted tetrahedral structures and Al extra-framework or amorphous alumina occluded in the pores. The MCM-41 type mesoporous materials obtained in this work show similar characteristics of those synthesized by conventional hydrothermal methods. (author)

  9. Metallization of ion beam synthesized Si/3C-SiC/Si layer systems by high-dose implantation of transition metal ions

    International Nuclear Information System (INIS)

    Lindner, J.K.N.; Wenzel, S.; Stritzker, B.

    2001-01-01

    The formation of metal silicide layers contacting an ion beam synthesized buried 3C-SiC layer in silicon by means of high-dose titanium and molybdenum implantations is reported. Two different strategies to form such contact layers are explored. The titanium implantation aims to convert the Si top layer of an epitaxial Si/SiC/Si layer sequence into TiSi 2 , while Mo implantations were performed directly into the SiC layer after selectively etching off all capping layers. Textured and high-temperature stable C54-TiSi 2 layers with small additions of more metal-rich silicides are obtained in the case of the Ti implantations. Mo implantations result in the formation of the high-temperature phase β-MoSi 2 , which also grows textured on the substrate. The formation of cavities in the silicon substrate at the lower SiC/Si interface due to the Si consumption by the growing silicide phase is observed in both cases. It probably constitutes a problem, occurring whenever thin SiC films on silicon have to be contacted by silicide forming metals independent of the deposition technique used. It is shown that this problem can be solved with ion beam synthesized contact layers by proper adjustment of the metal ion dose

  10. Influence of aluminum addition in the framework of MCM-41 mesoporous molecular sieve synthesized by non-hydrothermal method in an alkali-free system

    International Nuclear Information System (INIS)

    La-Salvia, Nathália; Lovón-Quintana, Juan José; Lovón, Adriana Siviero Pagani; Valença, Gustavo Paim

    2017-01-01

    Purely siliceous MCM-41 and Al-containing MCM-41 (Al-MCM-41) mesoporous materials were synthesized by non-hydrothermal method in alkali-free ions medium at room temperature and short reaction times. Under these synthesis conditions, it was also investigated the influence of Al incorporation in the crystal structure of MCM-41. The solids were characterized by ICP-OES, AAS, N 2 adsorption at 77 K, XRD, TEM, NH3 -TPD, 27 Al and 29 Si-MAS-NMR, FT-IR and TGA. The resulting mesoporous materials showed a well-defined hexagonally ordered pore geometry maintaining a uniform and unimodal pore size distribution with high specific surface areas (1000-1400 m 2 g -1 ). The Al +3 ions were introduced successfully in the structure of the purely siliceous MCM-41 expanding the unit cell parameter and forming four-coordinated Al species, and in a less extent, forming six-coordinated Al species. In addition, the surface acidity of the MCM-41 increased with Al loading. Contrary, the presence of Al in the MCM-41 mesoporous structure resulted in a decrease of the crystallinity and specific surface area possibly due to the presence of Al species in highly distorted tetrahedral structures and Al extra-framework or amorphous alumina occluded in the pores. The MCM-41 type mesoporous materials obtained in this work show similar characteristics of those synthesized by conventional hydrothermal methods. (author)

  11. A comparison of cell-free placental messenger ribonucleic acid and color Doppler ultrasound for the prediction of placental invasion in patients with placenta accreta

    Directory of Open Access Journals (Sweden)

    Elham Naghshineh

    2015-01-01

    Conclusions: Cell-free mRNA is an acceptable, easy made, functional test with sensitivity, specificity, PPV and NPV more than Doppler ultrasound for diagnosis and prediction of incidence of placenta accrete and we recommend the use of cell-free mRNA test for diagnosis of placenta accreta.

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

    Science.gov (United States)

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

    2017-10-01

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

  13. Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus

    Energy Technology Data Exchange (ETDEWEB)

    Fogeron, Marie-Laure [Université de Lyon, Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS (France); Jirasko, Vlastimil; Penzel, Susanne [ETH Zurich, Physical Chemistry (Switzerland); Paul, David [Heidelberg University, Department of Infectious Diseases, Molecular Virology (Germany); Montserret, Roland; Danis, Clément; Lacabanne, Denis; Badillo, Aurélie [Université de Lyon, Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS (France); Gouttenoire, Jérôme; Moradpour, Darius [University of Lausanne, Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois (Switzerland); Bartenschlager, Ralf [Heidelberg University, Department of Infectious Diseases, Molecular Virology (Germany); Penin, François [Université de Lyon, Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS (France); Meier, Beat H., E-mail: beme@ethz.ch [ETH Zurich, Physical Chemistry (Switzerland); and others

    2016-06-15

    We describe the expression of the hepatitis C virus nonstructural protein 4B (NS4B), which is an integral membrane protein, in a wheat germ cell-free system, the subsequent purification and characterization of NS4B and its insertion into proteoliposomes in amounts sufficient for multidimensional solid-state NMR spectroscopy. First spectra of the isotopically [{sup 2}H,{sup 13}C,{sup 15}N]-labeled protein are shown to yield narrow {sup 13}C resonance lines and a proper, predominantly α-helical fold. Clean residue-selective leucine, isoleucine and threonine-labeling is demonstrated. These results evidence the suitability of the wheat germ-produced integral membrane protein NS4B for solid-state NMR. Still, the proton linewidth under fast magic angle spinning is broader than expected for a perfect sample and possible causes are discussed.

  14. Comparison of Eight Cell-Free Media for Maintenance of Toxoplasma gondii Tachyzoites

    Directory of Open Access Journals (Sweden)

    Hamed KALANI

    2016-03-01

    Full Text Available Background: Toxoplasmosis is considered as one of the most common infectious diseases caused by the protozoan parasite Toxoplasma gondii. Tachyzoite is the main form of Toxoplasma and continuously is maintained in cell culture or injected into the mice peritoneal cavity. This study was designed to evaluate the survival rate of RH strain of T. gondii tachyzoites in different cell free, nutrient and biological media at different temperatures.Methods: This experimental study was performed at the Toxoplasmosis Research Center, Mazandaran University of Medical Sciences, Sari, Iran, in 2010. One ml of each solution including hypotonic saline (0.3%, normal saline (0.85%, RPMI-1640 (RPMI, RPMI with 10% fetal bovine serum (FBS, RPMI with 20% FBS, ovine hydatid cyst fluid, pasteurized milk of cow, and phosphate buffered saline (PBS along with 4×104 T. gondii tachyzoites were added to plate wells and incubated in 4 °C, 22 °C, 37 °C, and 37 °C under 5% CO2. The survival rate and viability as­sessment of parasites were performed daily and the results were analyzed using Univariate tests.Result: Tachyzoites survival rate in PBS (4 °C and normal saline (4 °C were con­siderably high, compared to other solutions in different conditions (P<0.001. The best temperature for Toxoplasma maintenance was 4 °C (P<0.001.Conclusion: This study introduces two available and economical solutions, PBS (4 °C and normal saline (4 °C media, for maintenance of Toxoplasma tachyzoites as appropriate choice media for a noticeable period of time (11 days in vitro.

  15. Effect of microbial cell-free meat extract on the growth of spoilage bacteria.

    Science.gov (United States)

    Nychas, G-J E; Dourou, D; Skandamis, P; Koutsoumanis, K; Baranyi, J; Sofos, J

    2009-12-01

    This study examined the effect of microbial cell-free meat extract (CFME) derived from spoiled meat, in which quorum sensing (QS) compounds were present, on the growth kinetics (lag phase, and growth rate) of two spoilage bacteria, Pseudomonas fluorescens and Serratia marcescens. Aliquots of CFME from spoiled meat were transferred to Brain Heart Infusion broth inoculated with 10(3) CFU ml(-1) of 18 h cultures of Ps. fluorescens or Ser. marcescens, both fresh meat isolates; CFME derived from unspoiled fresh meat ('clean' meat) served as a control. Changes in impedance measurements were monitored for 48 h, and the detection time (Tdet) was recorded. It was found that in the absence of CFME containing QS compounds the Tdet was shorter (P meat. The rate of growth of Ps. fluorescens, recorded as the maximum slope rate of conductance changes (MSrCC), after Tdet, was higher (P meat. Similar results in MSrCC of impedance changes were obtained for Ser. marcescens. The study indicated that the growth rate (expressed in MSrCC units) of meat spoilage bacteria in vitro was enhanced in samples supplemented with CFME containing QS compounds compared to control samples (i.e., without CFME or with CFME from 'clean' meat). This behaviour may explain the dominant role of these two bacteria in the spoilage of meat. These results illustrate the potential effect of signalling compounds released during storage of meat on the behaviour of meat spoilage bacteria. Understanding such interactions may assist in the control of fresh meat quality and the extension of its shelf life.

  16. Clinical utility of circulating cell-free DNA in advanced colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Allan A Lima Pereira

    Full Text Available Circulating cell-free DNA (cfDNA isolated from the plasma of cancer patients (pts has been shown to reflect the genomic mutation profile of the tumor. However, physician and patient assessment of clinical utility of these assays in patients with metastatic colorectal cancer (mCRC has not been previously described.Patients were prospectively consented to a prospective genomic matching protocol (Assessment of Targeted Therapies Against Colorectal Cancer [ATTACC], with collection of blood for cfDNA extraction and sequencing of a 54-gene panel in a CLIA-certified lab. Formalin-fixed, paraffin-embedded (FFPE tissue from prior resections or biopsies underwent 50-gene sequencing. Results from both assays were returned to the treating physicians for patient care and clinical trial selection. Follow-up surveys of treating physicians and chart reviews assessed clinical utility.128 mCRC pts were enrolled between 6/2014 and 1/2015. Results were returned in median of 13 and 26 days for cfDNA and FFPE sequencing, respectively. With cfDNA sequencing, 78% (100/128 of samples had a detectable somatic genomic alteration. 50% of cfDNA cases had potentially actionable alterations, and 60% of these could be genomically matched to at least one clinical trial in our institution. 50% (15/30 of these pts enrolled onto an identified matched trial. Physicians reported that the cfDNA testing improved the quality of care they could provide in 73% of the cases, and that 89% of pts reported greater satisfaction with the efforts to personalize experimental therapeutic agents.cfDNA sequencing can provide timely information on potentially actionable mutations and amplifications, thereby facilitating clinical trial enrollment and improving the perceived quality of care.

  17. Tumor Cell-Free DNA Copy Number Instability Predicts Therapeutic Response to Immunotherapy.

    Science.gov (United States)

    Weiss, Glen J; Beck, Julia; Braun, Donald P; Bornemann-Kolatzki, Kristen; Barilla, Heather; Cubello, Rhiannon; Quan, Walter; Sangal, Ashish; Khemka, Vivek; Waypa, Jordan; Mitchell, William M; Urnovitz, Howard; Schütz, Ekkehard

    2017-09-01

    Purpose: Chromosomal instability is a fundamental property of cancer, which can be quantified by next-generation sequencing (NGS) from plasma/serum-derived cell-free DNA (cfDNA). We hypothesized that cfDNA could be used as a real-time surrogate for imaging analysis of disease status as a function of response to immunotherapy and as a more reliable tool than tumor biomarkers. Experimental Design: Plasma cfDNA sequences from 56 patients with diverse advanced cancers were prospectively collected and analyzed in a single-blind study for copy number variations, expressed as a quantitative chromosomal number instability (CNI) score versus 126 noncancer controls in a training set of 23 and a blinded validation set of 33. Tumor biomarker concentrations and a surrogate marker for T regulatory cells (Tregs) were comparatively analyzed. Results: Elevated CNI scores were observed in 51 of 56 patients prior to therapy. The blinded validation cohort provided an overall prediction accuracy of 83% (25/30) and a positive predictive value of CNI score for progression of 92% (11/12). The combination of CNI score before cycle (Cy) 2 and 3 yielded a correct prediction for progression in all 13 patients. The CNI score also correctly identified cases of pseudo-tumor progression from hyperprogression. Before Cy2 and Cy3, there was no significant correlation for protein tumor markers, total cfDNA, or surrogate Tregs. Conclusions: Chromosomal instability quantification in plasma cfDNA can serve as an early indicator of response to immunotherapy. The method has the potential to reduce health care costs and disease burden for cancer patients following further validation. Clin Cancer Res; 23(17); 5074-81. ©2017 AACR . ©2017 American Association for Cancer Research.

  18. Investigation of cell-free DNA in canine plasma and its relation to disease.

    Science.gov (United States)

    Burnett, Deborah L; Cave, Nicholas J; Gedye, Kristene R; Bridges, Janis P

    2016-09-01

    DNA is released from dying cells during apoptosis and necrosis. This cell-free DNA (cfDNA) diffuses into the plasma where it can be measured. In humans, an increase in cfDNA correlates with disease severity and prognosis. It was hypothesized that when DNA in canine plasma was measured by emission fluorometry without prior DNA extraction, the concentration of cfDNA would increase with disease severity. The diseased population consisted of 97 client-owned dogs. The clinically normal population consisted of nine client-owned dogs presenting for 'wellness screens', and 15 colony-owned Harrier Hounds. Plasma cfDNA was measured by fluorometry without prior DNA extraction. The effects of ex vivo storage conditions were evaluated in plasma from two clinically normal dogs. In all other dogs, plasma was separated within two hours of collection. The association between the cfDNA concentration in hospitalized dogs and a variety of clinical, clinicopathological and outcome variables was tested. The concentration of cfDNA was reliably measured when plasma was separated within two hours of blood collection. The diseased dogs had significantly higher cfDNA than clinically normal dogs (P Dogs that did not survive to discharge had significantly higher cfDNA concentrations than survivors (P = 0.02). Conclusions/Clinical Importance: The concentration of cfDNA in the plasma of diseased dogs is associated with disease severity and prognosis. Measurement of canine cfDNA could be a useful non-specific disease indicator and prognostic tool.

  19. Quantification of Maternal Serum Cell-Free Fetal DNA in Early-Onset Preeclampsia

    Directory of Open Access Journals (Sweden)

    Mulan Ren

    2013-04-01

    Full Text Available The aim of this study was to determine whether the increased serum cell-free fetal DNA (cffDNA level of gravidas developed into early-onset preeclampsia (EOPE subsequently in the early second trimesters is related to prenatal screening markers. Serum was collected from 1011 gravidas. The level of cffDNA and prenatal screening markers were analyzed in 20 cases with EOPE and 20 controls. All fetuses were male. The maternal serum cffDNA level was assessed by amplification of the Y chromosome specific gene. Correlations between the variables were examined. (Logged cffDNA in EOPE (median, 3.08; interquartile range, 2.93–3.68 was higher than controls (median, 1.79; interquartile range, 1.46–2.53. The increased level of (logged cffDNA was correlated significantly with the increased human chorionic gonadotropin (HCG level (r = 0.628, p < 0.001. Significant reciprocal correlations between cffDNA and babies’ birth weight as well as gestation weeks at delivery were noted (r = −0.516, p = 0.001; r = −0.623, p < 0.001, respectively. The sensitivity and specificity of cffDNA to discriminate between the EOPE cases and the controls were 90% and 85%, respectively. CffDNA is a potential marker for EOPE, which had a significant reciprocal correlation with babies’ birth weight and gestation weeks at delivery. Moreover, it may help in indicating the underlying hypoxic condition in the placenta.

  20. Noninvasive Prenatal Diagnosis of Congenital Adrenal Hyperplasia Using Cell-Free Fetal DNA in Maternal Plasma

    Science.gov (United States)

    Tong, Yu K.; Yuen, Tony; Jiang, Peiyong; Pina, Christian; Chan, K. C. Allen; Khattab, Ahmed; Liao, Gary J. W.; Yau, Mabel; Kim, Se-Min; Chiu, Rossa W. K.; Sun, Li; Zaidi, Mone

    2014-01-01

    Context: Congenital adrenal hyperplasia (CAH) is an autosomal recessive condition that arises from mutations in CYP21A2 gene, which encodes for the steroidogenic enzyme 21-hydroxylase. To prevent genital ambiguity in affected female fetuses, prenatal treatment with dexamethasone must begin on or before gestational week 9. Currently used chorionic villus sampling and amniocentesis provide genetic results at approximately 14 weeks of gestation at the earliest. This means that mothers who want to undergo prenatal dexamethasone treatment will be unnecessarily treating seven of eight fetuses (males and three of four unaffected females), emphasizing the desirability of earlier genetic diagnosis in utero. Objective: The objective of the study was to develop a noninvasive method for early prenatal diagnosis of fetuses at risk for CAH. Patients: Fourteen families, each with a proband affected by phenotypically classical CAH, were recruited. Design: Cell-free fetal DNA was obtained from 3.6 mL of maternal plasma. Using hybridization probes designed to capture a 6-Mb region flanking CYP21A2, targeted massively parallel sequencing (MPS) was performed to analyze genomic DNA samples from parents and proband to determine parental haplotypes. Plasma DNA from pregnant mothers also underwent targeted MPS to deduce fetal inheritance of parental haplotypes. Results: In all 14 families, the fetal CAH status was correctly deduced by targeted MPS of DNA in maternal plasma, as early as 5 weeks 6 days of gestation. Conclusions: MPS on 3.6 mL plasma from pregnant mothers could potentially provide the diagnosis of CAH, noninvasively, before the ninth week of gestation. Only affected female fetuses will thus be treated. Our strategy represents a generic approach for noninvasive prenatal testing for an array of autosomal recessive disorders. PMID:24606108

  1. Ascorbic acid attenuates endothelial permeability triggered by cell-free hemoglobin.

    Science.gov (United States)

    Kuck, Jamie L; Bastarache, Julie A; Shaver, Ciara M; Fessel, Joshua P; Dikalov, Sergey I; May, James M; Ware, Lorraine B

    2018-01-01

    Increased endothelial permeability is central to shock and organ dysfunction in sepsis but therapeutics targeted to known mediators of increased endothelial permeability have been unsuccessful in patient studies. We previously reported that cell-free hemoglobin (CFH) is elevated in the majority of patients with sepsis and is associated with organ dysfunction, poor clinical outcomes and elevated markers of oxidant injury. Others have shown that Vitamin C (ascorbate) may have endothelial protective effects in sepsis. In this study, we tested the hypothesis that high levels of CFH, as seen in the circulation of patients with sepsis, disrupt endothelial barrier integrity. Human umbilical vein endothelial cells (HUVEC) were grown to confluence and treated with CFH with or without ascorbate. Monolayer permeability was measured by Electric Cell-substrate Impedance Sensing (ECIS) or transfer of 14 C-inulin. Viability was measured by trypan blue exclusion. Intracellular ascorbate was measured by HPLC. CFH increased permeability in a dose- and time-dependent manner with 1 mg/ml of CFH increasing inulin transfer by 50% without affecting cell viability. CFH (1 mg/ml) also caused a dramatic reduction in intracellular ascorbate in the same time frame (1.4 mM without CFH, 0.23 mM 18 h after 1 mg/ml CFH, p < 0.05). Pre-treatment of HUVECs with ascorbate attenuated CFH induced permeability. CFH increases endothelial permeability in part through depletion of intracellular ascorbate. Supplementation of ascorbate can attenuate increases in permeability mediated by CFH suggesting a possible therapeutic approach in sepsis. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Investigation of structure and magnetic properties of cobalt-nickel and manganese ferrites nanoparticles synthesized in direct micelles of sodium dodecyl sulphate system

    International Nuclear Information System (INIS)

    Fedosyuk, V.M.; Mirgorod, Yu.A.

    2016-01-01

    Results of investigation of the crystal structure and magnetic properties of the nanoparticles of transition metals ferrites (cobalt, nickel, manganese) synthesized by unified methods using direct sodium dodecyl sulfate micelles are presented. Crystal structure of the samples was investigated by X-ray diffraction on DRON-3M (in the CuKa-radiation). Particle size was investigated by transmission electron microscopy on microscope JEOL JEM-1011 (accelerating voltage 100 kV). All powders contain nanoparticles of the same size in the range 2-6 nm. Magnetic properties of the samples were estimated from temperature and field dependences of the magnetization. All samples exhibit properties of superparamagnets with different blocking temperatures below 45 K. (authors).

  3. Evolutionary selection of enzymatically synthesized semiconductors from biomimetic mineralization vesicles.

    Science.gov (United States)

    Bawazer, Lukmaan A; Izumi, Michi; Kolodin, Dmitriy; Neilson, James R; Schwenzer, Birgit; Morse, Daniel E

    2012-06-26

    The way nature evolves and sculpts materials using proteins inspires new approaches to materials engineering but is still not completely understood. Here, we present a cell-free synthetic biological platform to advance studies of biologically synthesized solid-state materials. This platform is capable of simultaneously exerting many of the hierarchical levels of control found in natural biomineralization, including genetic, chemical, spatial, structural, and morphological control, while supporting the evolutionary selection of new mineralizing proteins and the corresponding genetically encoded materials that they produce. DNA-directed protein expression and enzymatic mineralization occur on polystyrene microbeads in water-in-oil emulsions, yielding synthetic surrogates of biomineralizing cells that are then screened by flow sorting, with light-scattering signals used to sort the resulting mineralized composites differentially. We demonstrate the utility of this platform by evolutionarily selecting newly identified silicateins, biomineralizing enzymes previously identified from the silica skeleton of a marine sponge, for enzyme variants capable of synthesizing silicon dioxide (silica) or titanium dioxide (titania) composites. Mineral composites of intermediate strength are preferentially selected to remain intact for identification during cell sorting, and then to collapse postsorting to expose the encoding genes for enzymatic DNA amplification. Some of the newly selected silicatein variants catalyze the formation of crystalline silicates, whereas the parent silicateins lack this ability. The demonstrated bioengineered route to previously undescribed materials introduces in vitro enzyme selection as a viable strategy for mimicking genetic evolution of materials as it occurs in nature.

  4. Enterococcus faecium LKE12 Cell-Free Extract Accelerates Host Plant Growth via Gibberellin and Indole-3-Acetic Acid Secretion.

    Science.gov (United States)

    Lee, Ko-Eun; Radhakrishnan, Ramalingam; Kang, Sang-Mo; You, Young-Hyun; Joo, Gil-Jae; Lee, In-Jung; Ko, Jae-Hwan; Kim, Jin-Ho

    2015-09-01

    The use of microbial extracts containing plant hormones is a promising technique to improve crop growth. Little is known about the effect of bacterial cell-free extracts on plant growth promotion. This study, based on phytohormonal analyses, aimed at exploring the potential mechanisms by which Enterococcus faecium LKE12 enhances plant growth in oriental melon. A bacterial strain, LKE12, was isolated from soil, and further identified as E. faecium by 16S rDNA sequencing and phylogenetic analysis. The plant growth-promoting ability of an LKE12 bacterial culture was tested in a gibberellin (GA)-deficient rice dwarf mutant (waito-C) and a normal GA biosynthesis rice cultivar (Hwayongbyeo). E. faecium LKE12 significantly improved the length and biomass of rice shoots in both normal and dwarf cultivars through the secretion of an array of gibberellins (GA1, GA3, GA7, GA8, GA9, GA12, GA19, GA20, GA24, and GA53), as well as indole-3-acetic acid (IAA). To the best of our knowledge, this is the first study indicating that E. faecium can produce GAs. Increases in shoot and root lengths, plant fresh weight, and chlorophyll content promoted by E. faecium LKE12 and its cell-free extract inoculated in oriental melon plants revealed a favorable interaction of E. faecium LKE12 with plants. Higher plant growth rates and nutrient contents of magnesium, calcium, sodium, iron, manganese, silicon, zinc, and nitrogen were found in cell-free extract-treated plants than in control plants. The results of the current study suggest that E. faecium LKE12 promotes plant growth by producing GAs and IAA; interestingly, the exogenous application of its cell-free culture extract can be a potential strategy to accelerate plant growth.

  5. Evaluation of Synergistic Interactions Between Cell-Free Supernatant of Lactobacillus Strains and Amikacin and Genetamicin Against Pseudomonas aeruginosa

    OpenAIRE

    Aminnezhad, Sargol; Kermanshahi, Rouha Kasra; Ranjbar, Reza

    2015-01-01

    Background: The indiscriminate use of antibiotics in the treatment of infectious diseases can increase the development of antibiotic resistance. Therefore, there is a big demand for new sources of antimicrobial agents and alternative treatments for reduction of antibiotic dosage required to decrease the associated side effects. Objectives: In this study, the synergistic action of aminoglycoside antibiotics and cell-free supernatant (CFS) of probiotic (Lactobacillus rahmnosus and L. casei) aga...

  6. Ultrastructural Histopathology of Vervet Monkey Colonic Epithelium After In Vitro Exposure to Cell-free Supernatants of Shigella Cultures

    OpenAIRE

    Hill, R. R.; Collins, N. E.; Cowley, H. M.

    2011-01-01

    The full dysentery syndrome of human shigellosis is often preceded by a transient diarrhoea that may be induced by bacterial extracellular products before invasion of the colonic mucosa and development of subsequent pathology. To examine this hypothesis, we studied the effects of cell-free cultures of Shigella sp. on the ultrastructure of monkey colonic epithelium in vitro. Clinical isolates of shigella strains were grown in a niche-simulating medium. Sheets of colon wall collected from verve...

  7. Fully automated parallel oligonucleotide synthesizer

    Czech Academy of Sciences Publication Activity Database

    Lebl, M.; Burger, Ch.; Ellman, B.; Heiner, D.; Ibrahim, G.; Jones, A.; Nibbe, M.; Thompson, J.; Mudra, Petr; Pokorný, Vít; Poncar, Pavel; Ženíšek, Karel

    2001-01-01

    Roč. 66, č. 8 (2001), s. 1299-1314 ISSN 0010-0765 Institutional research plan: CEZ:AV0Z4055905 Keywords : automated oligonucleotide synthesizer Subject RIV: CC - Organic Chemistry Impact factor: 0.778, year: 2001

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

    Directory of Open Access Journals (Sweden)

    A. Konti

    2017-10-01

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

  9. An Extraordinary Accumulation of (-)-Pinoresinol in Cell-Free Extracts of Forsythia intermedia: Evidence for Enantiospecific Reduction of (+)-Pinoresinol

    Science.gov (United States)

    Katayama, Takeshi; Davin, Laurence B.; Lewis, Norman G.

    1992-01-01

    Stereoselective and enantiospecific transformation mechanisms in lignan biogenesis are only now yielding to scientific inquiry: it has been shown that soluble cell-free preparations from Forsythia intermedia catalysis the formation of the enantiomerically pure lignan, (-)-secoisolariciresinol, when incubated with coniferyl alcohol in the presence of NAD(P)H and H2O2. Surprisingly, (-)-pinoresinol also accumulates in this soluble cell-free assay mixture in greater than 96% enantiomeric excess, even though it is not the naturally occurring antipode present in Forsythia sp. But these soluble cell-free preparations do not engender stereoselective coupling; instead, racemic pinoresinols are first formed, catalysed by an H2O2-dependent peroxidase reaction. An enantiospecific NAD(P)H reductase then converts (+)- pinoresinol, and not the (-)-antipode, into (-)-secoisolariciresinol. Stereoselective syntheis of(+)-pinoresinol from E-coniferyl alcohol is, however, catalysed by an insoluble enzyme preparation in F. suspensa, obtained following removal of readily soluble and ionically bound enzymes; no exogenously supplied cofactors were required other than oxygen, although the reaction was stimulated by NAD-malate addition. Thus, the overall biochemical pathway to enantiomerically pure (-)-secoisolariciresinol has been delineated.

  10. Structural study of the membrane protein MscL using cell-free expression and solid-state NMR

    Science.gov (United States)

    Abdine, Alaa; Verhoeven, Michiel A.; Park, Kyu-Ho; Ghazi, Alexandre; Guittet, Eric; Berrier, Catherine; Van Heijenoort, Carine; Warschawski, Dror E.

    2010-05-01

    High-resolution structures of membrane proteins have so far been obtained mostly by X-ray crystallography, on samples where the protein is surrounded by detergent. Recent developments of solid-state NMR have opened the way to a new approach for the study of integral membrane proteins inside a membrane. At the same time, the extension of cell-free expression to the production of membrane proteins allows for the production of proteins tailor made for NMR. We present here an in situ solid-state NMR study of a membrane protein selectively labeled through the use of cell-free expression. The sample consists of MscL (mechano-sensitive channel of large conductance), a 75 kDa pentameric α-helical ion channel from Escherichia coli, reconstituted in a hydrated lipid bilayer. Compared to a uniformly labeled protein sample, the spectral crowding is greatly reduced in the cell-free expressed protein sample. This approach may be a decisive step required for spectral assignment and structure determination of membrane proteins by solid-state NMR.

  11. Generation of hydrogen peroxide from San Joaquin Valley particles in a cell-free solution

    Directory of Open Access Journals (Sweden)

    H. Shen

    2011-01-01

    Full Text Available Epidemiological studies have shown a correlation between exposure to ambient particulate matter (PM and adverse health effects. One proposed mechanism of PM-mediated health effects is the generation of reactive oxygen species (ROS – e.g., superoxide (O2, hydrogen peroxide (HOOH, and hydroxyl radical (OH – followed by oxidative stress. There are very few quantitative, specific measures of individual ROS generated from PM, but this information would help to more quantitatively address the link between ROS and the health effects of PM. To address this gap, we quantified the generation of HOOH by PM collected at an urban (Fresno and rural (Westside site in the San Joaquin Valley (SJV of California during summer and winter from 2006 to 2009. HOOH was quantified by HPLC after extracting the PM in a cell-free, phosphate-buffered saline (PBS solution with or without 50 μM ascorbate (Asc. Our results show that the urban PM generally generates much more HOOH than the rural PM but that there is no apparent seasonal difference in HOOH generation. In nearly all of the samples the addition of a physiologically relevant concentration of Asc greatly enhances HOOH formation, but a few of the coarse PM samples were able to generate a considerable amount of HOOH in the absence of added Asc, indicating the presence of unknown reductants. Normalized by air volume, the fine PM (PM2.5 generally makes more HOOH than the corresponding coarse PM (PMcf, i.e., 2.5 to 10 μm, primarily because the mass concentration of PM2.5 is much higher than that of PMcf. However, normalized by PM mass, the coarse PM typically generates more HOOH than the fine PM. The amount of HOOH produced by SJV PM is reduced on average by (78 ± 15% when the transition metal chelator desferoxamine (DSF is added to the extraction solution, indicating that transition metals play a dominant role in HOOH

  12. Direct quantification of cell-free, circulating DNA from unpurified plasma.

    Science.gov (United States)

    Breitbach, Sarah; Tug, Suzan; Helmig, Susanne; Zahn, Daniela; Kubiak, Thomas; Michal, Matthias; Gori, Tommaso; Ehlert, Tobias; Beiter, Thomas; Simon, Perikles

    2014-01-01

    Cell-free DNA (cfDNA) in body tissues or fluids is extensively investigated in clinical medicine and other research fields. In this article we provide a direct quantitative real-time PCR (qPCR) as a sensitive tool for the measurement of cfDNA from plasma without previous DNA extraction, which is known to be accompanied by a reduction of DNA yield. The primer sets were designed to amplify a 90 and 222 bp multi-locus L1PA2 sequence. In the first module, cfDNA concentrations in unpurified plasma were compared to cfDNA concentrations in the eluate and the flow-through of the QIAamp DNA Blood Mini Kit and in the eluate of a phenol-chloroform isoamyl (PCI) based DNA extraction, to elucidate the DNA losses during extraction. The analyses revealed 2.79-fold higher cfDNA concentrations in unpurified plasma compared to the eluate of the QIAamp DNA Blood Mini Kit, while 36.7% of the total cfDNA were found in the flow-through. The PCI procedure only performed well on samples with high cfDNA concentrations, showing 87.4% of the concentrations measured in plasma. The DNA integrity strongly depended on the sample treatment. Further qualitative analyses indicated differing fractions of cfDNA fragment lengths in the eluate of both extraction methods. In the second module, cfDNA concentrations in the plasma of 74 coronary heart disease patients were compared to cfDNA concentrations of 74 healthy controls, using the direct L1PA2 qPCR for cfDNA quantification. The patient collective showed significantly higher cfDNA levels (mean (SD) 20.1 (23.8) ng/ml; range 5.1-183.0 ng/ml) compared to the healthy controls (9.7 (4.2) ng/ml; range 1.6-23.7 ng/ml). With our direct qPCR, we recommend a simple, economic and sensitive procedure for the quantification of cfDNA concentrations from plasma that might find broad applicability, if cfDNA became an established marker in the assessment of pathophysiological conditions.

  13. Electrospun nanofibers decorated with bio-sonochemically synthesized gold nanoparticles as an ultrasensitive probe in amalgam-based mercury (II) detection system.

    Science.gov (United States)

    Parsaee, Zohreh

    2018-06-01

    In this study, bio-ultrasound-assisted synthesized gold nanoparticles using Gracilaria canaliculata algae have been immobilized on a polymeric support and used as a glassy probe chemosensor for detection and rapid removal of Hg 2+ ions. The function of the suggested chemosensor has been explained based on gold-amalgam formation and its catalytic role on the reaction of sodium borohydride and rhodamine B (RhB) with fluorescent and colorimetric sensing function. The catalyzed reduction of RhB by the gold amalgam led to a distinguished color change from red and yellow florescence to colorless by converting the amount of Hg 2+ deposited on Au-NPs. The detection limit of the colorimetric and fluorescence assays for Hg 2+ was 2.21 nM and 1.10 nM respectively. By exposing the mentioned colorless solution to air for at least 2 h, unexpectedly it was observed that the color and fluorescence of RhB were restored. Have the benefit of the above phenomenon a recyclable and portable glass-based sensor has been provided by immobilizing the Au-NPs and RB on the glass slide using electrospinning. Moreover, the introduced combinatorial membrane has facilitated the detection and removal of Hg 2+ ions in various Hg (II)-contaminated real water samples with efficiency of up to 99%. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. Technical Performance and Clinical Effectiveness of Drop Type With Adjustable Concentrator-Cell Free and Concentrated Ascites Reinfusion Therapy.

    Science.gov (United States)

    Yamada, Yosuke; Harada, Makoto; Yamaguchi, Akinori; Kobayashi, Yasuko; Chino, Takashi; Minowa, Takashi; Kosuge, Takashi; Tsukada, Wataru; Hashimoto, Koji; Kamijo, Yuji

    2017-12-01

    Cell-free and concentrated ascites reinfusion therapy (CART) is a very useful treatment method for refractory ascites but is difficult for many hospitals to employ due to its need for specialized equipment. We have therefore developed drop-type with adjustable concentrator CART (DC-CART) that uses a drop-type filtration mechanism and requires only a simple pump and pressure monitor for its concentration process. Easy adjustment of ascites concentration is possible through a recirculation loop, and filter membrane washing is aided by DC-CART's external pressure-type filtration to enable the processing of any quality or quantity of ascites. Moreover, the absence of a roller pump before filtration avoids inflammatory substance release from compressed cells. A total of 268 sessions of DC-CART using ascites from 98 patients were performed with good clinical results at our hospitals between January 2012 and June 2016. This report presents the detailed methods of DC-CART and summarizes its clinical effectiveness using patient ascites and blood data obtained from 59 sessions between March 2015 and February 2016. This novel technique successfully processed refractory ascites in numerous diseases with no serious adverse events. DC-CART could concentrate large amounts of ascites (from median weight: 4900 g [max: 20 200 g] to median weight: 695 g; median concentration ratio: 7.4), and a high amount of protein (median weight: 73 g [max: 294 g]) could be reinfused. Serum albumin levels were significantly increased (P = 0.010) and kidney function and systemic hemodynamics were well maintained in treated subjects. Additional concentration of ascites and adjustment of ascites volume were easily performed by recirculation (from median weight: 615 g to median weight: 360 g; median concentration ratio: 1.5). Time was needed during DC-CART for filter membrane cleaning, especially for viscous ascites. Overall, DC-CART represents a safe and useful treatment method for various forms

  15. Positive predictive value estimates for cell-free noninvasive prenatal screening from data of a large referral genetic diagnostic laboratory.

    Science.gov (United States)

    Petersen, Andrea K; Cheung, Sau Wai; Smith, Janice L; Bi, Weimin; Ward, Patricia A; Peacock, Sandra; Braxton, Alicia; Van Den Veyver, Ignatia B; Breman, Amy M

    2017-12-01

    Since its debut in 2011, cell-free fetal DNA screening has undergone rapid expansion with respect to both utilization and coverage. However, conclusive data regarding the clinical validity and utility of this screening tool, both for the originally included common autosomal and sex-chromosomal aneuploidies as well as the more recently added chromosomal microdeletion syndromes, have lagged behind. Thus, there is a continued need to educate clinicians and patients about the current benefits and limitations of this screening tool to inform pre- and posttest counseling, pre/perinatal decision making, and medical risk assessment/management. The objective of this study was to determine the positive predictive value and false-positive rates for different chromosomal abnormalities identified by cell-free fetal DNA screening using a large data set of diagnostic testing results on invasive samples submitted to the laboratory for confirmatory studies. We tested 712 patient samples sent to our laboratory to confirm a cell-free fetal DNA screening result, indicating high risk for a chromosome abnormality. We compiled data from all cases in which the indication for confirmatory testing was a positive cell-free fetal DNA screen, including the common trisomies, sex chromosomal aneuploidies, microdeletion syndromes, and other large genome-wide copy number abnormalities. Testing modalities included fluorescence in situ hybridization, G-banded karyotype, and/or chromosomal microarray analysis performed on chorionic villus samples, amniotic fluid, or postnatally obtained blood samples. Positive predictive values and false-positive rates were calculated from tabulated data. The positive predictive values for trisomy 13, 18, and 21 were consistent with previous reports at 45%, 76%, and 84%, respectively. For the microdeletion syndrome regions, positive predictive values ranged from 0% for detection of Cri-du-Chat syndrome and Prader-Willi/Angelman syndrome to 14% for 1p36 deletion

  16. Magnetic and structural properties of Cu{sub 0.85}Fe{sub 0.15}O system synthesized by co-precipitation

    Energy Technology Data Exchange (ETDEWEB)

    Colorado, H. D., E-mail: herdacom@gmail.com; Perez Alcazar, G. A. [Universidad del Valle, Departamento de Fisica (Colombia)

    2011-11-15

    Cu{sub 0.94}Fe{sub 0.06}O and Cu{sub 0.85}Fe{sub 0.15}O samples were synthesized by using the co-precipitation chemical method. Starting from aqueous solutions of copper nitrate, CuO (NO{sub 3}){sub 2} 3H{sub 2}O, iron nitrate, Fe (NO{sub 3}){sub 3} 9H{sub 2}O and sodium hydroxide as precipitating agent, NaOH. The precipitate of three samples for Cu{sub 0.94}Fe{sub 0.06}O and five for Cu{sub 0.85}Fe{sub 0.15}O of fine powder were calcined for 5 h at different temperatures. The obtained X rays diffraction patterns refined by the Rietveld method show the CuO characteristic pattern, showing that the Fe atoms enter to replace Cu atoms. Furthermore, it was obtained that the crystallite size decreases with calcination temperatures for Cu{sub 0.94}Fe{sub 0.06}O. The transmission Moessbauer spectroscopy showed that the samples present a disordered paramagnetic behavior due to the big value of the half-width of line of the quadrupolar splitting. Vibrating sample magnetometry confirms the paramagnetic character. The XRD results indicate that the material is nanostructured, due that the crystallite sizes are of the order of 10 nm for Cu{sub 0.94}Fe{sub 0.06}O and 40 nm for Cu{sub 0.85}Fe{sub 0.15}O.

  17. A comparison of cell-free placental messenger ribonucleic acid and color Doppler ultrasound for the prediction of placental invasion in patients with placenta accreta

    OpenAIRE

    Naghshineh, Elham; Khorvash, Elahe; Kamali, Sara

    2015-01-01

    Background: The aim of the present study was to comparison between cell-free placental messenger ribonucleic acid (mRNA) and Doppler ultrasound for the prediction of placental invasion in women with placenta accreta. Materials and Methods: In this cross-sectional study, 50 pregnant women at risk for placenta accreta underwent color Doppler and assessment of cell-free placental mRNA. Real-time reverse-transcription polymerase chain reaction was used for measurement of cell-free placental m...

  18. Circulating cell-free DNA: an up-coming molecular marker in exercise physiology.

    Science.gov (United States)

    Breitbach, Sarah; Tug, Suzan; Simon, Perikles

    2012-07-01

    The phenomenon of circulating cell-free DNA (cfDNA) concentrations is of importance for many biomedical disciplines including the field of exercise physiology. Increases of cfDNA due to exercise are described to be a potential hallmark for the overtraining syndrome and might be related to, or trigger adaptations of, immune function induced by strenuous exercise. At the same time, exercise provides a practicable model for studying the phenomenon of cfDNA that is described to be of pathophysiological relevance for different topics in clinical medicine like autoimmune diseases and cancer. In this review, we are summarizing the current knowledge of exercise-based acute and chronic alterations in cfDNA levels and their physiological significance. The effects of acute exercise on cfDNA concentrations have been investigated in resistance exercises and in continuous, stepwise and interval endurance exercises of different durations. cfDNA concentrations peaked immediately after acute exercise and showed a rapid return to baseline levels. Typical markers of skeletal muscle damage (creatine kinase, uric acid, C-reactive protein) show delayed kinetics compared with the cfDNA peak response. Exercise parameters such as intensity, duration or average energy expenditure do not explain the extent of increasing cfDNA concentrations after strenuous exercise. This could be due to complex processes inside the human organism during and after physical activity. Therefore, we hypothesize composite effects of different physiological stress parameters that come along with exercise to be responsible for increasing cfDNA concentrations. We suggest that due to acute stress, cfDNA levels increase rapidly by a spontaneous active or passive release mechanism that is not yet known. As a result of the rapid and parallel increase of cfDNA and lactate in an incremental treadmill test leading to exhaustion within 15-20 minutes, it is unlikely that cfDNA is released into the plasma by typical necrosis

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

    Science.gov (United States)

    Nomoto, Mika; Tada, Yasuomi

    2018-01-01

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

  20. Information Retrieval for Ecological Syntheses

    Science.gov (United States)

    Bayliss, Helen R.; Beyer, Fiona R.

    2015-01-01

    Research syntheses are increasingly being conducted within the fields of ecology and environmental management. Information retrieval is crucial in any synthesis in identifying data for inclusion whilst potentially reducing biases in the dataset gathered, yet the nature of ecological information provides several challenges when compared with…

  1. Method of synthesizing pyrite nanocrystals

    Science.gov (United States)

    Wadia, Cyrus; Wu, Yue

    2013-04-23

    A method of synthesizing pyrite nanocrystals is disclosed which in one embodiment includes forming a solution of iron (III) diethyl dithiophosphate and tetra-alkyl-ammonium halide in water. The solution is heated under pressure. Pyrite nanocrystal particles are then recovered from the solution.

  2. MgCo2-D2 and MgCoNi-D2 systems synthesized at high pressures and interaction mechanism during the HDDR processing

    Directory of Open Access Journals (Sweden)

    Chubin Wan

    2017-02-01

    MgCo2 is a new example of the hydrogen storage alloy, in which a successful HDDR processing results in the reversible formation of the initial intermetallic at much lower temperatures than in the equilibrium phase diagram of the Mg-Co system.

  3. Radiation syntheses of Pectin/acrylamide (PEC/PAM) and Pectin/Diethylaminoethylmethacrylate (PEC/DEAMA) hydrogels as drug delivery systems

    International Nuclear Information System (INIS)

    Abou El Fadl, F.I.; Maziad, N.A.

    2015-01-01

    Different pH responsive copolymer hydrogels based on pectin were prepared by the effect of radiation. The physical and chemical properties of prepared hydrogels were studied by FTIR, and TGA. Also, the prepared hydrogels were evaluated for the possible use as drug delivery system for chlortetracycline HCL as model drug. The results revealed that the swelling ratios and the release behavior of hydrogels depend mainly on the pH of the medium and the hydrogel composition. (author)

  4. In Vivo Evaluation of Biocompatibility and Chondrogenic Potential of a Cell-Free Collagen-Based Scaffold

    Directory of Open Access Journals (Sweden)

    Giovanna Calabrese

    2017-11-01

    Full Text Available Injured articular cartilage has a limited innate regenerative capacity, due to the avascular nature and low cellularity of the tissue itself. Although several approaches have been proposed to repair the joint cartilage, none of them has proven to be effective. The absence of suitable therapeutic options has encouraged tissue-engineering approaches combining specific cell types and biomaterials. In the present work, we have evaluated the potential of a cell-free Collagen I-based scaffold to promote the augmentation of cartilage-like phenotype after subcutaneous implantation in the mouse. Forty female mice were grafted subcutaneously with scaffolds, while four additional mice without scaffold were used as negative controls. The effects of scaffold were evaluated at 1, 2, 4, 8, or 16 weeks after implantation. Immunohistochemical analysis shows the expression of typical cartilage markers, including type-II Collagen, Aggrecan, Matrilin-1 and Sox 9. These data are also confirmed by qRT-PCR that further show that both COL2A1 and COL1A1 increase over time, but the first one increases more rapidly, thus suggesting a typical cartilage-like address. Histological analysis shows the presence of some pericellular lacunae, after 8 and 16 weeks. Results suggest that this scaffold (i is biocompatible in vivo, (ii is able to recruit host cells (iii induce chondrogenic differentiation of host cells. Such evidences suggest that this cell-free scaffold is promising and represents a potential approach for cartilage regeneration.

  5. Immobilization of anaerobic thermophilic bacteria for the production of cell-free thermostable. alpha. -amylases and pullulanases

    Energy Technology Data Exchange (ETDEWEB)

    Klingeberg, M [Goettingen Univ. (Germany, F.R.). Inst. fuer Mikrobiologie; Vorlop, K D [Technische Univ. Braunschweig (Germany, F.R.). Inst. fuer Technische Chemie; Antranikian, G [Technische Univ. Hamburg-Harburg, Hamburg (Germany, F. R.). Arbeitsbereich Biotechnologie 1

    1990-08-01

    For the production of cell-free thermostable {alpha}-amylases and pullulanases various anaerobic thermophilic bacteria that belong to the genera Clostridium and Thermoanaerobacter were immobilized in calcium alginate gel beads. The entrapment of bacteria was performed in full was well as in hollow spheres. An optimal limited medium, which avoided bacterial outgrowth, was developed for the cultivation of immobilized organisms at 60deg C using 0.4% starch as substrate. Compared to non-immobilized cells these techniques allowed a significant increase (up to 5.6-fold) in the specific activities of the extracellular enzymes formed. An increase in the productivity of extracellular enzymes was observed after immobilization of bacteria in full spheres. In the case of C. thermosaccharolyticum, for instance, the productivity was raised from 90 units (U)/10{sup 12} cells up to 700 U/10{sup 12} cells. Electrophoretic analysis of the secreted proteins showed that in all cases most of the amylolytic enzymes formed were released into the culture medium. Proteins that had a molecular mass of less than 450 000 daltons could easily diffuse through the gel matrix. Cultivation of immobilized bacteria in semi-continuous and fed-batch cultures was also accompanied by an elevation in the concentration of cell-free enzymes. (orig.).

  6. Microstructural evolution of the system Ni-ZrO{sub 2}-SiO{sub 2} synthesized by the sol-gel process

    Energy Technology Data Exchange (ETDEWEB)

    Garcia Murillo, A., E-mail: angarciam@ipn.m [Instituto Politecnico Nacional, CICATA Unidad Altamira, Km. 14.5, Carretera Tampico-Puerto Industrial Altamira, C.P. 89600 Altamira, Tamaulipas (Mexico); Instituto Politecnico Nacional, CIITEC, Cerrada CECATI S/N Col. Sta. Catarina, Del. Azcapotzalco, Mexico D.F. 02250 (Mexico); Carrillo Romo, F. de J. [Instituto Politecnico Nacional, CICATA Unidad Altamira, Km. 14.5, Carretera Tampico-Puerto Industrial Altamira, C.P. 89600 Altamira, Tamaulipas (Mexico); Instituto Politecnico Nacional, CIITEC, Cerrada CECATI S/N Col. Sta. Catarina, Del. Azcapotzalco, Mexico D.F. 02250 (Mexico); Torres Huerta, A.M.; Dominguez Crespo, M.A.; Ramirez Meneses, E. [Instituto Politecnico Nacional, CICATA Unidad Altamira, Km. 14.5, Carretera Tampico-Puerto Industrial Altamira, C.P. 89600 Altamira, Tamaulipas (Mexico); Terrones, H. [Instituto Potosino de Investigacion Cientifica y Tecnologica, Camino a la Presa San Jose 2055. Colonia Lomas 4 seccion S.L.P. (Mexico); Flores Vela, A. [Instituto Politecnico Nacional, CICATA Unidad Altamira, Km. 14.5, Carretera Tampico-Puerto Industrial Altamira, C.P. 89600 Altamira, Tamaulipas (Mexico)

    2010-04-16

    The analysis of the structural changes corresponding with composition and temperature is fundamental for understanding the applications of ceramic glass. In particular, the system ZrO{sub 2}-SiO{sub 2} is commonly used in the ceramic industry due to the high chemical stability and superior resistance for dissolution during firing in glazes. This work is focused on the synthesis of Ni doped ZrO{sub 2}-SiO{sub 2} powders with different Zr:Si molar ratios (20:80, 50:50 and 70:30) by sol-gel process. For purposes of comparison, un-doped ZrO{sub 2}-SiO{sub 2} systems were also prepared. TEOS, zirconium propoxide and nickel chloride hexahydrate were used as precursors of the SiO{sub 2}, ZrO{sub 2} and ion dopant, respectively. The obtained xerogels were thermally treated from 300 to 1300 {sup o}C in order to follow their structural evolution. FT-IR results show the corresponding bands of the M-O bonds related to the formation of zircon at high temperatures, while XRD analyses display t-ZrO{sub 2} with traces of m-ZrO{sub 2} at the evaluated temperatures; also, between 1200 and 1300 {sup o}C, the zircon compound (ZrSiO{sub 4}) was detected. It was observed that inserting nickel as a dopant has a significant effect on the structural and morphological characteristics. From the comparison of the doped and un-doped specimens, we hypothesize that the presence of Ni and the heat treatment promote the stabilization and crystallization of the zircon phase at molar ratios higher than 50Zr:50Si, or Ni is incorporating into the ZrO{sub 2} phase, provoking oxygen vacancies and leading to tetragonal phase stabilization.

  7. Solid phase syntheses of oligoureas

    Energy Technology Data Exchange (ETDEWEB)

    Burgess, K.; Linthicum, D.S.; Russell, D.H.; Shin, H.; Shitangkoon, A.; Totani, R.; Zhang, A.J.; Ibarzo, J. [Texas A& M Univ., College Station, TX (United States)

    1997-02-19

    Isocyanates 7 were formed from monoprotected diamines 3 or 6, which in turn can be easily prepared from commercially available N-BOC- or N-FMOC-protected amino acid derivatives. Isocyanates 7, formed in situ, could be coupled directly to a solid support functionalized with amine groups or to amino acids anchored on resins using CH{sub 2}Cl{sub 2} as solvent and an 11 h coupling time at 25 {degree}C. Such couplings afforded peptidomimetics with an N-phthaloyl group at the N-terminus. The optimal conditions identified for removal of the N-phthaloyl group were to use 60% hydrazine in DMF for 1-3 h. Several sequences of amino acids coupled to ureas (`peptidic ureas`) and of sequential urea units (`oligoureas`) were prepared via solid phase syntheses and isolated by HPLC. Partition coefficients were measured for two of these peptidomimetics, and their water solubilities were found to be similar to the corresponding peptides. A small library of 160 analogues of the YGGFL-amide sequence was prepared via Houghten`s tea bag methodology. This library was tested for binding to the anti-{beta}-endorphin monoclonal antibody. Overall, this paper describes methodology for solid phase syntheses of oligourea derivatives with side chains corresponding to some of the protein amino acids. The chemistry involved is ideal for high-throughput syntheses and screening operations. 51 refs., 3 figs., 2 tabs.

  8. Microscale to manufacturing scale-up of cell-free cytokine production--a new approach for shortening protein production development timelines.

    Science.gov (United States)

    Zawada, James F; Yin, Gang; Steiner, Alexander R; Yang, Junhao; Naresh, Alpana; Roy, Sushmita M; Gold, Daniel S; Heinsohn, Henry G; Murray, Christopher J

    2011-07-01

    Engineering robust protein production and purification of correctly folded biotherapeutic proteins in cell-based systems is often challenging due to the requirements for maintaining complex cellular networks for cell viability and the need to develop associated downstream processes that reproducibly yield biopharmaceutical products with high product quality. Here, we present an alternative Escherichia coli-based open cell-free synthesis (OCFS) system that is optimized for predictable high-yield protein synthesis and folding at any scale with straightforward downstream purification processes. We describe how the linear scalability of OCFS allows rapid process optimization of parameters affecting extract activation, gene sequence optimization, and redox folding conditions for disulfide bond formation at microliter scales. Efficient and predictable high-level protein production can then be achieved using batch processes in standard bioreactors. We show how a fully bioactive protein produced by OCFS from optimized frozen extract can be purified directly using a streamlined purification process that yields a biologically active cytokine, human granulocyte-macrophage colony-stimulating factor, produced at titers of 700 mg/L in 10 h. These results represent a milestone for in vitro protein synthesis, with potential for the cGMP production of disulfide-bonded biotherapeutic proteins. Copyright © 2011 Wiley Periodicals, Inc.

  9. Green Synthesized Zinc Oxide (ZnO Nanoparticles Induce Oxidative Stress and DNA Damage in Lathyrus sativus L. Root Bioassay System

    Directory of Open Access Journals (Sweden)

    Kamal K. Panda

    2017-05-01

    Full Text Available Zinc oxide nanoparticles (ZnONP-GS were synthesised from the precursor zinc acetate (Zn(CH3COO2 through the green route using the milky latex from milk weed (Calotropis gigantea L. R. Br by alkaline precipitation. Formation of the ZnONP-GS was monitored by UV-visible spectroscopy followed by characterization and confirmation by energy-dispersive X-ray spectroscopy (EDX, transmission electron microscopy (TEM, and X-ray diffraction (XRD. Both the ZnONP-GS and the commercially available ZnONP-S (Sigma-Aldrich and cationic Zn2+ from Zn(CH3COO2 were tested in a dose range of 0–100 mg·L−1 for their potency (i to induce oxidative stress as measured by the generation reactive oxygen species (ROS: O2•−, H2O2 and •OH, cell death, and lipid peroxidation; (ii to modulate the activities of antioxidant enzymes: catalase (CAT, superoxide dismutase (SOD, guaiacol peroxidase (GPX, and ascorbate peroxidase (APX; and (iii to cause DNA damage as determined by Comet assay in Lathyrus sativus L. root bioassay system. Antioxidants such as Tiron and dimethylthiourea significantly attenuated the ZnONP-induced oxidative and DNA damage, suggesting the involvement of ROS therein. Our study demonstrated that both ZnONP-GS and ZnONP-S induced oxidative stress and DNA damage to a similar extent but were significantly less potent than Zn2+ alone.

  10. Green Synthesized Zinc Oxide (ZnO) Nanoparticles Induce Oxidative Stress and DNA Damage in Lathyrus sativus L. Root Bioassay System.

    Science.gov (United States)

    Panda, Kamal K; Golari, Dambaru; Venugopal, A; Achary, V Mohan M; Phaomei, Ganngam; Parinandi, Narasimham L; Sahu, Hrushi K; Panda, Brahma B

    2017-05-18

    Zinc oxide nanoparticles (ZnONP-GS) were synthesised from the precursor zinc acetate (Zn(CH₃COO)₂) through the green route using the milky latex from milk weed ( Calotropis gigantea L. R. Br) by alkaline precipitation. Formation of the ZnONP-GS was monitored by UV-visible spectroscopy followed by characterization and confirmation by energy-dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM), and X-ray diffraction (XRD). Both the ZnONP-GS and the commercially available ZnONP-S (Sigma-Aldrich) and cationic Zn 2+ from Zn(CH₃COO)₂ were tested in a dose range of 0-100 mg·L -1 for their potency (i) to induce oxidative stress as measured by the generation reactive oxygen species (ROS: O₂ •- , H₂O₂ and • OH), cell death, and lipid peroxidation; (ii) to modulate the activities of antioxidant enzymes: catalase (CAT), superoxide dismutase (SOD), guaiacol peroxidase (GPX), and ascorbate peroxidase (APX); and (iii) to cause DNA damage as determined by Comet assay in Lathyrus sativus L. root bioassay system. Antioxidants such as Tiron and dimethylthiourea significantly attenuated the ZnONP-induced oxidative and DNA damage, suggesting the involvement of ROS therein. Our study demonstrated that both ZnONP-GS and ZnONP-S induced oxidative stress and DNA damage to a similar extent but were significantly less potent than Zn 2+ alone.

  11. Anatomical Organization of Urocortin 3-Synthesizing Neurons and Immunoreactive Terminals in the Central Nervous System of Non-Human Primates [Sapajus spp.

    Directory of Open Access Journals (Sweden)

    Daniella S. Battagello

    2017-07-01

    Full Text Available Urocortin 3 (UCN3 is a neuropeptide member of the corticotropin-releasing factor (CRF peptide family that acts as a selective endogenous ligand for the CRF, subtype 2 (CRF2 receptor. Immunohistochemistry and in situ hybridization data from rodents revealed UCN3-containing neurons in discrete regions of the central nervous system (CNS, such as the medial preoptic nucleus, the rostral perifornical area (PFA, the medial nucleus of the amygdala and the superior paraolivary nucleus. UCN3-immunoreactive (UCN3-ir terminals are distributed throughout regions that mostly overlap with regions of CRF2 messenger RNA (mRNA expression. Currently, no similar mapping exists for non-human primates. To better understand the role of this neuropeptide, we aimed to study the UCN3 distribution in the brains of New World monkeys of the Sapajus genus. To this end, we analyzed the gene and peptide sequences in these animals and performed immunohistochemistry and in situ hybridization to identify UCN3 synthesis sites and to determine the distribution of UCN3-ir terminals. The sequencing of the Sapajus spp. UCN3-coding gene revealed 88% and 65% identity to the human and rat counterparts, respectively. Additionally, using a probe generated from monkey cDNA and an antiserum raised against human UCN3, we found that labeled cells are mainly located in the hypothalamic and limbic regions. UCN3-ir axons and terminals are primarily distributed in the ventromedial hypothalamic nucleus (VMH and the lateral septal nucleus (LS. Our results demonstrate that UCN3-producing neurons in the CNS of monkeys are phylogenetically conserved compared to those of the rodent brain, that the distribution of fibers agrees with the distribution of CRF2 in other primates and that there is anatomical evidence for the participation of UCN3 in neuroendocrine control in primates.

  12. ANALISYS OF FRACTIONAL-N FREQUENCY SYNTHESIZERS

    Directory of Open Access Journals (Sweden)

    Boris I. Shakhtarin

    2018-01-01

    Full Text Available Modern information and control systems cannot be imagined without synchronization subsystems. These are the basic elements that provide tracking of the frequency and phase of reference and information signals, the evaluation of information parameters, and the synthesis of reference and clock signals. Frequency synthesizers (FS are widely used due to the high speed of frequency setting, a wide range of frequency grids and minimal phase noise in the operating frequency range. Since with the mass appearance of specialized microprocessors and with the improvement of automatic design systems, the feasibility and repeatability of products has become simpler, digital FS are increasingly being used. The most widely used are FS with a frequency divider on digital elements, which serves to convert the signal of a reference oscillator and a controlled generator. For FS using a divisor with an integer division factor in the feedback loop, there are a number of limitations, such as the lower frequency of the FS and the frequency step of the FS. To solve this problem, divisors with fractional-variable division factors in the feedback loop are used, which allow to obtain the required range and the grid frequency step of the FS. The methods of improving the quality of spectral and dynamic characteristics of digital synthesizers in a given band of frequency detuning are analyzed. The principles of the FS operation with a divisor with a fractionalvariable fission coefficient are described, and structural schemes are given. The results of imitation simulation in the Simulink system of the software package MATLAB of frequency synthesizers with a divisor with a fractional-variable fission factor implemented in various ways are presented, and a comparative analysis of the spectral characteristics of the obtained models is carried out. 

  13. Method of synthesizing tungsten nanoparticles

    Science.gov (United States)

    Thoma, Steven G; Anderson, Travis M

    2013-02-12

    A method to synthesize tungsten nanoparticles has been developed that enables synthesis of nanometer-scale, monodisperse particles that can be stabilized only by tetrahydrofuran. The method can be used at room temperature, is scalable, and the product concentrated by standard means. Since no additives or stabilizing surfactants are required, this method is particularly well suited for producing tungsten nanoparticles for dispersion in polymers. If complete dispersion is achieved due to the size of the nanoparticles, then the optical properties of the polymer can be largely maintained.

  14. Optical fusions and proportional syntheses

    Science.gov (United States)

    Albert-Vanel, Michel

    2002-06-01

    A tragic error is being made in the literature concerning matters of color when dealing with optical fusions. They are still considered to be of additive nature, whereas experience shows us somewhat different results. The goal of this presentation is to show that fusions are, in fact, of 'proportional' nature, tending to be additive or subtractive, depending on each individual case. Using the pointillist paintings done in the manner of Seurat, or the spinning discs experiment could highlight this intermediate sector of the proportional. So, let us try to examine more closely what occurs in fact, by reviewing additive, subtractive and proportional syntheses.

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

    Directory of Open Access Journals (Sweden)

    Masakatsu D Yanagimachi

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

  16. Peritoneal Cell-free DNA: an innovative method for determining acute cell damage in peritoneal membrane and for monitoring the recovery process after peritonitis.

    Science.gov (United States)

    Virzì, Grazia Maria; Milan Manani, Sabrina; Brocca, Alessandra; Cantaluppi, Vincenzo; de Cal, Massimo; Pastori, Silvia; Tantillo, Ilaria; Zambon, Roberto; Crepaldi, Carlo; Ronco, Claudio

    2016-02-01

    Cell-free DNA (cfDNA) is present in the peritoneal effluent of stable peritoneal dialysis (PD) patients, but there are no data on cfDNA in PD patients with peritonitis. We investigated the variation of peritoneal cfDNA levels subsequent to peritonitis in PD patients. We enrolled 53 PD patients: 30 without any history of systemic inflammation or peritonitis in the last 3 months (group A) and 23 with acute peritonitis (group B). CfDNA was quantified in the peritoneal effluent. Peritoneal samples on days 1, 3, 10, 30 and until day 120 from the start of peritonitis were collected for white blood cells (WBC) count and cfDNA evaluation in group B. Quantitative analysis of cfDNA showed significantly higher levels in group B on day 1, 3, 10 and 30 compared with group A (p peritoneal cfDNA levels tended to progressively decline during follow-up of peritonitis. From this decreasing curve, we estimated that 49 days are necessary to reach the value of 51 genome equivalents (GE)/ml (75th percentile in controls) and 63 days to reach 31 GE/ml (median). Our results demonstrate that cfDNA increases in peritoneal effluent of PD patients with peritonitis and tends to progressively decline in step with peritonitis resolution and membrane repair process. Peritoneal cfDNA quantification could be an innovative method to determine acute damage and an inverse index of the repair process.

  17. Cell-free H-cluster synthesis and [FeFe] hydrogenase activation: all five CO and CN⁻ ligands derive from tyrosine.

    Directory of Open Access Journals (Sweden)

    Jon M Kuchenreuther

    Full Text Available [FeFe] hydrogenases are promising catalysts for producing hydrogen as a sustainable fuel and chemical feedstock, and they also serve as paradigms for biomimetic hydrogen-evolving compounds. Hydrogen formation is catalyzed by the H-cluster, a unique iron-based cofactor requiring three carbon monoxide (CO and two cyanide (CN⁻ ligands as well as a dithiolate bridge. Three accessory proteins (HydE, HydF, and HydG are presumably responsible for assembling and installing the H-cluster, yet their precise roles and the biosynthetic pathway have yet to be fully defined. In this report, we describe effective cell-free methods for investigating H-cluster synthesis and [FeFe] hydrogenase activation. Combining isotopic labeling with FTIR spectroscopy, we conclusively show that each of the CO and CN⁻ ligands derive respectively from the carboxylate and amino substituents of tyrosine. Such in vitro systems with reconstituted pathways comprise a versatile approach for studying biosynthetic mechanisms, and this work marks a significant step towards an understanding of both the protein-protein interactions and complex reactions required for H-cluster assembly and hydrogenase maturation.

  18. Aeon: Synthesizing Scheduling Algorithms from High-Level Models

    Science.gov (United States)

    Monette, Jean-Noël; Deville, Yves; van Hentenryck, Pascal

    This paper describes the aeon system whose aim is to synthesize scheduling algorithms from high-level models. A eon, which is entirely written in comet, receives as input a high-level model for a scheduling application which is then analyzed to generate a dedicated scheduling algorithm exploiting the structure of the model. A eon provides a variety of synthesizers for generating complete or heuristic algorithms. Moreover, synthesizers are compositional, making it possible to generate complex hybrid algorithms naturally. Preliminary experimental results indicate that this approach may be competitive with state-of-the-art search algorithms.

  19. Current controversies in prenatal diagnosis 2: Cell-free DNA prenatal screening should be used to identify all chromosome abnormalities.

    Science.gov (United States)

    Chitty, Lyn S; Hudgins, Louanne; Norton, Mary E

    2018-02-01

    Noninvasive prenatal testing (NIPT) using cell-free DNA (cfDNA) from maternal serum has been clinically available since 2011. This technology has revolutionized our ability to screen for the common aneuploidies trisomy 21 (Down syndrome), trisomy 18, and trisomy 13. More recently, clinical laboratories have offered screening for other chromosome abnormalities including sex chromosome abnormalities and copy number variants (CNV) without little published data on the sensitivity, specificity, and positive predictive value. In this debate, the pros and cons of performing prenatal screening via cfDNA for all chromosome abnormalities is discussed. At the time of the debate in 2017, the general consensus was that the literature does not yet support using this technology to screen for all chromosome abnormalities and that education is key for both providers and the patients so that the decision-making process is as informed as possible. © 2018 John Wiley & Sons, Ltd.

  20. Preanalytical blood sample workup for cell-free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics.

    Science.gov (United States)

    van Ginkel, Joost H; van den Broek, Daan A; van Kuik, Joyce; Linders, Dorothé; de Weger, Roel; Willems, Stefan M; Huibers, Manon M H

    2017-10-01

    In current molecular cancer diagnostics, using blood samples of cancer patients for the detection of genetic alterations in plasma (cell-free) circulating tumor DNA (ctDNA) is an emerging practice. Since ctDNA levels in blood are low, highly sensitive Droplet Digital PCR (ddPCR) can be used for detecting rare mutational targets. In order to perform ddPCR on blood samples, a standardized procedure for processing and analyzing blood samples is necessary to facilitate implementation into clinical practice. Therefore, we assessed the technical sample workup procedure for ddPCR on blood plasma samples. Blood samples from healthy individuals, as well as lung cancer patients were analyzed. We compared different methods and protocols for sample collection, storage, centrifugation, isolation, and quantification. Cell-free DNA (cfDNA) concentrations of several wild-type targets and BRAF and EGFR-mutant ctDNA concentrations quantified by ddPCR were primary outcome measurements. Highest cfDNA concentrations were measured in blood collected in serum tubes. No significant differences in cfDNA concentrations were detected between various time points of up to 24 h until centrifugation. Highest cfDNA concentrations were detected after DNA isolation with the Quick cfDNA Serum & Plasma Kit, while plasma isolation using the QIAamp Circulating Nucleic Acid Kit yielded the most consistent results. DdPCR results on cfDNA are highly dependent on multiple factors during preanalytical sample workup, which need to be addressed during the development of this diagnostic tool for cancer diagnostics in the future. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  1. A cell-free scaffold-based cartilage repair provides improved function hyaline-like repair at one year.

    Science.gov (United States)

    Siclari, Alberto; Mascaro, Gennaro; Gentili, Chiara; Cancedda, Ranieri; Boux, Eugenio

    2012-03-01

    Bone marrow stimulation techniques in cartilage repair such as drilling are limited by the formation of fibrous to hyaline-like repair tissue. It has been suggested such techniques can be enhanced by covering the defect with scaffolds. We present an innovative approach using a polyglycolic acid (PGA)-hyaluronan scaffold with platelet-rich-plasma (PRP) in drilling. We asked whether (1) PRP immersed in a cell-free PGA-hyaluronan scaffold improves patient-reported 1-year outcomes for the Knee injury and Osteoarthritis Score (KOOS), and (2) implantation of the scaffold in combination with bone marrow stimulation leads to the formation of hyaline-like cartilage repair tissue. We reviewed 52 patients who had arthroscopic implantation of the PGA-hyaluronan scaffold immersed with PRP in articular cartilage defects of the knee pretreated with Pridie drilling. Patients were assessed by KOOS. At 9 months followup, histologic staining was performed in specimens obtained from five patients to assess the repair tissue quality. The KOOS subscores improved for pain (55 to 91), symptoms (57 to 88), activities of daily living (69 to 86), sports and recreation (36 to 70), and quality of life (38 to 73). The histologic evaluation showed a homogeneous hyaline-like cartilage repair tissue. The cell-free PGA-hyaluronan scaffold combined with PRP leads to cartilage repair and improved patient-reported outcomes (KOOS) during 12 months of followup. Histologic sections showed morphologic features of hyaline-like repair tissue. Long-term followup is needed to determine if the cartilage repair tissue is durable. Level IV, therapeutic study. See the Guidelines for Authors for a complete description of levels of evidence.

  2. Synthesizing lattice structures in phase space

    International Nuclear Information System (INIS)

    Guo, Lingzhen; Marthaler, Michael

    2016-01-01

    In one dimensional systems, it is possible to create periodic structures in phase space through driving, which is called phase space crystals (Guo et al 2013 Phys. Rev. Lett. 111 205303). This is possible even if for particles trapped in a potential without periodicity. In this paper we discuss ultracold atoms in a driven optical lattice, which is a realization of such a phase space crystals. The corresponding lattice structure in phase space is complex and contains rich physics. A phase space lattice differs fundamentally from a lattice in real space, because its coordinate system, i.e., phase space, has a noncommutative geometry, which naturally provides an artificial gauge (magnetic) field. We study the behavior of the quasienergy band structure and investigate the dissipative dynamics. Synthesizing lattice structures in phase space provides a new platform to simulate the condensed matter phenomena and study the intriguing phenomena of driven systems far away from equilibrium. (paper)

  3. Ultra-high-throughput screening of an in vitro-synthesized horseradish peroxidase displayed on microbeads using cell sorter.

    Directory of Open Access Journals (Sweden)

    Bo Zhu

    Full Text Available The C1a isoenzyme of horseradish peroxidase (HRP is an industrially important heme-containing enzyme that utilizes hydrogen peroxide to oxidize a wide variety of inorganic and organic compounds for practical applications, including synthesis of fine chemicals, medical diagnostics, and bioremediation. To develop a ultra-high-throughput screening system for HRP, we successfully produced active HRP in an Escherichia coli cell-free protein synthesis system, by adding disulfide bond isomerase DsbC and optimizing the concentrations of hemin and calcium ions and the temperature. The biosynthesized HRP was fused with a single-chain Cro (scCro DNA-binding tag at its N-terminal and C-terminal sites. The addition of the scCro-tag at both ends increased the solubility of the protein. Next, HRP and its fusion proteins were successfully synthesized in a water droplet emulsion by using hexadecane as the oil phase and SunSoft No. 818SK as the surfactant. HRP fusion proteins were displayed on microbeads attached with double-stranded DNA (containing the scCro binding sequence via scCro-DNA interactions. The activities of the immobilized HRP fusion proteins were detected with a tyramide-based fluorogenic assay using flow cytometry. Moreover, a model microbead library containing wild type hrp (WT and inactive mutant (MUT genes was screened using fluorescence-activated cell-sorting, thus efficiently enriching the WT gene from the 1:100 (WT:MUT library. The technique described here could serve as a novel platform for the ultra-high-throughput discovery of more useful HRP mutants and other heme-containing peroxidases.

  4. Cell-free propagation of Coxiella burnetii does not affect its relative virulence.

    Directory of Open Access Journals (Sweden)

    Runa Kuley

    Full Text Available Q fever is caused by the obligate intracellular bacterium Coxiella burnetii. In vitro growth of the bacterium is usually limited to viable eukaryotic host cells imposing experimental constraints for molecular studies, such as the identification and characterisation of major virulence factors. Studies of pathogenicity may benefit from the recent development of an extracellular growth medium for C. burnetii. However, it is crucial to investigate the consistency of the virulence phenotype of strains propagated by the two fundamentally different culturing systems. In the present study, we assessed the viability of C. burnetii and the lipopolysaccaride (LPS encoding region of the bacteria in both culture systems as indirect but key parameters to the infection potential of C. burnetii. Propidium monoazide (PMA treatment-based real-time PCR was used for enumeration of viable C. burnetii which were validated by fluorescent infectious focus forming unit counting assays. Furthermore, RNA isolated from C. burnetiipropagated in both the culture systems was examined for LPS-related gene expression. All thus far known LPS-related genes were found to be expressed in early passages in both culturing systems indicating the presence of predominantly the phase I form of C. burnetii. Finally, we used immune-competent mice to provide direct evidence, that the relative virulence of different C. burnetii strains is essentially the same for both axenic and cell-based methods of propagation.

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

    Science.gov (United States)

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

    2014-09-01

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

  6. Cell-free assay measuring repair DNA synthesis in human fibroblasts

    International Nuclear Information System (INIS)

    Ciarrocchi, G.; Linn, S.

    1978-01-01

    Osmotic disruption of confluent cultured human fibroblasts that have been irradiated or exposed to chemical carcinogens allows the specific measurement of repair DNA synthesis using dTTP as a precursor. Fibroblasts similarly prepared from various xeroderma pigmentosum cell lines show the deficiencies of uv-induced DNA synthesis predicted from in vivo studies, while giving normal responses to methylmethanesulfonate. A pyrimidine-dimer-specific enzyme, T4 endonuclease V, stimulated the rate of uv-induced repair synthesis with normal and xeroderma pigmentosum cell lines. This system should prove useful for identifying agents that induce DNA repair, and cells that respond abnormally to such induction. It should also be applicable to an in vitro complementation assay with repair-defective cells and proteins obtained from repair-proficient cells. Finally, by using actively growing fibroblasts and thymidine in the system, DNA replication can be measured and studied in vitro

  7. Streptomyces venezuelae TX-TL - a next generation cell-free synthetic biology tool.

    Science.gov (United States)

    Moore, Simon J; Lai, Hung-En; Needham, Hannah; Polizzi, Karen M; Freemont, Paul S

    2017-04-01

    Streptomyces venezuelae is a promising chassis in synthetic biology for fine chemical and secondary metabolite pathway engineering. The potential of S. venezuelae could be further realized by expanding its capability with the introduction of its own in vitro transcription-translation (TX-TL) system. TX-TL is a fast and expanding technology for bottom-up design of complex gene expression tools, biosensors and protein manufacturing. Herein, we introduce a S. venezuelae TX-TL platform by reporting a streamlined protocol for cell-extract preparation, demonstrating high-yield synthesis of a codon-optimized sfGFP reporter and the prototyping of a synthetic tetracycline-inducible promoter in S. venezuelae TX-TL based on the tetO-TetR repressor system. The aim of this system is to provide a host for the homologous production of exotic enzymes from Actinobacteria secondary metabolism in vitro. As an example, the authors demonstrate the soluble synthesis of a selection of enzymes (12-70 kDa) from the Streptomyces rimosus oxytetracycline pathway. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Quantitative characterization of pyrimidine dimer excision from UV-irradiated DNA (excision capacity) by cell-free extracts of the yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Bekker, M.L.; Kaboev, O.K.; Akhmedov, A.T.; Luchkina, L.A.

    1984-01-01

    Cell-free extracts from wild-type yeast (RAD + ) and from rad mutants belonging to the RAD3 epistatic group (rad1-1, rad2-1, rad3-1, rad4-1) contain activities catalyzing the excision of pyrimidine dimers (PD) from purified ultraviolet-irradiated DNA which was not pre-treated with exogenous UV-endonuclease. The level of these activities in cell-free extracts from rad mutants did not differ from that in wild-type extract and was close to the in vivo excision capacity of the latter calculated from the LD 37 (about 10 4 PD per haploid genome). (Auth.)

  9. Cell-free culture supernatant of Bifidobacterium breve CNCM I-4035 decreases pro-inflammatory cytokines in human dendritic cells challenged with Salmonella typhi through TLR activation.

    Science.gov (United States)

    Bermudez-Brito, Miriam; Muñoz-Quezada, Sergio; Gomez-Llorente, Carolina; Matencio, Esther; Bernal, Maria J; Romero, Fernando; Gil, Angel

    2013-01-01

    Dendritic cells (DCs) constitute the first point of contact between gut commensals and our immune system. Despite growing evidence of the immunomodulatory effects of probiotics, the interactions between the cells of the intestinal immune system and bacteria remain largely unknown. Indeed,, the aim of this work was to determine whether the probiotic Bifidobacterium breve CNCM I-4035 and its cell-free culture supernatant (CFS) have immunomodulatory effects in human intestinal-like dendritic cells (DCs) and how they respond to the pathogenic bacterium Salmonella enterica serovar Typhi, and also to elucidate the molecular mechanisms involved in these interactions. Human DCs were directly challenged with B. breve/CFS, S. typhi or a combination of these stimuli for 4 h. The expression pattern of genes involved in Toll-like receptor (TLR) signaling pathway and cytokine secretion was analyzed. CFS decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with S. typhi. In contrast, the B. breve CNCM I-4035 probiotic strain was a potent inducer of the pro-inflammatory cytokines and chemokines tested, i.e., TNF-α, IL-8 and RANTES, as well as anti-inflammatory cytokines including IL-10. CFS restored TGF-β levels in the presence of Salmonella. Live B.breve and its supernatant enhanced innate immune responses by the activation of TLR signaling pathway. These treatments upregulated TLR9 gene transcription. In addition, CFS was a more potent inducer of TLR9 expression than the probiotic bacteria in the presence of S. typhi. Expression levels of CASP8 and IRAK4 were also increased by CFS, and both treatments induced TOLLIP gene expression. Our results indicate that the probiotic strain B. breve CNCM I-4035 affects the intestinal immune response, whereas its supernatant exerts anti-inflammatory effects mediated by DCs. This supernatant may protect immune system from highly infectious agents such as Salmonella typhi and can down-regulate pro

  10. Cell-free culture supernatant of Bifidobacterium breve CNCM I-4035 decreases pro-inflammatory cytokines in human dendritic cells challenged with Salmonella typhi through TLR activation.

    Directory of Open Access Journals (Sweden)

    Miriam Bermudez-Brito

    Full Text Available Dendritic cells (DCs constitute the first point of contact between gut commensals and our immune system. Despite growing evidence of the immunomodulatory effects of probiotics, the interactions between the cells of the intestinal immune system and bacteria remain largely unknown. Indeed,, the aim of this work was to determine whether the probiotic Bifidobacterium breve CNCM I-4035 and its cell-free culture supernatant (CFS have immunomodulatory effects in human intestinal-like dendritic cells (DCs and how they respond to the pathogenic bacterium Salmonella enterica serovar Typhi, and also to elucidate the molecular mechanisms involved in these interactions. Human DCs were directly challenged with B. breve/CFS, S. typhi or a combination of these stimuli for 4 h. The expression pattern of genes involved in Toll-like receptor (TLR signaling pathway and cytokine secretion was analyzed. CFS decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with S. typhi. In contrast, the B. breve CNCM I-4035 probiotic strain was a potent inducer of the pro-inflammatory cytokines and chemokines tested, i.e., TNF-α, IL-8 and RANTES, as well as anti-inflammatory cytokines including IL-10. CFS restored TGF-β levels in the presence of Salmonella. Live B.breve and its supernatant enhanced innate immune responses by the activation of TLR signaling pathway. These treatments upregulated TLR9 gene transcription. In addition, CFS was a more potent inducer of TLR9 expression than the probiotic bacteria in the presence of S. typhi. Expression levels of CASP8 and IRAK4 were also increased by CFS, and both treatments induced TOLLIP gene expression. Our results indicate that the probiotic strain B. breve CNCM I-4035 affects the intestinal immune response, whereas its supernatant exerts anti-inflammatory effects mediated by DCs. This supernatant may protect immune system from highly infectious agents such as Salmonella typhi and can down

  11. Diurnal Variations of Human Circulating Cell-Free Micro-RNA

    DEFF Research Database (Denmark)

    Heegaard, Niels H H; Carlsen, Anting Liu; Lilje, Berit

    2016-01-01

    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...

  12. Applicability of Ion Torrent Colon and Lung sequencing panel on circulating cell-free DNA

    DEFF Research Database (Denmark)

    Demuth, Christina; Tranberg Madsen, Anne; Larsen, Anne Winther

    of targeted sequencing have been optimised for clinical use on FFPE, e.g. the Ion Torrent Colon and Lung panel. The size of DNA extracted from FFPE tissue is comparable with that from cfDNA. We therefore investigated the performance of the clinically relevant Ion Torrent Colon and Lung panel on cfDNA. Methods...... a baseline for the panel. Lastly, the panel was tested on 52 patient samples. Patient plasma samples are from a previously collected cohort of EGFR wild-type non-small cell lung cancer patients (ClinicalTrial.gov: NCT02043002) All samples were sequenced using the Ion Torrent Oncomine Solid Tumor DNA kit...... (Colon and Lung panel) from Thermo Fisher. Sample preparation was performed using the Ion Torrent Chef and sequencing was performed on the Personal Genome Machine (PGM) system. Data was analyzed using the Torrent Suite software, and variants called by Ion Reporter. Results: No somatic mutations were...

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

    Directory of Open Access Journals (Sweden)

    Roberta Troia

    2018-04-01

    Full Text Available Canine gastric dilatation–volvulus (GDV is a life-threatening disease characterized by extensive tissue ischemia, tissue hypoperfusion, and systemic inflammation. Biomarkers that better reflect the severity of gastric necrosis and systemic inflammation would aid clinicians in the management of these patients. This study aimed to investigate the prognostic significance of cell-free DNA (cfDNA, high-mobility group box-1 (HMGB1, and procalcitonin (PCT in dogs with GDV. Concentrations of cfDNA, HMGB1, and PCT were measured in citrated plasma samples collected from 29 dogs with GDV at hospital admission. Additional data collected included baseline lactate concentrations, APPLEfast score, evidence of gastric necrosis, occurrence of postoperative complications, and outcome. Twenty-four healthy dogs were sampled as controls. Continuous variables between groups were compared with the Mann–Whitney U and correlations between continuous variables were assessed by calculation of Spearman’s correlation coefficient. Alpha was set at 0.05. Dogs with GDV had significantly greater concentrations of cfDNA, HMGB1, and PCT compared to controls (P = 0.0009, P = 0.004, and P = 0.009, respectively. PCT concentrations were significantly higher in non-survivors compared to survivors (P = 0.008. Dogs with gastric necrosis had significantly greater lactate concentrations compared to dogs without gastric necrosis (P = 0.0005. The APPLEfast score was not prognostic. Lactate and PCT concentrations were moderately, positively correlated (rs 0.51, P = 0.0005. Concentrations of the inflammatory biomarkers cfDNA, HMGB1, and PCT are increased in canine GDV. Only lactate and PCT concentrations were prognostic in this population of GDV dogs and were predictive of the presence of gastric necrosis and of non-survival to hospital discharge, respectively.

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

    Science.gov (United States)

    Troia, Roberta; Giunti, Massimo; Calipa, Stefano; Goggs, Robert

    2018-01-01

    Canine gastric dilatation-volvulus (GDV) is a life-threatening disease characterized by extensive tissue ischemia, tissue hypoperfusion, and systemic inflammation. Biomarkers that better reflect the severity of gastric necrosis and systemic inflammation would aid clinicians in the management of these patients. This study aimed to investigate the prognostic significance of cell-free DNA (cfDNA), high-mobility group box-1 (HMGB1), and procalcitonin (PCT) in dogs with GDV. Concentrations of cfDNA, HMGB1, and PCT were measured in citrated plasma samples collected from 29 dogs with GDV at hospital admission. Additional data collected included baseline lactate concentrations, APPLE fast score, evidence of gastric necrosis, occurrence of postoperative complications, and outcome. Twenty-four healthy dogs were sampled as controls. Continuous variables between groups were compared with the Mann-Whitney U and correlations between continuous variables were assessed by calculation of Spearman's correlation coefficient. Alpha was set at 0.05. Dogs with GDV had significantly greater concentrations of cfDNA, HMGB1, and PCT compared to controls ( P  = 0.0009, P  = 0.004, and P  = 0.009, respectively). PCT concentrations were significantly higher in non-survivors compared to survivors ( P  = 0.008). Dogs with gastric necrosis had significantly greater lactate concentrations compared to dogs without gastric necrosis ( P  = 0.0005). The APPLE fast score was not prognostic. Lactate and PCT concentrations were moderately, positively correlated ( r s 0.51, P  = 0.0005). Concentrations of the inflammatory biomarkers cfDNA, HMGB1, and PCT are increased in canine GDV. Only lactate and PCT concentrations were prognostic in this population of GDV dogs and were predictive of the presence of gastric necrosis and of non-survival to hospital discharge, respectively.

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

    Science.gov (United States)

    Troia, Roberta; Giunti, Massimo; Calipa, Stefano; Goggs, Robert

    2018-01-01

    Canine gastric dilatation–volvulus (GDV) is a life-threatening disease characterized by extensive tissue ischemia, tissue hypoperfusion, and systemic inflammation. Biomarkers that better reflect the severity of gastric necrosis and systemic inflammation would aid clinicians in the management of these patients. This study aimed to investigate the prognostic significance of cell-free DNA (cfDNA), high-mobility group box-1 (HMGB1), and procalcitonin (PCT) in dogs with GDV. Concentrations of cfDNA, HMGB1, and PCT were measured in citrated plasma samples collected from 29 dogs with GDV at hospital admission. Additional data collected included baseline lactate concentrations, APPLEfast score, evidence of gastric necrosis, occurrence of postoperative complications, and outcome. Twenty-four healthy dogs were sampled as controls. Continuous variables between groups were compared with the Mann–Whitney U and correlations between continuous variables were assessed by calculation of Spearman’s correlation coefficient. Alpha was set at 0.05. Dogs with GDV had significantly greater concentrations of cfDNA, HMGB1, and PCT compared to controls (P = 0.0009, P = 0.004, and P = 0.009, respectively). PCT concentrations were significantly higher in non-survivors compared to survivors (P = 0.008). Dogs with gastric necrosis had significantly greater lactate concentrations compared to dogs without gastric necrosis (P = 0.0005). The APPLEfast score was not prognostic. Lactate and PCT concentrations were moderately, positively correlated (rs 0.51, P = 0.0005). Concentrations of the inflammatory biomarkers cfDNA, HMGB1, and PCT are increased in canine GDV. Only lactate and PCT concentrations were prognostic in this population of GDV dogs and were predictive of the presence of gastric necrosis and of non-survival to hospital discharge, respectively. PMID:29686994

  16. Cell-free expressed bacteriorhodopsin in different soluble membrane mimetics: biophysical properties and NMR accessibility.

    Science.gov (United States)

    Etzkorn, Manuel; Raschle, Thomas; Hagn, Franz; Gelev, Vladimir; Rice, Amanda J; Walz, Thomas; Wagner, Gerhard

    2013-03-05

    Selecting a suitable membrane-mimicking environment is of fundamental importance for the investigation of membrane proteins. Nonconventional surfactants, such as amphipathic polymers (amphipols) and lipid bilayer nanodiscs, have been introduced as promising environments that may overcome intrinsic disadvantages of detergent micelle systems. However, structural insights into the effects of different environments on the embedded protein are limited. Here, we present a comparative study of the heptahelical membrane protein bacteriorhodopsin in detergent micelles, amphipols, and nanodiscs. Our results confirm that nonconventional environments can increase stability of functional bacteriorhodopsin, and demonstrate that well-folded heptahelical membrane proteins are, in principle, accessible by solution-NMR methods in amphipols and phospholipid nanodiscs. Our data distinguish regions of bacteriorhodopsin that mediate membrane/solvent contacts in the tested environments, whereas the protein's functional inner core remains almost unperturbed. The presented data allow comparing the investigated membrane mimetics in terms of NMR spectral quality and thermal stability required for structural studies. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Analysis of human blood plasma cell-free DNA fragment size distribution using EvaGreen chemistry based droplet digital PCR assays.

    Science.gov (United States)

    Fernando, M Rohan; Jiang, Chao; Krzyzanowski, Gary D; Ryan, Wayne L

    2018-04-12

    Plasma cell-free DNA (cfDNA) fragment size distribution provides important information required for diagnostic assay development. We have developed and optimized droplet digital PCR (ddPCR) assays that quantify short and long DNA fragments. These assays were used to analyze plasma cfDNA fragment size distribution in human blood. Assays were designed to amplify 76,135, 490 and 905 base pair fragments of human β-actin gene. These assays were used for fragment size analysis of plasma cell-free, exosome and apoptotic body DNA obtained from normal and pregnant donors. The relative percentages for 76, 135, 490 and 905 bp fragments from non-pregnant plasma and exosome DNA were 100%, 39%, 18%, 5.6% and 100%, 40%, 18%,3.3%, respectively. The relative percentages for pregnant plasma and exosome DNA were 100%, 34%, 14%, 23%, and 100%, 30%, 12%, 18%, respectively. The relative percentages for non-pregnant plasma pellet (obtained after 2nd centrifugation step) were 100%, 100%, 87% and 83%, respectively. Non-pregnant Plasma cell-free and exosome DNA share a unique fragment distribution pattern which is different from pregnant donor plasma and exosome DNA fragment distribution indicating the effect of physiological status on cfDNA fragment size distribution. Fragment distribution pattern for plasma pellet that includes apoptotic bodies and nuclear DNA was greatly different from plasma cell-free and exosome DNA. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  18. Elevated levels of circulating cell-free DNA and neutrophil proteins are associated with neonatal sepsis and necrotizing enterocolitis in immature mice, pigs and infants

    DEFF Research Database (Denmark)

    Nguyen, Duc Ninh; Stensballe, Allan; Lai, Jacqueline C.Y.

    2017-01-01

    Preterm infants are highly susceptible to late-onset sepsis (LOS) and necrotizing enterocolitis (NEC), but disease pathogenesis and specific diagnostic markers are lacking. Circulating cell-free DNA (cfDNA) and immune cell-derived proteins are involved in multiple immune diseases in adults but ha...

  19. Quantitative cell-free DNA, KRAS, and BRAF mutations in plasma from patients with metastatic colorectal cancer during treatment with cetuximab and irinotecan

    DEFF Research Database (Denmark)

    Spindler, Karen-Lise Garm; Pallisgaard, Niels; Vogelius, Ivan Storgaard

    2012-01-01

    The present study investigated the levels of circulating cell-free DNA (cfDNA) in plasma from patients with metastatic colorectal cancer (mCRC) in relation to third-line treatment with cetuximab and irinotecan and the quantitative relationship of cfDNA with tumor-specific mutations in plasma....

  20. Synthesizing biomolecule-based Boolean logic gates.

    Science.gov (United States)

    Miyamoto, Takafumi; Razavi, Shiva; DeRose, Robert; Inoue, Takanari

    2013-02-15

    One fascinating recent avenue of study in the field of synthetic biology is the creation of biomolecule-based computers. The main components of a computing device consist of an arithmetic logic unit, the control unit, memory, and the input and output devices. Boolean logic gates are at the core of the operational machinery of these parts, and hence to make biocomputers a reality, biomolecular logic gates become a necessity. Indeed, with the advent of more sophisticated biological tools, both nucleic acid- and protein-based logic systems have been generated. These devices function in the context of either test tubes or living cells and yield highly specific outputs given a set of inputs. In this review, we discuss various types of biomolecular logic gates that have been synthesized, with particular emphasis on recent developments that promise increased complexity of logic gate circuitry, improved computational speed, and potential clinical applications.

  1. Synthesizing Biomolecule-based Boolean Logic Gates

    Science.gov (United States)

    Miyamoto, Takafumi; Razavi, Shiva; DeRose, Robert; Inoue, Takanari

    2012-01-01

    One fascinating recent avenue of study in the field of synthetic biology is the creation of biomolecule-based computers. The main components of a computing device consist of an arithmetic logic unit, the control unit, memory, and the input and output devices. Boolean logic gates are at the core of the operational machinery of these parts, hence to make biocomputers a reality, biomolecular logic gates become a necessity. Indeed, with the advent of more sophisticated biological tools, both nucleic acid- and protein-based logic systems have been generated. These devices function in the context of either test tubes or living cells and yield highly specific outputs given a set of inputs. In this review, we discuss various types of biomolecular logic gates that have been synthesized, with particular emphasis on recent developments that promise increased complexity of logic gate circuitry, improved computational speed, and potential clinical applications. PMID:23526588

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

    Directory of Open Access Journals (Sweden)

    Evangelia Papadimou

    2015-04-01

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

  3. Comparison of first-tier cell-free DNA screening for common aneuploidies with conventional publically funded screening.

    Science.gov (United States)

    Langlois, Sylvie; Johnson, JoAnn; Audibert, François; Gekas, Jean; Forest, Jean-Claude; Caron, André; Harrington, Keli; Pastuck, Melanie; Meddour, Hasna; Tétu, Amélie; Little, Julian; Rousseau, François

    2017-12-01

    This study evaluates the impact of offering cell-free DNA (cfDNA) screening as a first-tier test for trisomies 21 and 18. This is a prospective study of pregnant women undergoing conventional prenatal screening who were offered cfDNA screening in the first trimester with clinical outcomes obtained on all pregnancies. A total of 1198 pregnant women were recruited. The detection rate of trisomy 21 with standard screening was 83% with a false positive rate (FPR) of 5.5% compared with 100% detection and 0% FPR for cfDNA screening. The FPR of cfDNA screening for trisomies 18 and 13 was 0.09% for each. Two percent of women underwent an invasive diagnostic procedure based on screening or ultrasound findings; without the cfDNA screening, it could have been as high as 6.8%. Amongst the 640 women with negative cfDNA results and a nuchal translucency (NT) ultrasound, only 3 had an NT greater or equal to 3.5 mm: one had a normal outcome and two lost their pregnancy before 20 weeks. cfDNA screening has the potential to be a highly effective first-tier screening approach leading to a significant reduction of invasive diagnostic procedures. For women with a negative cfDNA screening result, NT measurement has limited clinical utility. © 2017 John Wiley & Sons, Ltd.

  4. Determining the role of mother race in neonatal outcome of trisomies 21, 18 and 13 using cell free DNA analysis

    Directory of Open Access Journals (Sweden)

    Najmie Saadati

    2016-12-01

    Full Text Available To determine the role of mother race in neonatal outcome of trisomies 21, 18 and 13 using cell free DNA (cf-DNA analysis. All women administered for a sonographic imaging at their 10-22 weeks’ gestation which were qualified for cf-DNA testing were suggested for increasing aneuploidy risk, between March 1, 2015 to March 30 , 2016. The cf-DNA analysis was conducted after women received genetic counseling in a specialty laboratory. The results were validated by amniocentesis. A total of 1992 women were screened using cf-DNA analysis. The participants were 1631 Non Arabs (Fars, Kurd, and Lor and 361 Arabs. The fetus risk for trisomy 21 in the Arab women of Arab race was two as much as Non Arab race, but trisomies 18 and 13 in women of Non Arab race were more than Arab race. The role of mother race (such as Arab and Non Arab in neonatal outcome is very important.

  5. Contingent first-trimester screening for aneuploidies with cell-free DNA in a Danish clinical setting

    DEFF Research Database (Denmark)

    Miltoft, Caroline Borregaard; Rode, Line; Ekelund, Charlotte Kvist

    2017-01-01

    OBJECTIVES: The primary aim was to compare the screening performance for Trisomy 21, of standard combined first trimester screening with referral to invasive testing at a cut-off at 1 in 300, with a contingent testing, consisting of referral to invasive testing at a 1 in 100 cut-off and referral...... to cell-free DNA (cfDNA) testing for a risk between 1 in 100 and 1 in 1000. METHODS: Singleton pregnant women with a combined first trimester risk ≥ 1 in 1000 were consecutively recruited from two Danish hospitals between August 2014 and May 2015. First trimester combined screening was based on maternal...... these there were 15 cases of Trisomy 21, one case of Trisomy 18 and two cases of Trisomy 13. The sensitivity for Trisomy 21 was 100% using both screening scenarios, while specificity increased significantly from 97.0% to 98.8% (p contingent approach. The sensitivity for Trisomy 21, 18 and 13...

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

    Science.gov (United States)

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

    2015-12-29

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

  7. Quantification of circulating cell-free DNA in the serum of patients with obstructive sleep apnea-hypopnea syndrome.

    Science.gov (United States)

    Ye, Liang; Ma, Guan-Hua; Chen, Ling; Li, Min; Liu, Jia-Lin; Yang, Kun; Li, Qing-Yun; Li, Ning; Wan, Huan-Ying

    2010-12-01

    Serum cell-free DNA concentrations have been reported to increase in many acute diseases as well as in some chronic conditions such as cancer and autoimmune diseases. The aim of this study was to examine whether serum DNA concentrations were elevated in patients with obstructive sleep apnea-hypopnea syndrome (OSAHS). The effects of nasal continuous positive airway pressure (nCPAP) on serum DNA were also investigated. One hundred twenty-seven people diagnosed with OSAHS by polysomnography (PSG) were admitted into the OSAHS group, and 52 subjects without OSAHS were recruited for the control group. The OSAHS group was further divided into mild, moderate, and severe OSAHS subgroups based on their apnea-hypopnea index (AHI) during sleep. Ten patients with moderate and severe OSAHS were treated with nCPAP. Serum DNA, interleukin-6 (IL-6), and malonaldehyde (MDA) concentrations were measured and were found to be significantly higher in patients with moderate and severe OSAHS groups than those in the mild OSAHS and control groups (p DNA correlated positively with AHI, oxygen desaturation index (ODI), IL-6, and MDA, and negatively correlated with minimal oxygen saturation (miniSaO(2)) (all p DNA concentrations. After 6 months of nCPAP therapy, serum concentrations of DNA, IL-6, and MDA were significantly decreased (p DNA in patients with OSAHS was positively correlated with disease severity. Serum DNA may become an important parameter for monitoring the severity of OSAHS and effectiveness of therapy.

  8. Epigenetic markers in circulating cell-free DNA as prognostic markers for survival of castration-resistant prostate cancer patients.

    Science.gov (United States)

    Hendriks, Rianne J; Dijkstra, Siebren; Smit, Frank P; Vandersmissen, Johan; Van de Voorde, Hendrik; Mulders, Peter F A; van Oort, Inge M; Van Criekinge, Wim; Schalken, Jack A

    2018-04-01

    Noninvasive biomarkers to guide personalized treatment for castration-resistant prostate cancer (CRPC) are needed. In this study, we analyzed hypermethylation patterns of two genes (GSTP1 and APC) in plasma cell-free DNA (cfDNA) of CRPC patients. The aim of this study was to analyze the cfDNA concentrations and levels of the epigenetic markers and to assess the value of these biomarkers for prognosis. In this prospective study, patients were included before starting new treatment after developing CRPC. The blood samples were collected prior to start of the treatment and at three time points thereafter. cfDNA was extracted from 1.5 mL of plasma and before performing a methylation-specific PCR, bisulfate modification was carried out. The median levels of cfDNA, GSTP1, and APC copies in the baseline samples of CRPC patients (n = 47) were higher than in controls (n = 30). In the survival analysis, the group with baseline marker levels below median had significant less PCa-related deaths (P-values Prostate Published by Wiley Periodicals, Inc.

  9. Determining antioxidant activities of lactobacilli cell-free supernatants by cellular antioxidant assay: a comparison with traditional methods.

    Directory of Open Access Journals (Sweden)

    Jiali Xing

    Full Text Available Antioxidant activity of lactic acid bacteria is associated with multiple health-protective effects. Traditional indexes of chemical antioxidant activities poorly reflect the antioxidant effects of these bacteria in vivo. Cellular antioxidant activity (CAA assay was used in this study to determine the antioxidant activity of cell-free supernatants (CFSs of 10 Lactobacillus strains. The performance of the CAA assay was compared with that of four chemical antioxidant activity assays, namely, DPPH radical scavenging, hydroxyl radical scavenging (HRS, reducing power (RP, and inhibition of linoleic acid peroxidation (ILAP. Results of the CAA assay were associated with those of DPPH and ILAP assays, but not with those of RP and HRS assays. The inter- and intra-specific antioxidant activities of CFS were characterized by chemical and CAA assays. L. rhamnosus CCFM 1107 displayed a high antioxidative effect similar to positive control L. rhamnosus GG ATCC 53103 in all of the assays. The CAA assay is a potential method for the detection of antioxidant activities of lactobacilli CFSs.

  10. Can the Roche hemolysis index be used for automated determination of cell-free hemoglobin? A comparison to photometric assays.

    Science.gov (United States)

    Petrova, Darinka Todorova; Cocisiu, Gabriela Ariadna; Eberle, Christoph; Rhode, Karl-Heinz; Brandhorst, Gunnar; Walson, Philip D; Oellerich, Michael

    2013-09-01

    The aim of this study was to develop a novel method for automated quantification of cell-free hemoglobin (fHb) based on the HI (Roche Diagnostics). The novel fHb method based on the HI was correlated with fHb measured using the triple wavelength methods of both Harboe [fHb, g/L = (0.915 * HI + 2.634)/100] and Fairbanks et al. [fHb, g/L = (0.917 * HI + 2.131)/100]. fHb concentrations were estimated from the HI using the Roche Modular automated platform in self-made and commercially available quality controls, as well as samples from a proficiency testing scheme (INSTAND). The fHb using Roche automated HI results were then compared to results obtained using the traditional spectrophotometric assays for one hundred plasma samples with varying degrees of hemolysis, lipemia and/or bilirubinemia. The novel method using automated HI quantification on the Roche Modular clinical chemistry platform correlated well with results using the classical methods in the 100 patient samples (Harboe: r = 0.9284; Fairbanks et al.: r = 0.9689) and recovery was good for self-made controls. However, commercially available quality controls showed poor recovery due to an unidentified matrix problem. The novel method produced reliable determination of fHb in samples without interferences. However, poor recovery using commercially available fHb quality control samples currently greatly limits its usefulness. © 2013.

  11. Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

    Directory of Open Access Journals (Sweden)

    Peng Xia

    2009-01-01

    Full Text Available Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf mitochondrial (mtDNA and nuclear (nDNA DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.

  12. Non-thermal radio frequency and static magnetic fields increase rate of hemoglobin deoxygenation in a cell-free preparation.

    Directory of Open Access Journals (Sweden)

    David Muehsam

    Full Text Available The growing body of clinical and experimental data regarding electromagnetic field (EMF bioeffects and their therapeutic applications has contributed to a better understanding of the underlying mechanisms of action. This study reports that two EMF modalities currently in clinical use, a pulse-modulated radiofrequency (PRF signal, and a static magnetic field (SMF, applied independently, increased the rate of deoxygenation of human hemoglobin (Hb in a cell-free assay. Deoxygenation of Hb was initiated using the reducing agent dithiothreitol (DTT in an assay that allowed the time for deoxygenation to be controlled (from several min to several hours by adjusting the relative concentrations of DTT and Hb. The time course of Hb deoxygenation was observed using visible light spectroscopy. Exposure for 10-30 min to either PRF or SMF increased the rate of deoxygenation occurring several min to several hours after the end of EMF exposure. The sensitivity and biochemical simplicity of the assay developed here suggest a new research tool that may help to further the understanding of basic biophysical EMF transduction mechanisms. If the results of this study were to be shown to occur at the cellular and tissue level, EMF-enhanced oxygen availability would be one of the mechanisms by which clinically relevant EMF-mediated enhancement of growth and repair processes could occur.

  13. SNPase-ARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients.

    Directory of Open Access Journals (Sweden)

    Julia Stadler

    Full Text Available Cell-free circulating tumor DNA in the plasma of cancer patients has become a common point of interest as indicator of therapy options and treatment response in clinical cancer research. Especially patient- and tumor-specific single nucleotide variants that accurately distinguish tumor DNA from wild type DNA are promising targets. The reliable detection and quantification of these single-base DNA variants is technically challenging. Currently, a variety of techniques is applied, with no apparent "gold standard". Here we present a novel qPCR protocol that meets the conditions of extreme sensitivity and specificity that are required for detection and quantification of tumor DNA. By consecutive application of two polymerases, one of them designed for extreme base-specificity, the method reaches unprecedented sensitivity and specificity. Three qPCR assays were tested with spike-in experiments, specific for point mutations BRAF V600E, PTEN T167A and NRAS Q61L of melanoma cell lines. It was possible to detect down to one copy of tumor DNA per reaction (Poisson distribution, at a background of up to 200 000 wild type DNAs. To prove its clinical applicability, the method was successfully tested on a small cohort of BRAF V600E positive melanoma patients.

  14. Increased Plasma Cell-Free DNA Level during HTNV Infection: Correlation with Disease Severity and Virus Load

    Directory of Open Access Journals (Sweden)

    Jing Yi

    2014-07-01

    Full Text Available Cell-free DNA (cf-DNA in blood represents a promising DNA damage response triggered by virus infection or trauma, tumor, etc. Hantavirus primarily causes two diseases: haemorrhagic fever with renal syndrome (HFRS and Hantavirus cardiopulmonary syndrome (HCPS, depending on different Hantavirus species. The aim of this study was to evaluate plasma cf-DNA levels in acute phase of HFRS, and to correlate plasma cf-DNA with disease severity and plasma Hanttan virus (HTNV load. We observed the appearance of cf-DNA in 166 plasma samples from 76 HFRS patients: the plasma cf-DNA levels peaked at the hypotensive stage of HFRS, and then decreased gradually. Until the diuretic stage, there was no significant difference in plasma cf-DNA level between patients and the healthy control. Exclusively in the febrile/hypotensive stage, the plasma cf-DNA levels of severe/critical patients were higher than those of the mild/moderate group. Moreover, the plasma cf-DNA value in the early stage of HFRS was correlated with HTNV load and disease severity. In most of the patients, plasma cf-DNA displayed a low-molecular weight appearance, corresponding to the size of apoptotic DNA. In conclusion, the plasma cf-DNA levels were dynamically elevated during HFRS, and correlated with disease severity, which suggests that plasma cf-DNA may be a potential biomarker for the pathogenesis and prognosis of HFRS.

  15. Rapid profiling of antimicrobial compounds characterising B. subtilis TR50 cell-free filtrate by high-performance liquid chromatography coupled to high-resolution Orbitrap™ mass spectrometry.

    Science.gov (United States)

    Monaci, Linda; Quintieri, Laura; Caputo, Leonardo; Visconti, Angelo; Baruzzi, Federico

    2016-01-15

    Several Bacillus strains, typically isolated from different food sources, represent renowned producers of a multitude of low and high molecular weight compounds, including lipopeptides and macrolactones, with an importance for their antimicrobial activity. The high homology shared by many of these compounds also occurring as closely related isoforms poses a challenge in their prompt detection. Identification and structural elucidation is generally achieved by matrix-assisted laser desorption/ionization (MALDI) or liquid chromatography (LC) coupled to mass spectrometry (MS) after a pre-fractionation and/or purification step of the extract. In this paper we report the application of a method based on LC separation and high-resolution Orbitrap™-based MS for the rapid screening of raw filtrate of the strain Bacillus subtilis TR50 endowed with antimicrobial activity, without requiring any sample pre-treatment. Upon direct analysis of the cell-free filtrate of Bacillus subtilis TR50 by high-resolution mass spectrometry (HRMS), different compounds families, that proved to exert a remarked antimicrobial activity against several foodborne pathogens, can be readily displayed along the chromatographic run. Among them, three different classes were identified and characterized belonging to the iturin, fengycin and surfactin groups. The high resolving power and accurate mass accuracy provided by the HRMS system in use ensured an enhanced selectivity compared to other mass spectrometers. In addition, after activation of the HCD cell, the HR-MS/MS spectra can provide insights in the structural elucidation of several compounds. The acquisition of HRMS spectra of raw filtrates of subtilis strains allows untargeted analysis of the major classes of compounds produced to be performed, thus facilitating identification of other unknown bioactive molecules after retrospective analysis. These features make this approach a fast tool applicable to the rapid screening and further

  16. Acute high-intensity interval exercise induces comparable levels of circulating cell-free DNA and Interleukin-6 in obese and normal-weight individuals.

    Science.gov (United States)

    Ferrandi, Peter J; Fico, Brandon G; Whitehurst, Michael; Zourdos, Michael C; Bao, Fanchen; Dodge, Katelyn M; Rodriguez, Alexandra L; Pena, Gabriel; Huang, Chun-Jung

    2018-06-01

    Obesity is associated with lipid aggregation in adipocytes and macrophage infiltration, leading to increased oxidative stress and inflammation. Increased cell-free DNA (cfDNA) concentrations have been observed in clinical conditions of systemic inflammation. While the beneficial effects of regular physical activity on the release of circulating cfDNA still remain unknown, acute intense exercise has been shown to increase inflammatory cytokines and cfDNA concentrations in normal-weight individuals. Therefore, the primary purpose of this study was to examine the effect of acute high-intensity interval Exercise (HIIE) on plasma cfDNA and interleukin-6 (IL-6) responses in obese and normal-weight subjects. Fourteen male subjects (7 obese and 7 normal-weight) participated in an acute HIIE protocol (30 min, 4x4min @ 80% - 90% of VO 2max ) on a treadmill. Between HIIE intervals, subjects performed 3 min of active recovery at 50-60% VO 2max . Blood samples were collected prior to, immediately following exercise, and one hour into recovery for measurements of plasma cfDNA and IL-6. Our results demonstrated a significant elevation in plasma cfDNA immediately following acute HIIE in both obese and normal-weight subjects. A comparable elevation in the concentration of plasma IL-6 was also found between two groups in response to acute HIIE. Furthermore, the level of plasma cfDNA was not correlated with IL-6 either at baseline or in response to acute HIIE. These findings may support the utilization of HIIE as a time-efficient exercise protocol to understand the obesity-associated cfDNA and inflammatory responses. Published by Elsevier Inc.

  17. Inhibition of chemiluminescence by carvedilol in the cell-free system, whole human blood and blood cells

    Czech Academy of Sciences Publication Activity Database

    Nosál, R.; Jančinová, V.; Číž, Milan; Drábiková, K.; Lojek, Antonín; Fábryová, V.

    2005-01-01

    Roč. 65, č. 1 (2005), s. 55-64 ISSN 0036-5513 Institutional research plan: CEZ:AV0Z50040507 Keywords : blood platelets * carvedilol * chemiluminescence Subject RIV: BO - Biophysics Impact factor: 0.946, year: 2005

  18. Calcium fluxes across the plasma membrane of Commelina communis L. assayed in a cell-free system

    International Nuclear Information System (INIS)

    Siebers, B.; Graef, P.; Weiler, E.W.

    1990-01-01

    The inside-out fraction of plasma membrane-rich vesicles prepared from leaves of Commelina communis L. by aqueous two-phase partitioning was loaded with 45 Ca 2+ through the action of the plasma membrane Ca 2+ -ATPase. Results suggest the presence of a Ca 2+ channel in the plasma membrane of C. communis. The channel is obtained in a Ca 2+ -inactivated state after preparation and Ca 2+ -loading of the vesicles. The inactivation is removed by TFP [trifluoperazine] or W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], presumably due to the Ca 2+ -mobilizing effect of these compounds. The activated Ca 2+ channel is La 3+ sensitive and, in the cell, would allow for passage of Ca 2+ into the cell. The possibility that TFP or W-7 act independent of CM, or through CM tightly associated with the plasma membrane, is discussed

  19. Designer lipid-like peptides: a class of detergents for studying functional olfactory receptors using commercial cell-free systems.

    Science.gov (United States)

    Corin, Karolina; Baaske, Philipp; Ravel, Deepali B; Song, Junyao; Brown, Emily; Wang, Xiaoqiang; Wienken, Christoph J; Jerabek-Willemsen, Moran; Duhr, Stefan; Luo, Yuan; Braun, Dieter; Zhang, Shuguang

    2011-01-01

    A crucial bottleneck in membrane protein studies, particularly G-protein coupled receptors, is the notorious difficulty of finding an optimal detergent that can solubilize them and maintain their stability and function. Here we report rapid production of 12 unique mammalian olfactory receptors using short designer lipid-like peptides as detergents. The peptides were able to solubilize and stabilize each receptor. Circular dichroism showed that the purified olfactory receptors had alpha-helical secondary structures. Microscale thermophoresis suggested that the receptors were functional and bound their odorants. Blot intensity measurements indicated that milligram quantities of each olfactory receptor could be produced with at least one peptide detergent. The peptide detergents' capability was comparable to that of the detergent Brij-35. The ability of 10 peptide detergents to functionally solubilize 12 olfactory receptors demonstrates their usefulness as a new class of detergents for olfactory receptors, and possibly other G-protein coupled receptors and membrane proteins.

  20. Designer lipid-like peptides: a class of detergents for studying functional olfactory receptors using commercial cell-free systems.

    Directory of Open Access Journals (Sweden)

    Karolina Corin

    Full Text Available A crucial bottleneck in membrane protein studies, particularly G-protein coupled receptors, is the notorious difficulty of finding an optimal detergent that can solubilize them and maintain their stability and function. Here we report rapid production of 12 unique mammalian olfactory receptors using short designer lipid-like peptides as detergents. The peptides were able to solubilize and stabilize each receptor. Circular dichroism showed that the purified olfactory receptors had alpha-helical secondary structures. Microscale thermophoresis suggested that the receptors were functional and bound their odorants. Blot intensity measurements indicated that milligram quantities of each olfactory receptor could be produced with at least one peptide detergent. The peptide detergents' capability was comparable to that of the detergent Brij-35. The ability of 10 peptide detergents to functionally solubilize 12 olfactory receptors demonstrates their usefulness as a new class of detergents for olfactory receptors, and possibly other G-protein coupled receptors and membrane proteins.

  1. Synthesizing Earth's geochemical data for hydrogeochemical analysis

    Science.gov (United States)

    Brantley, S. L.; Kubicki, J.; Miller, D.; Richter, D.; Giles, L.; Mitra, P.

    2007-12-01

    For over 200 years, geochemical, microbiological, and chemical data have been collected to describe the evolution of the surface earth. Many of these measurements are data showing variations in time or in space. To forward predict hydrologic response to changing tectonic, climatic, or anthropogenic forcings requires synthesis of these data and utilization in hydrogeochemical models. Increasingly, scientists are attempting to synthesize such data in order to make predictions for new regions or for future time periods. However, to make such complex geochemical data accessible requires development of sophisticated cyberinfrastructures that both invite uploading as well as usage of data. Two such cyberinfrastructure (CI) initiatives are currently developing, one to invite and promote the use of environmental kinetics data (laboratory time course data) through ChemxSeer, and the other to invite and promote the use of spatially indexed geochemical data for the Earth's Critical Zone through CZEN.org. The vision of these CI initiatives is to provide cyber-enhanced portals that encourage domain scientists to upload their data before publication (in private cyberspace), and to make these data eventually publicly accessible (after an embargo period). If the CI can be made to provide services to the domain specialist - e.g. to provide data analysis services or data comparison services - we envision that scientists will upload data. In addition, the CI can promote the use and comparison of datasets across disciplines. For example, the CI can facilitate the use of spatially indexed geochemical data by scientists more accustomed to dealing with time-course data for hydrologic flow, and can provide user-friendly interfaces with CI established to facilitate the use of hydrologic data. Examples of the usage of synthesized data to predict soil development over the last 13ky and its effects on active hydrological flow boundaries in surficial systems will be discussed for i) a N

  2. Plasma mSEPT9: A Novel Circulating Cell-free DNA-Based Epigenetic Biomarker to Diagnose Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Abderrahim Oussalah

    2018-04-01

    Full Text Available Summary: Background: Patients with cirrhosis are at high risk of hepatocellular carcinoma (HCC. The SEPT9 gene is a key regulator of cell division and tumor suppressor whose hypermethylation is associated with liver carcinogenesis. The primary aim of this study was to evaluate the diagnostic accuracy of a PCR-based assay for the analysis of SEPT9 promoter methylation in circulating cell-free DNA (mSEPT9 for diagnosing HCC among cirrhotic patients. Methods: We report two phase II biomarker studies that included cirrhotic patients with or without HCC from France (initial study and Germany (replication study. All patients received clinical and biological evaluations, and liver imaging according to current recommendations. The primary outcome was defined as the presence of HCC according to guidelines from the American Association for the Study of Liver Diseases. The diagnosis of HCC was confirmed by abdominal contrast-enhanced computed tomography scan and systematically discussed in a multidisciplinary consultation meeting. HCC-free cirrhotic patients were recruited if the screening abdominal ultrasound showed no evidence of HCC at the time of blood sampling for the mSEPT9 test and on the next visit six months later. The adjudicating physicians were blinded to patient results associated with the mSEPT9 test. Findings: We included 289 patients with cirrhosis (initial: 186; replication: 103, among whom 98 had HCC (initial: 51; replication: 47. The mSEPT9 test exhibited high diagnostic accuracy for HCC diagnosis, with an area under the receiver operating characteristic curve (AUROC of 0.944 (0.900–0.970, p < 0.0001 in the initial study (replication: 0.930 [0.862–0.971, p < 0.0001]; meta-analysis: AUROC = 0.940 [0.910–0.970, p < 0.0001], no heterogeneity: I2 = 0%, p = 0.67; and no publication bias. In multivariate logistic regression analysis, the number of positive mSEPT9 triplicates was the only independent variable significantly

  3. Genome-wide DNA Methylation Profiling of Cell-Free Serum DNA in Esophageal Adenocarcinoma and Barrett Esophagus

    Directory of Open Access Journals (Sweden)

    Rihong Zhai

    2012-01-01

    Full Text Available Aberrant DNA methylation (DNAm is a feature of most types of cancers. Genome-wide DNAm profiling has been performed successfully on tumor tissue DNA samples. However, the invasive procedure limits the utility of tumor tissue for epidemiological studies. While recent data indicate that cell-free circulating DNAm (cfDNAm profiles reflect DNAm status in corresponding tumor tissues, no studies have examined the association of cfDNAm with cancer or precursors on a genome-wide scale. The objective of this pilot study was to evaluate the putative significance of genome-wide cfDNAm profiles in esophageal adenocarcinoma (EA and Barrett esophagus (BE, EA precursor. We performed genome-wide DNAm profiling in EA tissue DNA (n = 8 and matched serum DNA (n = 8, in serum DNA of BE (n = 10, and in healthy controls (n = 10 using the Infinium HumanMethylation27 BeadChip that covers 27,578 CpG loci in 14,495 genes. We found that cfDNAm profiles were highly correlated to DNAm profiles in matched tumor tissue DNA (r = 0.92 in patients with EA. We selected the most differentially methylated loci to perform hierarchical clustering analysis. We found that 911 loci can discriminate perfectly between EA and control samples, 554 loci can separate EA from BE samples, and 46 loci can distinguish BE from control samples. These results suggest that genome-wide cfDNAm profiles are highly consistent with DNAm profiles detected in corresponding tumor tissues. Differential cfDNAm profiling may be a useful approach for the noninvasive screening of EA and EA premalignant lesions.

  4. Effect of L. plantarum cell-free extract and co-trimoxazole against Salmonella Typhimurium: a possible adjunct therapy

    Directory of Open Access Journals (Sweden)

    Kaur Prabhjot

    2011-02-01

    Full Text Available Abstract Background Frequent and indiscriminate use of antibiotics has led to the development of multi-drug resistant bacterial strains. It necessitates the exploitation of alternative therapeutic strategies. In order to reduce the dose of antibiotic required and to decrease the associated side effects, the present study was aimed at evaluating the synergism, if any, between a conventional antibiotic, co-trimoxazole (CTZ and cell free supernatant (CFS of a probiotic (L. plantarum against S. Typhimurium NCTC 74. This antimicrobial combination was selected on the basis of antibiotic susceptibility pattern of Salmonella and L. plantarum. Methods The synergy was evaluated in terms of size of zone of inhibition, fractional inhibitory concentration index, time-kill assay (in-vitro as well as macrophage functions (ex-vivo. Results The concentration producing the same or higher antibacterial effect (size of zone of inhibition was reduced to half when both the agents were used in combination with respect to the concentrations required when used separately. CTZ and CFS exhibited synergetic activity against Salmonella by checkerboard microtitre test and the time-kill test. Ex-vivo studies demonstrated a significantly higher intracellular killing of bacteria by macrophages treated with CFS (80 AU/ml + (CTZ (2 μg/ml as compared to when treated with both separately at higher concentrations. Significant reduction in the extent of lipid peroxidation and nitrite levels generated by macrophages in presence of CFS and CTZ, in conjunction, further substantiated the synergistic efficacy of the combination. Conclusions The antimicrobial efficacy of this combination indicates that it may serve as the basis in developing alternative strategies to combat Salmonella infections.

  5. Perception of Paralinguistic Traits in Synthesized Voices

    DEFF Research Database (Denmark)

    Baird, Alice Emily; Hasse Jørgensen, Stina; Parada-Cabaleiro, Emilia

    2017-01-01

    Along with the rise of artificial intelligence and the internet-of-things, synthesized voices are now common in daily–life, providing us with guidance, assistance, and even companionship. From formant to concatenative synthesis, the synthesized voice continues to be defined by the same traits we...

  6. Composites comprising biologically-synthesized nanomaterials

    Science.gov (United States)

    Curran, Seamus; Dias, Sampath; Blau, Werner; Wang, Jun; Oremland, Ronald S; Baesman, Shaun

    2013-04-30

    The present disclosure describes composite materials containing a polymer material and a nanoscale material dispersed in the polymer material. The nanoscale materials may be biologically synthesized, such as tellurium nanorods synthesized by Bacillus selenitireducens. Composite materials of the present disclosure may have optical limiting properties and find use in optical limiting devices.

  7. Rapid point-of-care testing for epidermal growth factor receptor gene mutations in patients with lung cancer using cell-free DNA from cytology specimen supernatants.

    Science.gov (United States)

    Asaka, Shiho; Yoshizawa, Akihiko; Saito, Kazusa; Kobayashi, Yukihiro; Yamamoto, Hiroshi; Negishi, Tatsuya; Nakata, Rie; Matsuda, Kazuyuki; Yamaguchi, Akemi; Honda, Takayuki

    2018-06-01

    Epidermal growth factor receptor (EGFR) mutations are associated with responses to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small-cell lung cancer (NSCLC). Our previous study revealed a rapid point-of-care system for detecting EGFR mutations. This system analyzes cell pellets from cytology specimens using droplet-polymerase chain reaction (d-PCR), and has a reaction time of 10 min. The present study aimed to validate the performance of the EGFR d-PCR assay using cell-free DNA (cfDNA) from supernatants obtained from cytology specimens. Assay results from cfDNA supernatant analyses were compared with those from cell pellets for 90 patients who were clinically diagnosed with, or suspected of having, lung cancer (80 bronchial lavage fluid samples, nine pleural effusion samples and one spinal fluid sample). EGFR mutations were identified in 12 and 15 cases using cfDNA supernatants and cell pellets, respectively. The concordance rates between cfDNA-supernatant and cell‑pellet assay results were 96.7% [kappa coefficient (K)=0.87], 98.9% (K=0.94), 98.9% (K=0.79) and 98.9% (K=0.79) for total EGFR mutations, L858R, E746_A750del and T790M, respectively. All 15 patients with EGFR mutation-positive results, as determined by EGFR d-PCR assay using cfDNA supernatants or cell pellets, also displayed positive results by conventional EGFR assays using tumor tissue or cytology specimens. Notably, EGFR mutations were even detected in five cfDNA supernatants for which the cytological diagnoses of the corresponding cell pellets were 'suspicious for malignancy', 'atypical' or 'negative for malignancy.' In conclusion, this rapid point-of-care system may be considered a promising novel screening method that may enable patients with NSCLC to receive EGFR-TKI therapy more rapidly, whilst also reserving cell pellets for additional morphological and molecular analyses.

  8. The correlation between cell-free DNA and tumour burden was estimated by PET/CT in patients with advanced NSCLC

    DEFF Research Database (Denmark)

    Nygaard, A D; Holdgaard, Paw; Spindler, K-L G

    2014-01-01

    Background:Cell-free DNA (cfDNA) circulating in the blood holds a possible prognostic value in malignant diseases. Under malignant conditions, the level of cfDNA increases but the biological mechanism remains to be fully understood. We aimed to examine the correlation between cfDNA and total tumour...... burden defined by positron emission tomography (PET) parameters.Methods:Patients with advanced non-small cell lung cancer (NSCLC) were enrolled into a prospective biomarker trial. Before treatment, plasma was extracted and the level of cfDNA was determined by qPCR. An (18)F-fluorodeoxyglucose ((18)F...... analysis. MTV>the median was associated with a significantly shorter OS (P=0.02). There was no significant difference in OS according to TLG (P=0.08).Conclusion:Cell-free DNA may not be a simple measure of tumour burden, but seems to reflect more complex mechanisms of tumour biology, making it attractive...

  9. Wheat germ cell-free expression: Two detergents with a low critical micelle concentration allow for production of soluble HCV membrane proteins.

    Science.gov (United States)

    Fogeron, Marie-Laure; Badillo, Aurélie; Jirasko, Vlastimil; Gouttenoire, Jérôme; Paul, David; Lancien, Loick; Moradpour, Darius; Bartenschlager, Ralf; Meier, Beat H; Penin, François; Böckmann, Anja

    2015-01-01

    Membrane proteins are notoriously difficult to express in a soluble form. Here, we use wheat germ cell-free expression in the presence of various detergents to produce the non-structural membrane proteins 2, 4B and 5A of the hepatitis C virus (HCV). We show that lauryl maltose neopentyl glycol (MNG-3) and dodecyl octaethylene glycol ether (C12E8) detergents can yield essentially soluble membrane proteins at detergent concentrations that do not inhibit the cell-free reaction. This finding can be explained by the low critical micelle concentration (CMC) of these detergents, which keeps the monomer concentrations low while at the same time providing the necessary excess of detergent concentration above CMC required for full target protein solubilization. We estimate that a tenfold excess of detergent micelles with respect to the protein concentration is sufficient for solubilization, a number that we propose as a guideline for detergent screening assays. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Observer design for non linear systems: application to automatic fault detection in process engineering; Synthese d'observateurs pour les systemes non lineaires. Application a la detection automatique de pannes en genie des procedes

    Energy Technology Data Exchange (ETDEWEB)

    Armanet, F.

    1999-04-01

    This thesis describes some theoretical contributions in state observer design for non linear systems and the conception of an automatic fault detector system for a petrochemical process. The first chapter is an overview of the observer theory for non linear systems. The second chapter presents a new methodology of high gain observer design for single-output U-uniformly observable systems. It consists in calculate a symmetric positive definite matrix which allows the design of an high gain observer which is exponentially converging. This observer is applied to estimate the concentrations in a perfectly mixed tank reactor with a kinetic scheme corresponding to the conversion of a product A onto a product B which is also converting onto a product C. In the third chapter, the use of high gain observer is extended for systems which are not uniformly observable but all admissible inputs are locally regularly persistent. A characterization of some of this class of inputs is given and an application for the preceding reactor illustrates this theory. The fourth chapter includes a summary of the observer used in residual generator design for linear and non linear systems. Two examples of automatic fault detector using these methods are describes. In annexed documents, a detailed study of the process modeling and the main observability properties are presented. (author)

  11. Validation of liquid biopsy: plasma cell-free DNA testing in clinical management of advanced non-small cell lung cancer

    OpenAIRE

    Veldore,Vidya; Choughule,Anuradha; Routhu,Tejaswi; Mandloi,Nitin; Noronha,Vanita; Joshi,Amit; Dutt,Amit; Gupta,Ravi; Vedam,Ramprasad; Prabhash,Kumar

    2018-01-01

    Vidya H Veldore,1,* Anuradha Choughule,2,* Tejaswi Routhu,1 Nitin Mandloi,1 Vanita Noronha,2 Amit Joshi,2 Amit Dutt,3 Ravi Gupta,1 Ramprasad Vedam,1 Kumar Prabhash2 1MedGenome Labs Private Ltd,, Bangalore, India; 2Tata Memorial Centre, Parel, Mumbai, India; 3The Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Center, Kharghar, Navi Mumbai, Maharashtra, India *These authors contributed equally to this work Abstract: Plasma cell-free tumor DNA, or circulating tumo...

  12. Inhibition of the early stage of Salmonella enterica serovar Enteritidis biofilm development on stainless steel by cell-free supernatant of a Hafnia alvei culture.

    Science.gov (United States)

    Chorianopoulos, Nikos G; Giaouris, Efstathios D; Kourkoutas, Yiannis; Nychas, George-John E

    2010-03-01

    Compounds present in Hafnia alvei cell-free culture supernatant cumulatively negatively influence the early stage of biofilm development by Salmonella enterica serovar Enteritidis on stainless steel while they also reduce the overall metabolic activity of S. Enteritidis planktonic cells. Although acylhomoserine lactones (AHLs) were detected among these compounds, the use of several synthetic AHLs was not able to affect the initial stage of biofilm formation by this pathogen.

  13. Quantitative analysis of plasma cell-free DNA and its DNA integrity in patients with metastatic prostate cancer using ALU sequence

    International Nuclear Information System (INIS)

    Fawzy, A.; Sweify, K.M.; Nofal, N.; El-Fayoumy, H.M.

    2016-01-01

    Background: Prostate cancer (PC) is the most common cancer affecting men, it accounts for 29% of all male cancer and 11% of all male cancer related death. DNA is normally released from an apoptotic source which generates small fragments of cell-free DNA, whereas cancer patients have cell-free circulating DNA that originated from necrosis, autophagy, or mitotic catastrophe, which produce large fragments. Aim of work: Differentiate the cell free DNA levels (cfDNA) and its integrity in prostate cancer patients and control group composed of benign prostate hyperplasia (BPH) and healthy persons. Methodology: cf-DNA levels were quantified by real-time PCR amplification in prostate cancer patients ( n = 50), (BPH) benign prostate hyperplasia ( n = 25) and healthy controls ( n = 30) using two sets of ALU gene (product size of 115 bp and 247-bp) and its integrity was calculated as a ratio of qPCR results of 247 bp ALU over 115 bp ALU. Results: Highly significant levels of cf-DNA and its integrity in PC patients compared to BPH. Twenty-eight (56%) patients with prostate cancer had bone metastasis. ALU115 qpcr is superior to the other markers in discriminating metastatic patients with a sensitivity of 96.4% and a specificity of 86.4% and (AUC = 0.981) Conclusion: ALU115 qpcr could be used as a valuable biomarker helping in identifying high risk patients, indicating early spread of tumor cells as a potential seed for future metastases

  14. Formation of hydroxyl radical from San Joaquin Valley particles extracted in a cell-free surrogate lung fluid

    Directory of Open Access Journals (Sweden)

    H. Shen

    2011-09-01

    Full Text Available Previous studies have suggested that the adverse health effects from ambient particulate matter (PM are linked to the formation of reactive oxygen species (ROS by PM in cardiopulmonary tissues. While hydroxyl radical (OH is the most reactive of the ROS species, there are few quantitative studies of OH generation from PM. Here we report on OH formation from PM collected at an urban (Fresno and rural (Westside site in the San Joaquin Valley (SJV of California. We quantified OH in PM extracts using a cell-free, phosphate-buffered saline (PBS solution with or without 50 μM ascorbate (Asc. The results show that generally the urban Fresno PM generates much more OH than the rural Westside PM. The presence of Asc at a physiologically relevant concentration in the extraction solution greatly enhances OH formation from all the samples. Fine PM (PM2.5 generally makes more OH than the corresponding coarse PM (PMcf, i.e. with diameters of 2.5 to 10 μm normalized by air volume collected, while the coarse PM typically generates more OH normalized by PM mass. OH production by SJV PM is reduced on average by (97 ± 6 % when the transition metal chelator desferoxamine (DSF is added to the extraction solution, indicating a dominant role of transition metals. By measuring calibration curves of OH generation from copper and iron, and quantifying copper and iron concentrations in our particle extracts, we find that PBS-soluble copper is primarily responsible for OH production by the SJV PM, while iron often makes a significant contribution. Extrapolating our results to expected burdens of PM-derived OH in human lung lining fluid suggests that typical daily PM exposures in the San Joaquin Valley are unlikely to result in a high amount of pulmonary OH, although high

  15. Cell Free DNA of Tumor Origin Induces a 'Metastatic' Expression Profile in HT-29 Cancer Cell Line.

    Directory of Open Access Journals (Sweden)

    István Fűri

    Full Text Available Epithelial cells in malignant conditions release DNA into the extracellular compartment. Cell free DNA of tumor origin may act as a ligand of DNA sensing mechanisms and mediate changes in epithelial-stromal interactions.To evaluate and compare the potential autocrine and paracrine regulatory effect of normal and malignant epithelial cell-related DNA on TLR9 and STING mediated pathways in HT-29 human colorectal adenocarcinoma cells and normal fibroblasts.DNA isolated from normal and tumorous colonic epithelia of fresh frozen surgically removed tissue samples was used for 24 and 6 hour treatment of HT-29 colon carcinoma and HDF-α fibroblast cells. Whole genome mRNA expression analysis and qRT-PCR was performed for the elements/members of TLR9 signaling pathway. Immunocytochemistry was performed for epithelial markers (i.e. CK20 and E-cadherin, DNA methyltransferase 3a (DNMT3a and NFκB (for treated HDFα cells.Administration of tumor derived DNA on HT29 cells resulted in significant (p<0.05 mRNA level alteration in 118 genes (logFc≥1, p≤0.05, including overexpression of metallothionein genes (i.e. MT1H, MT1X, MT1P2, MT2A, metastasis-associated genes (i.e. TACSTD2, MACC1, MALAT1, tumor biomarker (CEACAM5, metabolic genes (i.e. INSIG1, LIPG, messenger molecule genes (i.e. DAPP, CREB3L2. Increased protein levels of CK20, E-cadherin, and DNMT3a was observed after tumor DNA treatment in HT-29 cells. Healthy DNA treatment affected mRNA expression of 613 genes (logFc≥1, p≤0.05, including increased expression of key adaptor molecules of TLR9 pathway (e.g. MYD88, IRAK2, NFκB, IL8, IL-1β, STING pathway (ADAR, IRF7, CXCL10, CASP1 and the FGF2 gene.DNA from tumorous colon epithelium, but not from the normal epithelial cells acts as a pro-metastatic factor to HT-29 cells through the overexpression of pro-metastatic genes through TLR9/MYD88 independent pathway. In contrast, DNA derived from healthy colonic epithelium induced TLR9 and STING signaling

  16. Non-invasive prenatal cell-free fetal DNA testing for down syndrome and other chromosomal abnormalities

    Directory of Open Access Journals (Sweden)

    Darija Strah

    2015-12-01

    Full Text Available Background: Chorionic villus sampling and amniocentesis as definitive diagnostic procedures represent a gold standard for prenatal diagnosis of chromosomal abnormalities. The methods are invasive and lead to a miscarriage and fetal loss in approximately 0.5–1 %. Non-invasive prenatal DNA testing (NIPT is based on the analysis of cell-free fetal DNA from maternal blood. It represents a highly accurate screening test for detecting the most common fetal chromosomal abnormalities. In our study we present the results of NIPT testing in the Diagnostic Center Strah, Slovenia, over the last 3 years.Methods: In our study, 123 pregnant women from 11th to 18th week of pregnancy were included. All of them had First trimester assessment of risk for trisomy 21, done before NIPT testing.Results: 5 of total 6 high-risk NIPT cases (including 3 cases of Down syndrome and 2 cases of Klinefelter’s syndrome were confirmed by fetal karyotyping. One case–Edwards syndrome was false positive. Patau syndrome, triple X syndrome or Turner syndrome were not observed in any of the cases. Furthermore, there were no false negative cases reported. In general, NIPT testing had 100 % sensitivity (95 % confidence interval: 46.29 %–100.00 % and 98.95 % specificity (95 % confidence interval: 93.44 %–99.95 %. In determining Down syndrome alone, specificity (95 % confidence interval: 95.25 %- 100.00 % and sensitivity (95 % confidence interval: 31.00 %–100.00 % turned out to be 100 %. In 2015, the average turnaround time for analysis was 8.3 days from the day when the sample was taken. Repeated blood sampling was required in 2 cases (redraw rate = 1.6 %.Conclusions: Our results confirm that NIPT represents a fast, safe and highly accurate advanced screening test for most common chromosomal abnormalities. In current clinical practice, NIPT would significantly decrease the number of unnecessary invasive procedures and the rate of fetal

  17. A cell-free fluorometric high-throughput screen for inhibitors of Rtt109-catalyzed histone acetylation.

    Directory of Open Access Journals (Sweden)

    Jayme L Dahlin

    Full Text Available The lysine acetyltransferase (KAT Rtt109 forms a complex with Vps75 and catalyzes the acetylation of histone H3 lysine 56 (H3K56ac in the Asf1-H3-H4 complex. Rtt109 and H3K56ac are vital for replication-coupled nucleosome assembly and genotoxic resistance in yeast and pathogenic fungal species such as Candida albicans. Remarkably, sequence homologs of Rtt109 are absent in humans. Therefore, inhibitors of Rtt109 are hypothesized as potential and minimally toxic antifungal agents. Herein, we report the development and optimization of a cell-free fluorometric high-throughput screen (HTS for small-molecule inhibitors of Rtt109-catalyzed histone acetylation. The KAT component of the assay consists of the yeast Rtt109-Vps75 complex, while the histone substrate complex consists of full-length Drosophila histone H3-H4 bound to yeast Asf1. Duplicated assay runs of the LOPAC demonstrated day-to-day and plate-to-plate reproducibility. Approximately 225,000 compounds were assayed in a 384-well plate format with an average Z' factor of 0.71. Based on a 3σ cut-off criterion, 1,587 actives (0.7% were identified in the primary screen. The assay method is capable of identifying previously reported KAT inhibitors such as garcinol. We also observed several prominent active classes of pan-assay interference compounds such as Mannich bases, catechols and p-hydroxyarylsulfonamides. The majority of the primary active compounds showed assay signal interference, though most assay artifacts can be efficiently removed by a series of straightforward counter-screens and orthogonal assays. Post-HTS triage demonstrated a comparatively small number of confirmed actives with IC50 values in the low micromolar range. This assay, which utilizes five label-free proteins involved in H3K56 acetylation in vivo, can in principle identify compounds that inhibit Rtt109-catalyzed H3K56 acetylation via different mechanisms. Compounds discovered via this assay or adaptations thereof could

  18. Implications of failure to achieve a result from prenatal maternal serum cell-free DNA testing: a historical cohort study.

    Science.gov (United States)

    Chan, N; Smet, M-E; Sandow, R; da Silva Costa, F; McLennan, A

    2017-11-01

    To investigate the pregnancy outcomes in a cohort of women who failed to obtain a result in non-invasive prenatal testing (NIPT). Historical cohort study. A multicentre private practice in Sydney, Australia. Women who failed to obtain a result from NIPT (n = 131). The maternal characteristics, antenatal investigations and pregnancy outcomes for these women were compared with those who obtained a result at the same practice and to the general Australian obstetric population. Antenatal investigations: pregnancy-associated plasma protein-A (PAPP-A), free β-human chorionic gonadotrophin (β-hCG), placental growth factor (PlGF), uterine artery pulsatility index (PI), mean arterial pressure (MAP). Pregnancy outcomes: chromosomal abnormality, pre-eclampsia, gestational diabetes, small-for-gestational-age (SGA), preterm delivery. Only 1.1% of NIPT samples failed to return a result. This cohort was significantly older and had significantly increased weight compared with the general Australian obstetric population. Pregnancy outcomes were available for 94% of the cohort. There were significantly higher rates of chromosomal aneuploidies (6.5% versus 0.2%, P < 0.0001), pre-eclampsia (11% versus 1.5%, P < 0.0001) and gestational diabetes (23% versus 7.5%, P < 0.0001) compared with the general obstetric population. Rates of preterm delivery and SGA were elevated but did not reach significance. Antenatal investigations demonstrated decreased PAPP-A MoM (0.75 versus 1.14, P < 0.0001), decreased free β-hCG (0.71 versus 1.01, P < 0.0001) and increased uterine artery PI (1.79 versus 1.65, P = 0.02). Women who fail to obtain a result from NIPT are at increased risk of adverse pregnancy outcomes, in particular chromosomal aneuploidy, gestational diabetes and pre-eclampsia. None received. Women who fail to obtain a result from cell-free DNA NIPT are at increased risk of adverse pregnancy outcomes. © 2017 Royal College of Obstetricians and Gynaecologists.

  19. Evaluation of Synergistic Interactions Between Cell-Free Supernatant of Lactobacillus Strains and Amikacin and Genetamicin Against Pseudomonas aeruginosa.

    Science.gov (United States)

    Aminnezhad, Sargol; Kermanshahi, Rouha Kasra; Ranjbar, Reza

    2015-04-01

    The indiscriminate use of antibiotics in the treatment of infectious diseases can increase the development of antibiotic resistance. Therefore, there is a big demand for new sources of antimicrobial agents and alternative treatments for reduction of antibiotic dosage required to decrease the associated side effects. In this study, the synergistic action of aminoglycoside antibiotics and cell-free supernatant (CFS) of probiotic (Lactobacillus rahmnosus and L. casei) against Pseudomonas aeruginosa PTCC 1430 was evaluated. A growth medium for culturing of probiotic bacteria was separated by centrifugation. The antimicrobial effects of CFS of probiotic bacteria were evaluated using the agar well diffusion assay. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were evaluated using the micro dilution method. Finally, an interaction between CFS and amikacin or gentamicin against P. aeruginosa PTCC 1430 was examined through the checkerboard method and fractional inhibitory concentration (FIC). Furthermore, CFSs from Lactobacillus strains were analyzed by reversed phase HPLC (RP-HPLC) for antimicrobial compounds. The results showed a significant effect of CFS on the growth of P. aeruginosa. The MIC and MBC of CFS from L. casei were 62.5 µL⁄mL while the MIC and MBC of CFS from L. rhamnosus were 62.5 μL⁄mL and 125 μL⁄mL, respectively. Using the FIC indices, synergistic interactions were observed in combination of CFS and antibiotics. Fractional Inhibitory Concentration indices of CFS from L. casei and aminoglycoside antibiotics were 0.124 and 0.312 while FIC indices of CFS from L. rhamnosus and aminoglycoside antibiotics were 0.124 and 0.56, respectively showing a synergism effect. The results of RP-HPLC showed that CFS of Lactobacillus strains contained acetic acid, lactic acid and hydrogen peroxide (H2O2). Our findings indicate that probiotic bacterial strains of Lactobacillus have a significant inhibitory effect on the

  20. Synthesizing controllers from duration calculus

    DEFF Research Database (Denmark)

    Fränzle, Martin

    1996-01-01

    Duration Calculus is a logic for reasoning about requirements for real-time systems at a high level of abstraction from operational detail, which qualifies it as an interesting starting point for embedded controller design. Such a design activity is generally thought to aim at a control device...... the physical behaviours of which satisfy the requirements formula, i.e. the refinement relation between requirements and implementations is taken to be trajectory inclusion. Due to the abstractness of the vocabulary of Duration Calculus, trajectory inclusion between control requirements and controller designs...... for embedded controller design and exploit this fact for developing an automatic procedure for controller synthesis from specifications formalized in Duration Calculus. As far as we know, this is the first positive result concerning feasibility of automatic synthesis from dense-time Duration Calculus....

  1. RESEARCH FOR THE AEROSPACE SYSTEMS DIRECTORATE (R4RQ) Delivery Order 0006: Airbreathing Propulsion Fuels and Energy Exploratory Research and Development (APFEERD) Sub Task: Review of Materials Compatibility Tests of Synthesized Hydrocarbon Kerosenes and Blends

    Science.gov (United States)

    2017-07-31

    concentrations used in the reported test programs. 15. SUBJECT TERMS synthesized jet fuels ; alternative jet fuels ; renewable jet fuel ; fuel physical...resources for producing jet fuel , there have been complaints from the producers about the time and cost of approving these products for use. Alternately ...Aviation Alternate Fuels Initiative (CAAFI), frustration was exhibited by many of the prospective producers who complained about the time and cost of the

  2. A comparison of cell-free placental messenger ribonucleic acid and color Doppler ultrasound for the prediction of placental invasion in patients with placenta accreta.

    Science.gov (United States)

    Naghshineh, Elham; Khorvash, Elahe; Kamali, Sara

    2015-01-01

    The aim of the present study was to comparison between cell-free placental messenger ribonucleic acid (mRNA) and Doppler ultrasound for the prediction of placental invasion in women with placenta accreta. In this cross-sectional study, 50 pregnant women at risk for placenta accreta underwent color Doppler and assessment of cell-free placental mRNA. Real-time reverse-transcription polymerase chain reaction was used for measurement of cell-free placental mRNA in maternal plasma. Based on the findings at cesarean delivery and histological examination, patients were divided into two groups of women with and without placenta accrete. To compare of the mean of mRNA levels between the two groups we used independent t-test and to compare of the mean of age and gestational age at sonography we used Mann-Whitney test. For determination of sensitivity and specificity and the cut-off point of mRNA levels we used the receiver operating characteristic curve. A total of 50 women with a mean age of 30.24 ± 4.905 years entered the study and 12 (24%) patients were diagnosed with placenta accreta. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of Doppler ultrasound were 83.3%, 78.9%, 56% and 94%, respectively. Results of our study showed if we consider a cut-off point equal to 3.325, with sensitivity and specificity of 0.917 and 0.789, respectively and the sensitivity, specificity, PPV and NPV of mRNA with were cut-off point of 3.325 were 91.7%, 78.9%, 57.9% and 96.8%, respectively. Cell-free mRNA is an acceptable, easy made, functional test with sensitivity, specificity, PPV and NPV more than Doppler ultrasound for diagnosis and prediction of incidence of placenta accrete and we recommend the use of cell-free mRNA test for diagnosis of placenta accreta.

  3. Method to synthesize metal chalcogenide monolayer nanomaterials

    Science.gov (United States)

    Hernandez-Sanchez, Bernadette A.; Boyle, Timothy J.

    2016-12-13

    Metal chalcogenide monolayer nanomaterials can be synthesized from metal alkoxide precursors by solution precipitation or solvothermal processing. The synthesis routes are more scalable, less complex and easier to implement than other synthesis routes.

  4. nanoparticles synthesized by citrate precursor m

    African Journals Online (AJOL)

    user

    (M=Co, Cu) nanoparticles synthesized by citrate precursor method ... The structural characterization was carried out using an X-ray Diffractometer (Rikagu Miniflex, Japan) ..... His current area of interest includes magnetic nanomaterials.

  5. Syntheses, molecular and crystalline architectures, and ...

    Indian Academy of Sciences (India)

    Syntheses, molecular and crystalline architectures, and luminescence behaviour of terephthalate bridged heptacoordinated dinuclear lead(II) complexes containing a pentadentate N-donor Schiff base. SUBHASIS ROYa, SOMNATH CHOUBEYa, SUMITAVA KHANa, KISHALAY BHARa,. PARTHA MITRAb and BARINDRA ...

  6. Synthese en chemotherapeutisch onderzoek van sulfanilamidopyrimidinen

    NARCIS (Netherlands)

    Grevenstuk, Anton Bernard

    1942-01-01

    In order to investigate the influence of substitution in the pyrimidine nucleous on the activity of the three isomeric sulfanilamidopyrimidines (2, 5 and 6), a number of substituted sulfanilamidopyrimidines were synthesized and tested on chemotherapeutic activity. ... Zie: Summary

  7. CAMAC programmable-control frequency synthesizer

    International Nuclear Information System (INIS)

    Yumaguzin, T.Kh.; Vyazovkin, D.E.; Nazirov, Eh.P.; Tuktarov, R.F.

    1989-01-01

    Synthesizer allows to set frequency with 0.015% accuracy and to scan it with variable step. Frequency controlled divider with further summing-up of divided frequency with fundamental one is used in synthesizer, and it has allowed to use digit of the input code and to obtain 3-4 MHz frequency range. Variation of operation flowsheet in the other frequency range is possible. K-155 and K-531 series microcircuits were used during development

  8. Operational Design that Synthesizes Art and Science

    Science.gov (United States)

    2011-05-04

    FINAL 3. DATES COVERED (From - To) Feb - May 2011 4. TITLE AND SUBTITLE OPERATIONAL DESIGN THAT SYNTHESIZES ART AND SCIENCE 5a...TITLE AND SUBTITLE Operational Design That Synthesizes Art And Science 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR...proponents of EBO view warfare as only a science and not a combination of art and science . 9 Another main point of contention centered on the term

  9. Raman assisted lightwave synthesized frequency sweeper

    DEFF Research Database (Denmark)

    Pedersen, Anders Tegtmeier; Rottwitt, Karsten

    2010-01-01

    We present a Lightwave Synthesized Frequency Sweeper comprising a Raman amplifier for loss compensation. The generated pulse train contains 123 pulses and has a flat signal level as well as a low noise level.......We present a Lightwave Synthesized Frequency Sweeper comprising a Raman amplifier for loss compensation. The generated pulse train contains 123 pulses and has a flat signal level as well as a low noise level....

  10. Sequence determination of synthesized chondroitin sulfate dodecasaccharides.

    Science.gov (United States)

    Shioiri, Tatsumasa; Tsuchimoto, Jun; Watanabe, Hideto; Sugiura, Nobuo

    2016-06-01

    Chondroitin sulfate (CS) is a linear acidic polysaccharide composed of repeating disaccharide units of glucuronic acid and N-acetyl-d-galactosamine. The polysaccharide is modified with sulfate groups at different positions by a variety of sulfotransferases. CS chains exhibit various biological and pathological functions by interacting with cytokines and growth factors and regulating their signal transduction. The fine structure of the CS chain defines its specific biological roles. However, structural analysis of CS has been restricted to disaccharide analysis, hampering the understanding of the structure-function relationship of CS chains. Here, we chemo-enzymatically synthesized CS dodecasaccharides having various sulfate modifications using a bioreactor system of bacterial chondroitin polymerase mutants and various CS sulfotransferases. We developed a sequencing method for CS chains using the CS dodecasaccharides. The method consists of (i) labeling a reducing end with 2-aminopyridine (PA), (ii) partial digestion of CS with testicular hyaluronidase, followed by separation of PA-conjugated oligosaccharides with different chain lengths, (iii) limited digestion of these oligosaccharides with chondroitin lyase AC II into disaccharides, followed by labeling with 2-aminobenzamide, (iv) CS disaccharide analysis using a dual-fluorescence HPLC system (reversed-phase ion-pair and ion-exchange chromatography), and (v) estimation of the composition by calculating individual disaccharide ratios. This CS chain sequencing allows characterization of CS-modifying enzymes and provides a useful tool toward understanding the structure-function relationship of CS chains. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. Comparison of quantification algorithms for circulating cell-free DNA methylation biomarkers in blood plasma from cancer patients.

    Science.gov (United States)

    de Vos, Luka; Gevensleben, Heidrun; Schröck, Andreas; Franzen, Alina; Kristiansen, Glen; Bootz, Friedrich; Dietrich, Dimo

    2017-01-01

    SHOX2 and SEPT9 methylation in circulating cell-free DNA (ccfDNA) in blood are established powerful and clinically valuable biomarkers for diagnosis, staging, prognosis, and monitoring of cancer patients. The aim of the present study was to evaluate different quantification algorithms (relative quantification, absolute quantification, quasi-digital PCR) with regard to their clinical performance. Methylation analyses were performed in a training cohort (141 patients with head and neck squamous cell carcinoma [HNSCC], 170 control cases) and a testing cohort (137 HNSCC cases, 102 controls). DNA was extracted from plasma samples, bisulfite-converted, and analyzed via quantitative real-time PCR. SHOX2 and SEPT9 methylations were assessed separately and as panel [mean SEPT9 / SHOX2 ] using the ΔCT method for absolute quantification and the ΔΔCT-method for relative quantification. Quasi-digital PCR was defined as the number of amplification-positive PCR replicates. The diagnostic (sensitivity, specificity, area under the curve (AUC) of the receiver operating characteristic (ROC)) and prognostic accuracy (hazard ratio (HR) from Cox regression) were evaluated. Sporadic methylation in control samples necessitated the introduction of cutoffs resulting in 61-63% sensitivity/90-92% specificity ( SEPT9 /training), 53-57% sensitivity/87-90% specificity ( SHOX2 /training), and 64-65% sensitivity/90-91% specificity (mean SEPT9 / SHOX2 /training). Results were confirmed in a testing cohort with 54-56% sensitivity/88-90% specificity ( SEPT9 /testing), 43-48% sensitivity/93-95% specificity ( SHOX2 /testing), and 49-58% sensitivity/88-94% specificity (mean SEPT9 / SHOX2 /testing). All algorithms showed comparable cutoff-independent diagnostic accuracy with largely overlapping 95% confidence intervals ( SEPT9 : AUC training  = 0.79-0.80; AUC testing  = 0.74-0.75; SHOX2 : AUC training  = 0.78-0.81, AUC testing  = 0.77-0.79; mean SEPT9 / SHOX2 : AUC training  = 0

  12. Synthesizing genetic sequential logic circuit with clock pulse generator.

    Science.gov (United States)

    Chuang, Chia-Hua; Lin, Chun-Liang

    2014-05-28

    Rhythmic clock widely occurs in biological systems which controls several aspects of cell physiology. For the different cell types, it is supplied with various rhythmic frequencies. How to synthesize a specific clock signal is a preliminary but a necessary step to further development of a biological computer in the future. This paper presents a genetic sequential logic circuit with a clock pulse generator based on a synthesized genetic oscillator, which generates a consecutive clock signal whose frequency is an inverse integer multiple to that of the genetic oscillator. An analogous electronic waveform-shaping circuit is constructed by a series of genetic buffers to shape logic high/low levels of an oscillation input in a basic sinusoidal cycle and generate a pulse-width-modulated (PWM) output with various duty cycles. By controlling the threshold level of the genetic buffer, a genetic clock pulse signal with its frequency consistent to the genetic oscillator is synthesized. A synchronous genetic counter circuit based on the topology of the digital sequential logic circuit is triggered by the clock pulse to synthesize the clock signal with an inverse multiple frequency to the genetic oscillator. The function acts like a frequency divider in electronic circuits which plays a key role in the sequential logic circuit with specific operational frequency. A cascaded genetic logic circuit generating clock pulse signals is proposed. Based on analogous implement of digital sequential logic circuits, genetic sequential logic circuits can be constructed by the proposed approach to generate various clock signals from an oscillation signal.

  13. Cell free expression of hif1α and p21 in maternal peripheral blood as a marker for preeclampsia and fetal growth restriction.

    Directory of Open Access Journals (Sweden)

    Osnat Ashur-Fabian

    Full Text Available Preeclampsia, a severe unpredictable complication of pregnancy, occurs in 6% of pregnancies, usually in the second or third trimester. The specific etiology of preeclampsia remains unclear, although the pathophysiological hallmark of this condition appears to be an inadequate blood supply to the placenta. As a result of the impaired placental blood flow, intrauterine growth restriction (IUGR and consequential fetal oxidative stress may occur. Consistent with this view, pregnancies complicated by preeclampsia and IUGR are characterized by up-regulation of key transcriptional regulators of the hypoxic response including, hif1α and as well as p53 and its target genes. Recently, the presence of circulating cell-free fetal RNA has been documented in maternal plasma. We speculated that pregnancies complicated by preeclampsia and IUGR, will be associated with an abnormal expression of p53 and/or hif1α related genes in the maternal plasma. Maternal plasma from 113 singleton pregnancies (72 normal and 41 complicated pregnancies and 19 twins (9 normal and 10 complicated pregnancies were collected and cell free RNA was extracted. The expression of 18 genes was measured by one step real-time RT-PCR and was analyzed for prevalence of positive/negative expression levels. Results indicate that, among the genes examined, cell free plasma expressions of p21 and hif1α were more prevalent in pregnancies complicated by hypoxia and/or IUGR (p<0.001. To conclude, we present in this manuscript data to support the association between two possible surrogate markers of hypoxia and common complications of pregnancy. More work is needed in order to implement these findings in clinical practice.

  14. Characterization of cell-free extracts from fenpropathrin-degrading strain Bacillus cereus ZH-3 and its potential for bioremediation of pyrethroid-contaminated soils.

    Science.gov (United States)

    Liu, Jie; Huang, Wenwen; Han, Haitao; She, Changchun; Zhong, Guohua

    2015-08-01

    Synthetic pyrethroid fenpropathrin has received increasing attention because of its environmental contamination and toxic effects on non-target organisms including human beings. Here we report the degradation characteristics of cell-free extracts from fenpropathrin-degrading strain Bacillus cereus ZH-3 and its potential for pyrethroid bioremediation in soils. 50mg·L(-1) of fenpropathrin was decreased to 20.6mg·L(-1) by the enzymatic extracts (869.4mg·L(-1)) within 30min. Kinetic constants Km and Vm were determined to be 1006.7nmol·L(-1) and 56.8nmol·min(-1), respectively. Degradation products were identified as 3-phenoxybenzaldehyde, α-hydroxy-3-phenoxy-benzeneacetonitrile and phenol by gas chromatography-mass spectrometry (GC-MS). In addition to degradation of fenpropathrin, the cell-free extracts could degrade other pyrethroids including beta-cypermethrin, cyfluthrin, deltamethrin and cypermethrin. Additionally, the reaction conditions were optimized. In the sterile and non-sterile soils, 50mg·kg(-1) of fenpropathrin was reduced to 15.3 and 13.9mg·L(-1) in 1d, respectively. Sprayed 100 and 300mg·kg(-1) of fenpropathrin emulsifiable concentrate (EC), up to 84.6% and 92.1% of soil fenpropathrin were removed from soils within 7d, respectively. Taken together, our results depict the biodegradation characteristics of cell-free extracts from B. cereus ZH-3, highlight its promising potential in bioremediation of pyrethroid-contaminated soils and also provide new insights into the utilization of degrading microbes. Copyright © 2015. Published by Elsevier B.V.

  15. A Human Amnion-Derived Extracellular Matrix-Coated Cell-Free Scaffold for Cartilage Repair: In Vitro and In Vivo Studies.

    Science.gov (United States)

    Nogami, Makiko; Kimura, Tomoatsu; Seki, Shoji; Matsui, Yoshito; Yoshida, Toshiko; Koike-Soko, Chika; Okabe, Motonori; Motomura, Hiraku; Gejo, Ryuichi; Nikaido, Toshio

    2016-04-01

    Extracellular matrix (ECM) derived from human amniotic mesenchymal cells (HAMs) has various biological activities. In this study, we developed a novel HAM-derived ECM-coated polylactic-co-glycolic acid (ECM-PLGA) scaffold, examined its property on mesenchymal cells, and investigated its potential as a cell-free scaffold for cartilage repair. ECM-PLGA scaffolds were developed by inoculating HAM on a PLGA. After decellularization by irradiation, accumulated ECM was examined. Exogenous cell growth and differentiation of rat mesenchymal stem cells (MSCs) on the ECM-PLGA were analyzed in vitro by cell attachment/proliferation assay and reverse transcription-polymerase chain reaction. The cell-free ECM-PLGA scaffolds were implanted into osteochondral defects in the trochlear groove of rat knees. After 4, 12, or 24 weeks, the animals were sacrificed and the harvested tissues were examined histologically. The ECM-PLGA contained ECM that mimicked natural amniotic stroma that contains type I collagen, fibronectin, hyaluronic acid, and chondroitin sulfates. The ECM-PLGA showed excellent properties of cell attachment and proliferation. MSCs inoculated on the ECM-PLGA scaffold showed accelerated type II collagen mRNA expression after 3 weeks in culture. The ECM-PLGA implanted into an osteochondral defect in rat knees induced gradual tissue regeneration and resulted in hyaline cartilage repair, which was better than that in the empty control group. These in vitro and in vivo experiments show that the cell-free scaffold composed of HAM-derived ECM and PLGA provides a favorable growth environment for MSCs and facilitates the cartilage repair process. The ECM-PLGA may become a "ready-made" biomaterial for cartilage repair therapy.

  16. Uncertainty Evaluation for SMART Synthesized Power Distribution

    International Nuclear Information System (INIS)

    Cho, J. Y.; Song, J. S.; Lee, C. C.; Park, S. Y.; Kim, K. Y.; Lee, K. H.

    2010-07-01

    This report performs the uncertainty analysis for the SMART synthesis power distribution generated by a SSUN (SMART core SUpporting system coupled by Nuclear design code) code. SSUN runs coupled with the MASTER neutronics code and generates the core 3-D synthesis power distribution by using DPCM3D. The MASTER code plays a role to provide the DPCM3D constants to the SSUN code for the current core states. The uncertainties evaluated in this report are the form of 95%/95% probability/confidence one-sided tolerance limits and can be used in conjunction with Technical Specification limits on these quantities to establish appropriate LCO (Limiting Conditions of Operation) and LSSS (Limiting Safety System Settings) limits. This report is applicable to SMART nuclear reactor using fixed rhodium detector systems. The unknown true power distribution should be given for the uncertainty evaluation of the synthesis power distribution. This report produces virtual distributions for the true power distribution by imposing the CASMO-3/MASTER uncertainty to the MASTER power distribution. Detector signals are generated from these virtual distribution and the DPCM3D constants are from the MASTER power distribution. The SSUN code synthesizes the core 3-D power distribution by using these detector signals and the DPCM3D constants. The following summarizes the uncertainty evaluation procedure for the synthesis power distribution. (1) Generation of 3-D power distribution by MASTER -> Determination of the DPCM3D constants. (2) Generation of virtual power distribution (assumed to be true power distribution) -> Generation of detector signals. (3) Generation of synthesis power distribution. (4) Uncertainty evaluation for the synthesis power distribution. Chi-Square normality test rejects the hypothesis of normal distribution for the synthesis power error distribution. Therefore, the KRUSKAL WALLIS test and the non-parametric statistics are used for data pooling and the tolerance limits. The

  17. Synthesizing Modular Invariants for Synchronous Code

    Directory of Open Access Journals (Sweden)

    Pierre-Loic Garoche

    2014-12-01

    Full Text Available In this paper, we explore different techniques to synthesize modular invariants for synchronous code encoded as Horn clauses. Modular invariants are a set of formulas that characterizes the validity of predicates. They are very useful for different aspects of analysis, synthesis, testing and program transformation. We describe two techniques to generate modular invariants for code written in the synchronous dataflow language Lustre. The first technique directly encodes the synchronous code in a modular fashion. While in the second technique, we synthesize modular invariants starting from a monolithic invariant. Both techniques, take advantage of analysis techniques based on property-directed reachability. We also describe a technique to minimize the synthesized invariants.

  18. Pre-analytical variables of circulating cell-free nucleosomes containing 5-methylcytosine DNA or histone modification H3K9Me3

    DEFF Research Database (Denmark)

    Rasmussen, Louise; Herzog, Marielle; Rømer, Eva

    2016-01-01

    Aim: To evaluate pre-analytical variables of circulating cell-free nucleosomes containing 5-methylcytosine DNA (5mC) or histone modification H3K9Me3 (H3K9Me3). Materials and methods: Six studies were designed to assess the possible influence of pre-analytical variables. Study 1: influence of stasis...... significantly lower levels of 5mC or H3K9Me3 compared to levels in healthy individuals. Conclusion: Levels of 5mC or H3K9Me3 appear stable in most pre-analytical settings if blood samples are stored at room temperature until centrifugation....

  19. M10.6.6: Designed and manufactured Frequency Synthesizer Board (AMC)

    CERN Document Server

    Czuba, K

    2011-01-01

    The LLRF system require generation of highly stable clock and trigger signals for high precision data processing and synchronous system operation. This deliverable provides an updated AMC module designed to fulfill the LLRF system timing synchronization needs. The module contains three independent clock synthesizers that are able to generate LVDS clock signals in the range of 10 MHz to 100 MHz. The clock synthesizers can be synchronized either by an internal quartz oscillator or an external phase reference signal provided to the board from the FLASH Master Oscillator. Besides clock synthesizers the AMC card contains also an optical receiver suited to convert and decode FLASH timing signals.

  20. Syntheses of copper complexes of nicotinohydroxamic and ...

    African Journals Online (AJOL)

    Nicotinohydroxamic acid (NHA) and isonicotinohydroxamic acid (INHA) were synthesized, characterized by electronic and spectral studies,magnetic measurements and their pKa determined spectrophotometrically as 8.68 ± 0.02 in aqueous medium of 0.1mol dm-3 I=ionic strength. The composition of the complexes was ...

  1. SYNTHESES AND PROPERTIES OF SOME ORGANOSILANE POLYMERS

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xinghua; Robert West

    1984-01-01

    Some organosilane polymers with high molecular weights have been synthesized by cocondensation of organosilicon dihalide monomers with sodium metal in toluene. These polymers are both soluble in common solvents and meltable at lower temperatures, and can be molded, cast into films or drawn into fibers. Exposure of the solid polymers to ultraviolet light leads to degradation or crosslinking.

  2. Biological activities of synthesized silver nanoparticles

    Indian Academy of Sciences (India)

    The C. halicacabum leaf extract synthesized AgNPs efficiency were tested against different bacterial pathogens MTCC-426 Proteus vulgaris, MTCC-2453 Pseudomonas aeruginosa, MTCC-96 Staphylococcus aureus, MTCC-441 Bacillus subtilis andMTCC-735 Salmonella paratyphi, and fungal pathogens Alternaria solani ...

  3. Synthesizing chaotic maps with prescribed invariant densities

    International Nuclear Information System (INIS)

    Rogers, Alan; Shorten, Robert; Heffernan, Daniel M.

    2004-01-01

    The Inverse Frobenius-Perron Problem (IFPP) concerns the creation of discrete chaotic mappings with arbitrary invariant densities. In this Letter, we present a new and elegant solution to the IFPP, based on positive matrix theory. Our method allows chaotic maps with arbitrary piecewise-constant invariant densities, and with arbitrary mixing properties, to be synthesized

  4. Cytotoxicity of Nanoliposomal Cisplatin Coated with Synthesized ...

    African Journals Online (AJOL)

    Purpose: To evaluate the cytotoxicity of pegylated nanoliposomal cisplatin on human ovarian cancer cell line A2780CP. Methods: Synthesized methoxypolyethylene glycol (mPEG) propionaldehyde was characterized by 1Hnuclear magnetic resonance (1H-NMR) and Fourier transform infrared spectroscopy (FTIR) and used ...

  5. Biosynthesis of silver nanoparticles synthesized by Aspergillus

    Indian Academy of Sciences (India)

    In the present study, biosynthesis of silver nanoparticles and its antioxidant, antimicrobial and cytotoxic activities were investigated. Silver nanoparticles were extracellularly synthesized using Aspergillus flavus and the formation of nanoparticles was observed after 72 h of incubation. The results recorded from colour ...

  6. * Comparison of Autologous, Allogeneic, and Cell-Free Scaffold Approaches for Engineered Tendon Repair in a Rabbit Model-A Pilot Study.

    Science.gov (United States)

    Wang, Wenbo; Deng, Dan; Wang, Bin; Zhou, Guangdong; Zhang, WenJie; Cao, Yilin; Zhang, Peihua; Liu, Wei

    2017-08-01

    Tendons are subjected to high strength dynamic mechanical forces in vivo. Mechanical strength is an essential requirement for tendon scaffold materials. A composite scaffold was used in this study to provide mechanical strength, which was composed of an inter part of nonwoven polyglycolic acid (PGA) fibers and an outer part of the net knitted with PGA and polylactic acid (PLA) fibers in a ratio of 4:2. This study compared three different approaches for in vivo tendon engineering, that is, cell-free scaffold and allogeneic and autologous cell seeded scaffolds, using a rabbit Achilles tendon repair model. Dermal fibroblasts were, respectively, isolated from the dermis of regular rabbits or green fluorescence protein transgenic rabbits as the autologous and the allogeneic cell sources, respectively. The cell scaffolds and cell-free scaffolds were implanted to bridge a partial segmental defect of rabbit Achilles tendon. The engineered tendons were harvested at 7 and 13 months postsurgery for various examinations. The results showed that all three groups could achieve in vivo tendon regeneration similarly with slightly better tissue formation in autologous group than in other two groups, including better scaffold degradation and relatively thicker collagen fibrils. There were no statistically significant differences in mechanical parameters among three groups. This work demonstrated that allogeneic fibroblasts and scaffold alone are likely to be used for tendon tissue engineering.

  7. The healing of bony defects by cell-free collagen-based scaffolds compared to stem cell-seeded tissue engineered constructs.

    LENUS (Irish Health Repository)

    Lyons, Frank G

    2010-12-01

    One of the key challenges in tissue engineering is to understand the host response to scaffolds and engineered constructs. We present a study in which two collagen-based scaffolds developed for bone repair: a collagen-glycosaminoglycan (CG) and biomimetic collagen-calcium phosphate (CCP) scaffold, are evaluated in rat cranial defects, both cell-free and when cultured with MSCs prior to implantation. The results demonstrate that both cell-free scaffolds showed excellent healing relative to the empty defect controls and somewhat surprisingly, to the tissue engineered (MSC-seeded) constructs. Immunological analysis of the healing response showed higher M1 macrophage activity in the cell-seeded scaffolds. However, when the M2 macrophage response was analysed, both groups (MSC-seeded and non-seeded scaffolds) showed significant activity of these cells which are associated with an immunomodulatory and tissue remodelling response. Interestingly, the location of this response was confined to the construct periphery, where a capsule had formed, in the MSC-seeded groups as opposed to areas of new bone formation in the non-seeded groups. This suggests that matrix deposited by MSCs during in vitro culture may adversely affect healing by acting as a barrier to macrophage-led remodelling when implanted in vivo. This study thus improves our understanding of host response in bone tissue engineering.

  8. Syntheses of F-18 Labeled Fluoroalkyltyrosine Derivatives

    Energy Technology Data Exchange (ETDEWEB)

    Moon, Byung Seok; Lee, Kyo Chul; Yang, Seung Dae; Chun, Kwon Soo [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Chi, Dae Yoon [Inha Univ., Inchon (Korea, Republic of)

    2005-07-01

    Positron emission tomography (PET) offers the highest resolution of all nuclear medicine imaging modalities and allows quantitation of tracer concentration in tissues. For more than 60 years, some of C-11 or F-18 labeled amino acids have been synthesized and evaluated for potential use in oncology, neurology and psychiatric disorders. Besides, a variety of radioisotope labeled amino acids have proven to be useful for imaging tumors, especially for brain tumor, lung tumor and breast tumor. These amino acids can be subdivided into two categories. The first category is represented by radiolabled naturally occurring amino acids and structurally similar analogues. Although these radiolabeled amino acids have proven useful in detecting brain and systemic tumors, it is susceptible to in vivo metabolism through multiple pathways that give rise to numerous radiolabled metabolites. On the other side, structurally similar amino acid analogues have some significant advantages over the natural amino acids. These nonnatural amino acids are not metabolized, which simplifieds the kinetic analysis of their uptake. On the basis of the promising results obtained with these nonnatural amino acids in preclinical studies, recent efforts have focused on the development of new F-18 labeled nonnatural amino acids. Recently, O-(2-[{sup 18}F]Fluoroethyl)-L-tyrosine (FET), O-(3-[{sup 18}F]Fluoropropyl)-L-tyrosine (FPT) were developed and evaluated among structurally similar to a new amino acid analogue. FET has shown high uptake in activated inflammatory cells using an experimental acute abscess model and in inflammation within lymph nodes. FPT was superior to FDG and had a slight advantage over FET in the differentiation of tumor from inflammation, and, like FET, it appeared to be a potential amino acid tracer for tumor imaging with PET. In this paper, we elected to introduce fluoroethyl and fluoropropyl groups at the R{sub 1} positions and OCH{sub 3} at R{sub 2} position to the same effect

  9. Syntheses of F-18 Labeled Fluoroalkyltyrosine Derivatives

    International Nuclear Information System (INIS)

    Moon, Byung Seok; Lee, Kyo Chul; Yang, Seung Dae; Chun, Kwon Soo; Chi, Dae Yoon

    2005-01-01

    Positron emission tomography (PET) offers the highest resolution of all nuclear medicine imaging modalities and allows quantitation of tracer concentration in tissues. For more than 60 years, some of C-11 or F-18 labeled amino acids have been synthesized and evaluated for potential use in oncology, neurology and psychiatric disorders. Besides, a variety of radioisotope labeled amino acids have proven to be useful for imaging tumors, especially for brain tumor, lung tumor and breast tumor. These amino acids can be subdivided into two categories. The first category is represented by radiolabled naturally occurring amino acids and structurally similar analogues. Although these radiolabeled amino acids have proven useful in detecting brain and systemic tumors, it is susceptible to in vivo metabolism through multiple pathways that give rise to numerous radiolabled metabolites. On the other side, structurally similar amino acid analogues have some significant advantages over the natural amino acids. These nonnatural amino acids are not metabolized, which simplifieds the kinetic analysis of their uptake. On the basis of the promising results obtained with these nonnatural amino acids in preclinical studies, recent efforts have focused on the development of new F-18 labeled nonnatural amino acids. Recently, O-(2-[ 18 F]Fluoroethyl)-L-tyrosine (FET), O-(3-[ 18 F]Fluoropropyl)-L-tyrosine (FPT) were developed and evaluated among structurally similar to a new amino acid analogue. FET has shown high uptake in activated inflammatory cells using an experimental acute abscess model and in inflammation within lymph nodes. FPT was superior to FDG and had a slight advantage over FET in the differentiation of tumor from inflammation, and, like FET, it appeared to be a potential amino acid tracer for tumor imaging with PET. In this paper, we elected to introduce fluoroethyl and fluoropropyl groups at the R 1 positions and OCH 3 at R 2 position to the same effect of FET. Herein, we wish

  10. Enzymatic synthesizing of phytosterol oleic esters.

    Science.gov (United States)

    Pan, Xinxin; Chen, Biqiang; Wang, Juan; Zhang, Xinzhi; Zhul, Biyun; Tan, Tianwei

    2012-09-01

    A method of synthesizing the phytosterol esters from oleic acid and sterols was studied, using immobilized lipase Candida sp. 99-125 as catalyst. Molar ratio (oleic acid/phytosterols), temperature, reaction period, organic solvents, catalyst, and silica-gel drier were optimized, and the result showed that 93.4% of the sterols had been esterified under the optimal synthetic condition: the molar ratio of oleic acid/phytosterol is 1:1 in 10 mL iso-octane, immobilized lipase (w, 140% of the sterols), incubated in an orbital shaker (200 rpm) at a temperature of 45 °C for 24 h. The immobilized lipase could be reused for at least 13 times with limited loss of esterification activity. The conversion still maintained up to 86.6%. Hence, this developed process for synthesizing phytosterol esters could be considered as simple and low-energy consumption compared to existing chemical processes.

  11. Syntheses and studies of organosilicon compounds

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Ren [Iowa State Univ., Ames, IA (United States)

    1999-02-12

    The syntheses of polycarbosilanes and polysilanes as silicon carbide ceramic precursors have been active research areas in the Barton Research Group. In this thesis, the work is focused on the preparation of polycarbosilanes and polysilanes as stoichiometric silicon carbide precursor polymers. The syntheses of the precursor polymers are discussed and the conversions of these precursors to silicon carbide via pyrolysis are reported. The XRD pattern and elemental analyses of the resulting silicon carbide ceramics are presented. Silicon monoxide is an important intermediate in the production of silicon metal. The existence of silicon monoxide in gap phase has been widely accepted. In the second part of this thesis, the generation of gaseous silicon monoxide in four different reactors and the reactions of gaseous silicon monoxide towards organic compounds are discussed.

  12. SYNTHESES AND CHARACTERIZATIONS OF THE CYANIDE ...

    African Journals Online (AJOL)

    2015-10-28

    (Received October 28, 2015; revised June 25, 2016) ... suggest that the Ni(II) ion is four coordinate with four cyanide-carbon atoms in ... However, there have been many studies on octahedral [M(CN)6]n- but little ... were synthesized and investigated by vibrational spectral (FT-IR and ..... Karaağaç, D.; Kürkçüoğlu, G.S. Bull.

  13. Molecular trees: from syntheses towards applications

    International Nuclear Information System (INIS)

    Ardoin, N.; Astruc, D.

    1995-01-01

    Molecular trees, also called dendrimers, arborols, cauliflowers, cascades or hyperbranched molecules, have been synthesized since their first observation in 1978 by divergent, convergent or combined methods, with various functions on the branches. The potential applications of these nanoscopic molecules are in the fields of biology (gene therapy, virus mimicking an vectorization) and molecular materials sciences (new polymers, adhesion, liquid crystals, etc). (authors). 236 refs., 6 figs., 2 tabs., 8 schemes

  14. Novel stereocontrolled syntheses of tashiromine and epitashiromine

    Directory of Open Access Journals (Sweden)

    Loránd Kiss

    2015-04-01

    Full Text Available A novel stereocontrolled approach has been developed for the syntheses of tashiromine and epitashiromine alkaloids from cyclooctene β-amino acids. The synthetic concept is based on the azetidinone opening of a bicyclic β-lactam, followed by oxidative ring opening through ring C–C double bond and reductive ring-closure reactions of the cis- or trans-cyclooctene β-amino acids.

  15. Spurious in PLL-DDS frequency synthesizers

    Czech Academy of Sciences Publication Activity Database

    Kroupa, Věnceslav František; Štursa, Jarmil

    2002-01-01

    Roč. 2, č. 1 (2002), s. 48-51 ISSN 1335-8243. [Digital Signal Processing and Multimedia Communications DSP-MCOM 2001 /5./. Košice, 27.11.2001-29.11.2001] R&D Projects: GA ČR GA102/00/0958 Institutional research plan: CEZ:AV0Z2067918 Keywords : frequency synthesizers * phase locked loops * direct digital synthesis Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering

  16. Soft-Template-Synthesized Mesoporous Carbon for Oral Drug Delivery

    Energy Technology Data Exchange (ETDEWEB)

    Saha, Dipendu [ORNL; Warren, Kaitlyn E [ORNL; Naskar, Amit K [ORNL

    2014-01-01

    Template-synthesized mesoporous carbons were successfully used in in vitro investigations of controlled delivery of three model drugs, captopril, furosemide, and ranitidine hydrochloride. Captopril and furosemide exhibited desorption kinetics over 30 40 h, and ranitidine HCl had a complete release time of 5 10 h. As evident from the slow release kinetics, we contend that our mesoporous carbon is an improved drug-delivery medium compared to state-of-the-art porous silica-based substrates. The mesoporous carbons, synthesized from phloroglucinol and lignin, a synthetic and a sustainable precursor, respectively, exhibit BET surface area of 200 400 m2 g-1 and pore volume of 0.2 0.6 cm3 g-1. The phloroglucinol-based carbon has narrower pore widths and higher pore volume than the lignin-derived counterpart and maintains a longer release time. Numerical modeling of the release kinetics data reveals that the diffusivities of all the drugs from lignin-based carbon media are of equivalent magnitude (10-22 to 10-24 m2 s-1). However, a tailored reduction of pore width in the sorbent reduces the diffusivity of smaller drug molecules (captopril) by an order of magnitude. Thus, engineered pore morphology in our synthesized carbon sorbent, along with its potential to tailor the chemistry of its interaction with sorbet, can be exploited for optimal delivery system of a preferred drug within its therapeutic level and below the level of toxicity.

  17. Validation of liquid biopsy: plasma cell-free DNA testing in clinical management of advanced non-small cell lung cancer

    Directory of Open Access Journals (Sweden)

    Veldore VH

    2018-01-01

    Full Text Available Vidya H Veldore,1,* Anuradha Choughule,2,* Tejaswi Routhu,1 Nitin Mandloi,1 Vanita Noronha,2 Amit Joshi,2 Amit Dutt,3 Ravi Gupta,1 Ramprasad Vedam,1 Kumar Prabhash2 1MedGenome Labs Private Ltd,, Bangalore, India; 2Tata Memorial Centre, Parel, Mumbai, India; 3The Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Center, Kharghar, Navi Mumbai, Maharashtra, India *These authors contributed equally to this work Abstract: Plasma cell-free tumor DNA, or circulating tumor DNA (ctDNA, from liquid biopsy is a potential source of tumor genetic material, in the absence of tissue biopsy, for EGFR testing. Our validation study reiterates the clinical utility of ctDNA next generation sequencing (NGS for EGFR mutation testing in non-small cell lung cancer (NSCLC. A total of 163 NSCLC cases were included in the validation, of which 132 patients had paired tissue biopsy and ctDNA. We chose to validate ctDNA using deep sequencing with custom designed bioinformatics methods that could detect somatic mutations at allele frequencies as low as 0.01%. Benchmarking allele specific real time PCR as one of the standard methods for tissue-based EGFR mutation testing, the ctDNA NGS test was validated on all the plasma derived cell-free DNA samples. We observed a high concordance (96.96% between tissue biopsy and ctDNA for oncogenic driver mutations in Exon 19 and Exon 21 of the EGFR gene. The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic accuracy of the assay were 91.1%, 100% 100%, 95.6%, and 97%, respectively. A false negative rate of 3% was observed. A subset of mutations was also verified on droplet digital PCR. Sixteen percent EGFR mutation positivity was observed in patients where only liquid biopsy was available, thus creating options for targeted therapy. This is the first and largest study from India, demonstrating successful validation of circulating cell-free DNA as a clinically

  18. Synthesizing Smart Polymeric and Composite Materials

    Science.gov (United States)

    Gong, Chaokun

    Smart materials have been widely investigated to explore new functionalities unavailable to traditional materials or to mimic the multifunctionality of biological systems. Synthetic polymers are particularly attractive as they already possess some of the attributes required for smart materials, and there are vast room to further enhance the existing properties or impart new properties by polymer synthesis or composite formulation. In this work, three types of smart polymer and composites have been investigated with important new applications: (1) healable polymer composites for structural application and healable composite conductor for electronic device application; (2) conducting polymer polypyrrole actuator for implantable medical device application; and (3) ferroelectric polymer and ceramic nanoparticles composites for electrocaloric effect based solid state refrigeration application. These application entail highly challenging materials innovation, and my work has led to significant progress in all three areas. For the healable polymer composites, well known intrinsically healable polymer 2MEP4F (a Diels-Alder crosslinked polymer formed from a monomer with four furan groups and another monomer with two maleimide groups) was first chosen as the matrix reinforced with fiber. Glass fibers were successfully functionalized with maleimide functional groups on their surface. Composites from functionalized glass fibers and 2MEP4F healable polymer were made to compare with composites made from commercial carbon fibers and 2MEP4F polymer. Dramatically improved short beam shear strength was obtained from composite of functionalized glass fibers and 2MEP4F polymer. The high cost of 2MEP4F polymer can potentially limit the large-scale application of the developed healable composite, we further developed a new healable polymer with much lower cost. This new polymer was formed through the Diels-Alder crosslinking of poly(furfuryl alcohol) (PFA) and 1,1'-(Methylenedi-4

  19. Cell-Free and Cell-Based Approaches to Explore the Roles of Host Membranes and Lipids in the Formation of Viral Replication Compartment Induced by Tombusviruses.

    Science.gov (United States)

    Nagy, Peter D; Pogany, Judit; Xu, Kai

    2016-03-03

    Plant positive strand RNA viruses are intracellular infectious agents that take advantage of cellular lipids and membranes to support replication and protect viral RNA from degradation by host antiviral responses. In this review, we discuss how Tomato bushy stunt virus (TBSV) co-opts lipid transfer proteins and modulates lipid metabolism and transport to facilitate the assembly of the membrane-bound viral replicase complexes within intricate replication compartments. Identification and characterization of the proviral roles of specific lipids and proteins involved in lipid metabolism based on results from yeast (Saccharomyces cerevisiae) model host and cell-free approaches are discussed. The review also highlights the advantage of using liposomes with chemically defined composition to identify specific lipids required for TBSV replication. Remarkably, all the known steps in TBSV replication are dependent on cellular lipids and co-opted membranes.

  20. Positive view and increased likely uptake of follow-up testing with analysis of cell-free fetal DNA as alternative to invasive testing among Danish pregnant women

    DEFF Research Database (Denmark)

    Miltoft, Caroline B; Rode, Line; Tabor, Ann

    2018-01-01

    AND METHODS: Unselected and high-risk women attending first-trimester screening (Rigshospitalet, Copenhagen University Hospital) were invited to fill out the questionnaire Antenatal testing for Down syndrome as an online survey. RESULTS: The survey included 203 unselected and 50 high-risk women (response...... of follow-up testing without a corresponding rise in the termination rate of affected fetuses as some women test for information only. However, both unselected and high-risk women had overwhelmingly positive views underlining attention to avoid routinization.......INTRODUCTION: The aim of this study was to investigate the attitude (view, likely uptake and preferred strategy) towards cell-free fetal DNA (cfDNA) testing among pregnant women before a first-trimester risk assessment for trisomy 21 (unselected women) and after obtaining a high risk. MATERIAL...

  1. A rapid and cell-free assay to test the activity of lynch syndrome-associated MSH2 and MSH6 missense variants

    DEFF Research Database (Denmark)

    Drost, Mark; Zonneveld, José B M; van Hees, Sandrine

    2012-01-01

    amino acid alterations. The pathogenicity of these variants of uncertain significance (VUS) is difficult to assess, precluding diagnosis of carriers and their relatives. Here we present a rapid cell-free assay to investigate MMR activity of MSH2 or MSH6 VUS. We used this assay to analyze a series of MSH......2 and MSH6 VUS, selected from the Leiden Open Variation Database. Whereas a significant fraction of the MSH2 VUS has lost MMR activity, suggesting pathogenicity, the large majority of the MSH6 VUS appears MMR proficient. We anticipate that this assay will be an important tool in the development...... of a comprehensive and widely applicable diagnostic procedure for LS-associated VUS....

  2. Application of real-time PCR of sex-independent insertion-deletion polymorphisms to determine fetal sex using cell-free fetal DNA from maternal plasma.

    Science.gov (United States)

    Ho, Sherry Sze Yee; Barrett, Angela; Thadani, Henna; Asibal, Cecille Laureano; Koay, Evelyn Siew-Chuan; Choolani, Mahesh

    2015-07-01

    Prenatal diagnosis of sex-linked disorders requires invasive procedures, carrying a risk of miscarriage of up to 1%. Cell-free fetal DNA (cffDNA) present in cell-free DNA (cfDNA) from maternal plasma offers a non-invasive source of fetal genetic material for analysis. Detection of Y-chromosome sequences in cfDNA indicates presence of a male fetus; in the absence of a Y-chromosome signal a female fetus is inferred. We aimed to validate the clinical utility of insertion-deletion polymorphisms (INDELs) to confirm presence of a female fetus using cffDNA. Quantitative real-time PCR (qPCR) for the Y-chromosome-specific sequence, SRY, was performed on cfDNA from 82 samples at 6-39 gestational weeks. In samples without detectable SRY, qPCRs for eight INDELs were performed on maternal genomic DNA and cfDNA. Detection of paternally inherited fetal alleles in cfDNA negative for SRY confirmed a female fetus. Fetal sex was correctly determined in 77/82 (93.9%) cfDNA samples. SRY was detected in all 39 samples from male-bearing pregnancies, and none of the 43 female-bearing pregnancies (sensitivity and specificity of SRY qPCR is therefore 100%; 95% CI 91%-100%). Paternally inherited fetal alleles were detected in 38/43 samples with no SRY signal, confirming the presence of a female fetus (INDEL assay sensitivity is therefore 88.4%; 95% CI 74.1%-95.6%). Since paternally inherited fetal INDELs were not used in women bearing male fetuses, the specificity of INDELs cannot be calculated. Five cfDNA samples were negative for both SRY and INDELS. We have validated a non-invasive prenatal test to confirm fetal sex as early as 6 gestational weeks using cffDNA from maternal plasma.

  3. Biocompatibility of poly allylamine synthesized by plasma

    International Nuclear Information System (INIS)

    Colin, E.; Enriquez, M.A.; Olayo, M.G.; Cruz, G.J.; Morales, J.; Olayo, R.

    2007-01-01

    A study of the electric and hydrophilic properties of poly allylamine (PAI) synthesized by plasma whose structure contains N-H, C-H, C-O and O-H bonds is presented, that promote the biocompatibility with the human body. To study the PAI hydrolytic affinity, solutions of salt concentration similar to those of the human body were used. The results indicate that the solutions modify the charge balance in the surfaces reducing the hydrophobicity in the poly allylamine whose contact angle oscillates among 10 and 16 degrees and the liquid-solid surface tension between 4 and 8 dina/cm. (Author)

  4. Synthese monitoring mestmarkt 2006-2012

    OpenAIRE

    Koeijer, de, T.J.; Luesink, H.H.; Daatselaar, C.H.G.

    2014-01-01

    De aanvoer en afzet van dierlijke mest via de mestmarkt in Nederland zijn op verzoek van het ministerie van Economische Zaken (EZ) voor de periode 2006-2012 in beeld gebracht. Dit is gedaan op basis van analyses van de Vervoersbewijzen Dierlijke Mest (VDM’s) van RVO.nl (Rijksdienst voor Ondernemend Nederland) en op basis van modelberekeningen met MAMBO. Dit WOt-technical report geeft een synthese van de resultaten. Op basis van vergelijkbare meststromen is het aanbod op basis van de VDM’s 73 ...

  5. Biogenic synthesized nanoparticles and their applications

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Abhijeet, E-mail: abhijeet.singh@jaipur.manipal.edu; Sharma, Madan Mohan [Manipal University Jaipur (India)

    2016-05-06

    In the present scenario, there are growing concerns over the potential impacts of bioengineered nanoparticles in the health sector. However, our understanding of how bioengineered nanoparticles may affect organisms within natural ecosystems, lags far behind our rapidly increasing ability to engineer novel nanoparticles. To date, research on the biological impacts of bioengineered nanoparticles has primarily consisted of controlled lab studies of model organisms with single species in culture media. Here, we described a cost effective and environment friendly technique for green synthesis of silver nanoparticles. Silver nanoparticles were successfully synthesized from 1 mM AgNO{sub 3} via a green synthesis process using leaf extract as reducing as well as capping agent. Nanoparticles were characterized with the help of UV–vis absorption spectroscopy, X-ray diffraction and TEM analysis which revealed the size of nanoparticles of 30-40 nm size. Further the nanoparticles synthesized by green route are found highly toxic against pathogenic bacteria and plant pathogenic fungi viz. Escherichia coli, Pseudomonas syringae and Sclerotiniasclerotiorum. The most important outcome of this work will be the development of value-added products and protection of human health from pathogens viz., bacteria, virus, fungi etc.

  6. Biogenic synthesized nanoparticles and their applications

    International Nuclear Information System (INIS)

    Singh, Abhijeet; Sharma, Madan Mohan

    2016-01-01

    In the present scenario, there are growing concerns over the potential impacts of bioengineered nanoparticles in the health sector. However, our understanding of how bioengineered nanoparticles may affect organisms within natural ecosystems, lags far behind our rapidly increasing ability to engineer novel nanoparticles. To date, research on the biological impacts of bioengineered nanoparticles has primarily consisted of controlled lab studies of model organisms with single species in culture media. Here, we described a cost effective and environment friendly technique for green synthesis of silver nanoparticles. Silver nanoparticles were successfully synthesized from 1 mM AgNO_3 via a green synthesis process using leaf extract as reducing as well as capping agent. Nanoparticles were characterized with the help of UV–vis absorption spectroscopy, X-ray diffraction and TEM analysis which revealed the size of nanoparticles of 30-40 nm size. Further the nanoparticles synthesized by green route are found highly toxic against pathogenic bacteria and plant pathogenic fungi viz. Escherichia coli, Pseudomonas syringae and Sclerotiniasclerotiorum. The most important outcome of this work will be the development of value-added products and protection of human health from pathogens viz., bacteria, virus, fungi etc.

  7. Impact of 50% Synthesized Iso-Paraffins (SIP) on F-76 Fuel Coalescence

    Science.gov (United States)

    2013-12-16

    petroleum JP-5 and Synthesized Iso-Paraffins (SIP). SIP fuels are made from direct fermentation of sugar into olefinic hydrocarbons. The olefinic...manufactured scaled down filter/coalescer and separator to simulate the performance of a full-scale filter separator system. This test is designed to predict...5 and Synthesized Iso-Paraffins (SIP). SIP fuels are made from direct fermentation of sugar into olefinic hydrocarbons. The olefinic hydrocarbons

  8. Characterization of hematite nanoparticles synthesized via two different pathways

    Science.gov (United States)

    Das, Soumya; Hendry, M. Jim

    2014-08-01

    Hematite is one of the most common and thermodynamically stable iron oxides found in both natural and anthropogenic systems. Owing to its ubiquity, stability, moderate specific surface area, and ability to sequester metals and metalloids from aquatic systems, it has been the subject of a large number of adsorption studies published during the past few decades. Although preparation techniques are known to affect the surface morphology of hematite nanoparticles, the effects of aging under environmentally relevant conditions have yet to be tested with respect to surface morphology, surface area, and adsorptive capacity. We prepared hematite via two different pathways and aged it under highly alkaline conditions encountered in many mill tailings settings. Crystal habits and morphologies of the hematite nanoparticles were analyzed via scanning electron microscopy and transmission electron microscopy. X-ray diffraction, Raman spectroscopy, and Brunauer-Emmett-Teller surface area analyses were also conducted on the hematite nanoparticles before and after aging. The hematite synthesized via an Fe(III) salt solution (average particle size 37 nm) was morphologically and structurally different from the hematite synthesized via ferrihydrite aging (average particle size 144 nm). Overall, our data demonstrate that the crystallinity of hematite produced via ferrihydrite transformation is susceptible to morphological alterations/modifications. In contrast, the hematite formed via hydrolysis of an Fe(III) salt solution remains very stable in terms of structure, size, and morphology even under extreme experimental conditions.

  9. Enantioselective catalytic syntheses of alpha-branched chiral amines

    DEFF Research Database (Denmark)

    Brase, S.; Baumann, T.; Dahmen, S.

    2007-01-01

    Chiral amines play a pivotal role in fine chemical and natural product syntheses and the design of novel materials.......Chiral amines play a pivotal role in fine chemical and natural product syntheses and the design of novel materials....

  10. Methods for synthesizing metal oxide nanowires

    Science.gov (United States)

    Sunkara, Mahendra Kumar; Kumar, Vivekanand; Kim, Jeong H.; Clark, Ezra Lee

    2016-08-09

    A method of synthesizing a metal oxide nanowire includes the steps of: combining an amount of a transition metal or a transition metal oxide with an amount of an alkali metal compound to produce a mixture; activating a plasma discharge reactor to create a plasma discharge; exposing the mixture to the plasma discharge for a first predetermined time period such that transition metal oxide nanowires are formed; contacting the transition metal oxide nanowires with an acid solution such that an alkali metal ion is exchanged for a hydrogen ion on each of the transition metal oxide nanowires; and exposing the transition metal oxide nanowires to the plasma discharge for a second predetermined time period to thermally anneal the transition metal oxide nanowires. Transition metal oxide nanowires produced using the synthesis methods described herein are also provided.

  11. Sorption of mercury on chemically synthesized polyaniline

    International Nuclear Information System (INIS)

    Remya Devi, P.S.; Verma, R.; Sudersanan, M.

    2006-01-01

    Sorption of inorganic mercury (Hg 2+ ) and methyl mercury, on chemically synthesized polyaniline, in 0.1-10N HCl solutions has been studied. Hg 2+ is strongly sorbed at low acidities and the extent of sorption decreases with increase in acidity. The sorption of methyl mercury is very low in the HCl concentration range studied. Sorption of Hg 2+ on polyaniline in 0.1-10N LiCl and H 2 SO 4 solutions has also been studied. The analysis of the data indicates that the sorption of Hg 2+ depends on the degree of protonation of polyaniline and the nature of mercury(II) chloride complexes in solution. X-ray photoelectron spectroscopy analysis (XPS) of polyaniline sorbed with mercury show that mercury is bound as Hg 2+ . Sorbed mercury is quantitatively eluted from polyaniline with 0.5N HNO 3 . Polyaniline can be used for separation and pre-concentration of inorganic mercury from aqueous samples. (author)

  12. [Aerobic methylobacteria are capable of synthesizing auxins].

    Science.gov (United States)

    Ivanova, E G; Doronina, N V; Trotsenko, Iu A

    2001-01-01

    Obligately and facultatively methylotrophic bacteria with different pathways of C1 metabolism were found to be able to produce auxins, particularly indole-3-acetic acid (IAA), in amounts of 3-100 micrograms/ml. Indole-3-pyruvic acid and indole-3-acetamide were detected only in methylobacteria with the serine pathway of C1 metabolism, Methylobacterium mesophilicum and Aminobacter aminovorans. The production of auxins by methylobacteria was stimulated by the addition of tryptophan to the growth medium and was inhibited by ammonium ions. The methylobacteria under study lacked tryptophan decarboxylase and tryptophan side-chain oxidase. At the same time, they were found to contain several aminotransferases. IAA is presumably synthesized by methylobacteria through indole-3-pyruvic acid.

  13. Structure of the enzymatically synthesized fructan inulin

    International Nuclear Information System (INIS)

    Heyer, A.G.; Schroeer, B.; Radosta, S.; Wolff, D.; Czapla, S.; Springer, J.

    1998-01-01

    Construction, purification and characterization of a fusion protein of maltose-binding protein of Escherichia coli and the fructosyltransferase of Streptococcus mutans is described. With the purified protein, in vitro synthesis of inulin was performed. The obtained polysaccharide was characterized by high-performance size-exclusion chromatography (HPSEC) and static light scattering (SLS) in dilute aqueous and dimethyl sulfoxide solution. For all samples very high molecular weights between 60x10 6 and 90x10 6 g/mol and a remarkable small polydispersity index of 1.1 have been determined. Small root-mean-square radii of gyration point to a compact conformation in dilute solution. No difference between native and enzymatically synthesized inulin was observed by X-ray powder diffraction and thermoanalysis of solid samples. (Copyright (c) 1998 Elsevier Science B.V., Amsterdam. All rights reserved.)

  14. Structure of the enzymatically synthesized fructan inulin

    Energy Technology Data Exchange (ETDEWEB)

    Heyer, A.G.; Schroeer, B. [Max-Planck-Institut fuer Molekulare Pflanzenphysiologie, Karl-Liebknecht-Str. 25, 14476 Golm (Germany); Radosta, S. [Fraunhofer-Institut fuer Angewandte Polymerforschung, Postfach 126, 14504 Teltow (Germany); Wolff, D.; Czapla, S.; Springer, J. [Technische Universitaet Berlin, FG Makromolekulare Chemie, Str. des 17. Juni 135, 10623 Berlin (Germany)

    1998-12-15

    Construction, purification and characterization of a fusion protein of maltose-binding protein of Escherichia coli and the fructosyltransferase of Streptococcus mutans is described. With the purified protein, in vitro synthesis of inulin was performed. The obtained polysaccharide was characterized by high-performance size-exclusion chromatography (HPSEC) and static light scattering (SLS) in dilute aqueous and dimethyl sulfoxide solution. For all samples very high molecular weights between 60x10{sup 6} and 90x10{sup 6} g/mol and a remarkable small polydispersity index of 1.1 have been determined. Small root-mean-square radii of gyration point to a compact conformation in dilute solution. No difference between native and enzymatically synthesized inulin was observed by X-ray powder diffraction and thermoanalysis of solid samples. (Copyright (c) 1998 Elsevier Science B.V., Amsterdam. All rights reserved.)

  15. Synthesized Mammography: Clinical Evidence, Appearance, and Implementation

    Directory of Open Access Journals (Sweden)

    Melissa A. Durand

    2018-04-01

    Full Text Available Digital breast tomosynthesis (DBT has improved conventional mammography by increasing cancer detection while reducing recall rates. However, these benefits come at the cost of increased radiation dose. Synthesized mammography (s2D has been developed to provide the advantages of DBT with nearly half the radiation dose. Since its F.D.A. approval, multiple studies have evaluated the clinical performance of s2D. In clinical practice, s2D images are not identical to conventional 2D images and are designed for interpretation with DBT as a complement. This article reviews the present literature to assess whether s2D is a practical alternative to conventional 2D, addresses the differences in mammographic appearance of findings, and provides suggestions for implementation into clinical practice.

  16. Synthesized Mammography: Clinical Evidence, Appearance, and Implementation.

    Science.gov (United States)

    Durand, Melissa A

    2018-04-04

    Digital breast tomosynthesis (DBT) has improved conventional mammography by increasing cancer detection while reducing recall rates. However, these benefits come at the cost of increased radiation dose. Synthesized mammography (s2D) has been developed to provide the advantages of DBT with nearly half the radiation dose. Since its F.D.A. approval, multiple studies have evaluated the clinical performance of s2D. In clinical practice, s2D images are not identical to conventional 2D images and are designed for interpretation with DBT as a complement. This article reviews the present literature to assess whether s2D is a practical alternative to conventional 2D, addresses the differences in mammographic appearance of findings, and provides suggestions for implementation into clinical practice.

  17. [Facultative and obligate aerobic methylobacteria synthesize cytokinins].

    Science.gov (United States)

    Ivanova, E G; Doronina, N V; Shepeliakovskaia, A O; Laman, A G; Brovko, F A; Trotsenko, Iu A

    2000-01-01

    The presence and expression of genes controlling the synthesis and secretion of cytokinins by the pink-pigmented facultative methylotroph Methylobacterium mesophilicum VKM B-2143 with the serine pathway and nonpigmented obligate methylotroph Methylovorus mays VKM B-2221 with the ribulose monophosphate pathway of C1 metabolism were shown using the polymerase chain reaction (PCR) and reverse transcription-PCR methods. The presence of the corresponding mRNA in M. mesophilicum cells grown on methanol or succinate suggests that the expression of these genes is constitutive. The cytokinin activity of culture liquid and its fractions was determined by a biotest with Amarantus caudatus L. seedlings. Using enzyme-linked immunosorbent analysis, we detected zeatin (riboside) in the culture liquid of both bacteria studied. The data obtained show that the aerobic methylobacteria are phytosymbionts that are able to utilize the single- and polycarbon compounds secreted by symbiotic plants and to synthesize cytokinins.

  18. Method and apparatus for synthesizing hydrocarbons

    Science.gov (United States)

    Colmenares, C.A.; Somorjai, G.A.; Maj, J.J.

    1985-04-16

    A method and apparatus for synthesizing a mixture of aliphatic alcohols having five carbons or less is disclosed. An equal molar ratio of CO and H/sub 2/ gases is caused to pass through a ThO/sub 2/ catalyst having a surface area of about 80 to 125 m/sup 2//g. The catalyst further optionally includes Na ions present as substitutional cations in an amount of about 5 to 10 atom %. At a temperature of about 570 to 630/sup 0/K, and at pressures of about 20 to 50 atm, methanol and isobutanol are the predominant products and are produced in amounts of about 90 wt % of the total hydrocarbon mixture. 6 figs.

  19. National Gas Survey. Synthesized gaseous hydrocarbon fuels

    Energy Technology Data Exchange (ETDEWEB)

    None

    1978-06-01

    The supply-Technical Advisory Task Force-Synthesized Gaseous Hydrocarbon Fuels considered coal, hydrocarbon liquids, oil shales, tar sands, and bioconvertible materials as potential feedstocks for gaseous fuels. Current status of process technology for each feedstock was reviewed, economic evaluations including sensitivity analysis were made, and constraints for establishment of a synthesized gaseous hydrocarbon fuels industry considered. Process technology is presently available to manufacture gaseous hydrocarbon fuels from each of the feedstocks. In 1975 there were eleven liquid feedstock SNG plants in the United States having a capacity of 1.1 billion SCFD. There can be no contribution of SNG before 1982 from plants using feedstocks other than liquids because there are no plants in operation or under construction as of 1977. Costs for SNG are higher than current regulated prices for U.S. natural gas. Because of large reserves, coal is a prime feedstock candidate although there are major constraints in the area of coal leases, mining and water permits, and others. Commercial technology is available and several new gasification processes are under development. Oil shale is also a feedstock in large supply and commercial process technology is available. There are siting and permit constraints, and water availability may limit the ultimate size of an oil shale processing industry. Under projected conditions, bioconvertible materials are not expected to support the production of large quantities of pipeline quality gas during the next decade. Production of low or medium Btu gas from municipal solid wastes can be expected to be developed in urban areas in conjunction with savings in disposal costs. In the economic evaluations presented, the most significant factor for liquid feedstock plants is the anticipated cost of feedstock and fuel. The economic viability of plants using other feedstocks is primarily dependent upon capital requirements.

  20. Porous oxides synthesized by the combustion method

    International Nuclear Information System (INIS)

    Lugo L, V.

    2005-01-01

    The result of this work, seeks to be a contribution for the treatment of radioactive wastes, with base to the sorption properties that present those porous oxides, synthesized by a method that allows to increase the sorption capacity. The main objective of the present investigation has been the modification of the structural characteristics of the oxides of Fe, Mg and Zn to increase its capacity of sorption of 60 Co in particular. It was studied the effect of the synthesis method by combustion in the inorganic oxides; the obtained solids were characterized using the following techniques: X-ray diffraction (XRD), scanning electron microscopy (SEM), semiquantitative elementary analysis by Dispersive energy spectroscopy (EDS) and determination of surface area by the Brunauner-Emmett-Teller method (BET). Also was carried out batch type experiments for the sorption of Co 2+ , with the purpose of studying the sorption capacity of each one of the prepared oxides. In accordance with that previously exposed, the working plan that was carried out in this investigation is summarized in the following stages: 1. Preparation of inorganic oxides by two different methods, studying the effect of the temperature in the synthesis process. 2. Characterization of the inorganic oxides by XRD, by means of which those were chosen the solids with better properties. 3. Characterization of the inorganic oxides by SEM and EDS where it was studied the morphology of the synthesized materials and the semiquantitative elemental composition. 4. Realization of a sorption experiment type Batch with non radioactive Co 2+ to simulate the sorption of 60 Co and determination of the sorption capacity by means of neutron activation of the non radioactive cobalt. 5. Determination of the surface area by the (BET) technique of the inorganic oxides with better sorption properties. (Author)

  1. Effect of zinc oxide nanoparticles synthesized by a precipitation

    Indian Academy of Sciences (India)

    ZnO nanoparticles were synthesized by a precipitation method in aqueous media from zinc nitrate hexahydrate and sodium hydroxide. The synthesized ZnO nanoparticles exhibited a crystalline structure with hexagonal structure of the wurtzite. The morphology of the synthesized ZnO nanoparticles presented a spherical ...

  2. Methylation status of the APC and RASSF1A promoter in cell-free circulating DNA and its prognostic role in patients with colorectal cancer.

    Science.gov (United States)

    Matthaios, Dimitrios; Balgkouranidou, Ioanna; Karayiannakis, Anastasios; Bolanaki, Helen; Xenidis, Nikolaos; Amarantidis, Kyriakos; Chelis, Leonidas; Romanidis, Konstantinos; Chatzaki, Aikaterini; Lianidou, Evi; Trypsianis, Grigorios; Kakolyris, Stylianos

    2016-07-01

    DNA methylation is the most frequent epigenetic alteration. Using methylation-specific polymerase chain reaction (MSP), the methylation status of the adenomatous polyposis coli ( APC ) and Ras association domain family 1 isoform A ( RASSF1A ) genes was examined in cell-free circulating DNA from 155 plasma samples obtained from patients with early and advanced colorectal cancer (CRC). APC and RASSF1A hypermethylation was frequently observed in both early and advanced disease, and was significantly associated with a poorer disease outcome. The methylation status of the APC and RASSF1A promoters was investigated in cell-free DNA of patients with CRC. Using MSP, the promoter methylation status of APC and RASSF1A was examined in 155 blood samples obtained from patients with CRC, 88 of whom had operable CRC (oCRC) and 67 had metastatic CRC (mCRC). The frequency of APC methylation in patients with oCRC was 33%. Methylated APC promoter was significantly associated with older age (P=0.012), higher stage (P=0.014) and methylated RASSF1A status (P=0.050). The frequency of APC methylation in patients with mCRC was 53.7%. In these patients, APC methylation was significantly associated with methylated RASSF1A status (P=0.016). The frequency of RASSF1A methylation in patients with oCRC was 25%. Methylated RASSF1A in oCRC was significantly associated with higher stage (P=0.021). The frequency of RASSF1A methylation in mCRC was 44.8%. Methylated RASSF1A in mCRC was associated with moderate differentiation (P=0.012), high levels of carcinoembryonic antigen (P=0.023) and methylated APC status (P=0.016). Patients with an unmethylated APC gene had better survival in both early (81±5 vs. 27±4 months, PAPC . Patients with an unmethylated RASSF1A gene had better survival in both early (71±6 vs. 46±8 months, PAPC and RASSF1A promoter methylation status and survival may be indicative of a prognostic role for these genes in CRC, which requires additional testing in larger studies.

  3. New aids for the non-invasive prenatal diagnosis of achondroplasia: dysmorphic features, charts of fetal size and molecular confirmation using cell-free fetal DNA in maternal plasma

    NARCIS (Netherlands)

    Chitty, L. S.; Griffin, D. R.; Meaney, C.; Barrett, A.; Khalil, A.; Pajkrt, E.; Cole, T. J.

    2011-01-01

    To improve the prenatal diagnosis of achondroplasia by constructing charts of fetal size, defining frequency of sonographic features and exploring the role of non-invasive molecular diagnosis based on cell-free fetal deoxyribonucleic acid (DNA) in maternal plasma. Data on fetuses with a confirmed

  4. ACVP-05: Virus Genetic Analysis from Cell-Free Plasma, Virally Infected Cells or Tissues and Cultured Supernatant Via Single Genome Amplification and Direct Sequencing | Frederick National Laboratory for Cancer Research

    Science.gov (United States)

    The Viral Evolution Core within the AIDS and Cancer Virus Program will extract viral RNA/DNA from cell-free or cell-associated samples. Complementary (cDNA) will be generated as needed, and cDNA or DNA will be diluted to a single copy prior to nested

  5. Bio-inspired routes for synthesizing efficient nanoscale platinum electrocatalysts

    Energy Technology Data Exchange (ETDEWEB)

    Cha, Jennifer N. [Univ. of California, San Diego, CA (United States); Wang, Joseph [Univ. of California, San Diego, CA (United States)

    2014-08-31

    The overall objective of the proposed research is to use fundamental advances in bionanotechnology to design powerful platinum nanocrystal electrocatalysts for fuel cell applications. The new economically-viable, environmentally-friendly, bottom-up biochemical synthetic strategy will produce platinum nanocrystals with tailored size, shape and crystal orientation, hence leading to a maximum electrochemical reactivity. There are five specific aims to the proposed bio-inspired strategy for synthesizing efficient electrocatalytic platinum nanocrystals: (1) isolate peptides that both selectively bind particular crystal faces of platinum and promote the nucleation and growth of particular nanocrystal morphologies, (2) pattern nanoscale 2-dimensional arrays of platinum nucleating peptides from DNA scaffolds, (3) investigate the combined use of substrate patterned peptides and soluble peptides on nanocrystal morphology and growth (4) synthesize platinum crystals on planar and large-area carbon electrode supports, and (5) perform detailed characterization of the electrocatalytic behavior as a function of catalyst size, shape and morphology. Project Description and Impact: This bio-inspired collaborative research effort will address key challenges in designing powerful electrocatalysts for fuel cell applications by employing nucleic acid scaffolds in combination with peptides to perform specific, environmentally-friendly, simultaneous bottom-up biochemical synthesis and patterned assembly of highly uniform and efficient platinum nanocrystal catalysts. Bulk synthesis of nanoparticles usually produces a range of sizes, accessible catalytic sites, crystal morphologies, and orientations, all of which lead to inconsistent catalytic activities. In contrast, biological systems routinely demonstrate exquisite control over inorganic syntheses at neutral pH and ambient temperature and pressures. Because the orientation and arrangement of the templating biomolecules can be precisely

  6. Integrated digital superconducting logic circuits for the quantum synthesizer. Report

    International Nuclear Information System (INIS)

    Buchholz, F.I.; Kohlmann, J.; Khabipov, M.; Brandt, C.M.; Hagedorn, D.; Balashov, D.; Maibaum, F.; Tolkacheva, E.; Niemeyer, J.

    2006-11-01

    This report presents the results, which were reached in the framework of the BMBF cooperative plan ''Quantum Synthesizer'' in the partial plan ''Integrated Digital Superconducting Logic Circuits''. As essential goal of the plan a novel instrument on the base of quantum-coherent superconducting circuits should be developed. which allows to generate praxis-relevant wave forms with quantum accuracy, the quantum synthesizer. The main topics of development of the reported partial plan lied at the one hand in the development of integrated, digital, superconducting circuit in rapid-single-flux (RSFQ) quantum logics for the pattern generator of the quantum synthesizer, at the other hand in the further development of the fabrication technology for the aiming of high circuit complexity. In order to fulfil these requirements at the PTB a new design system was implemented, based on the software of Cadence. Together with the required RSFQ extensions for the design of digital superconducting circuits was a platform generated, on which the reachable circuit complexity is exclusively limited by the technology parameters of the available fabrication technology: Physical simulations are with PSCAN up to a complexity of more than 1000 circuit elements possible; furthermore VHDL allows the verification of arbitrarily large circuit architectures. In accordance for this the production line at the PTB was brought to a level, which allows in Nb/Al-Al x O y /Nb SIS technology implementation the fabrication of highly integrable RSFQ circuit architectures. The developed and fabricated basic circuits of the pattern generator have proved correct functionality and reliability in the measuring operation. Thereby for the circular RSFQ shift registers a key role as local memories in the construction of the pattern generator is devolved upon. The registers were realized with the aimed bit lengths up to 128 bit and with reachable signal-processing speeds of above 10 GHz. At the interface RSFQ

  7. Formulation and in vitro release evaluation of newly synthesized palm kernel oil esters-based nanoemulsion delivery system for 30% ethanolic dried extract derived from local Phyllanthus urinaria for skin antiaging

    Directory of Open Access Journals (Sweden)

    Mahdi ES

    2011-10-01

    Full Text Available Elrashid Saleh Mahdi1, Azmin Mohd Noor1, Mohamed Hameem Sakeena1, Ghassan Z Abdullah1, Muthanna F Abdulkarim1, Munavvar Abdul Sattar2 1Department of Pharmaceutical Technology, 2Department of Physiology, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Pulau Pinang, Malaysia Background: Recently there has been a remarkable surge of interest about natural products and their applications in the cosmetic industry. Topical delivery of antioxidants from natural sources is one of the approaches used to reverse signs of skin aging. The aim of this research was to develop a nanoemulsion cream for topical delivery of 30% ethanolic extract derived from local Phyllanthus urinaria (P. urinaria for skin antiaging. Methods: Palm kernel oil esters (PKOEs-based nanoemulsions were loaded with P. urinaria extract using a spontaneous method and characterized with respect to particle size, zeta potential, and rheological properties. The release profile of the extract was evaluated using in vitro Franz diffusion cells from an artificial membrane and the antioxidant activity of the extract released was evaluated using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH method. Results: Formulation F12 consisted of wt/wt, 0.05% P. urinaria extract, 1% cetyl alcohol, 0.5% glyceryl monostearate, 12% PKOEs, and 27% Tween® 80/Span® 80 (9/1 with a hydrophilic lipophilic balance of 13.9, and a 59.5% phosphate buffer system at pH 7.4. Formulation F36 was comprised of 0.05% P. urinaria extract, 1% cetyl alcohol, 1% glyceryl monostearate, 14% PKOEs, 28% Tween® 80/Span® 80 (9/1 with a hydrophilic lipophilic balance of 13.9, and 56% phosphate buffer system at pH 7.4 with shear thinning and thixotropy. The droplet size of F12 and F36 was 30.74 nm and 35.71 nm, respectively, and their nanosizes were confirmed by transmission electron microscopy images. Thereafter, 51.30% and 51.02% of the loaded extract was released from F12 and F36 through an artificial cellulose membrane

  8. Effect of lanthanide contraction on the mixed polyamine systems Ln/Sb/Se/(en+dien) and Ln/Sb/Se/(en+trien): Syntheses and characterizations of lanthanide complexes with a tetraelenidoantimonate ligand

    International Nuclear Information System (INIS)

    Zhao Jing; Liang Jingjing; Pan Yingli; Zhang Yong; Jia Dingxian

    2011-01-01

    Mixed polyamine systems Ln/Sb/Se/(en+dien) and Ln/Sb/Se/(en+trien) (Ln=lanthanide, en=ethylenediamine, dien=diethylenetriamine, trien=triethylenetetramine) were investigated under solvothermal conditions, and novel mixed-coordinated lanthanide(III) complexes [Ln(en) 2 (dien)(η 2 -SbSe 4 )] (Ln=Ce(1a), Nd(1b)), [Ln(en) 2 (dien)(SbSe 4 )] (Ln=Sm(2a), Gd(2b), Dy(2c)), [Ln(en)(trien)(μ-η 1 ,η 2 -SbSe 4 )] ∞ (Ln=Ce(3a), Nd(3b)) and [Sm(en)(trien)(η 2 -SbSe 4 )] (4a) were prepared. Two structural types of lanthanide selenidoantimonates were obtained across the lanthanide series in both en+dien and en+trien systems. The tetrahedral anion [SbSe 4 ] 3- acts as a monodentate ligand mono-SbSe 4 , a bidentate chelating ligand η 2 -SbSe 4 or a tridentate bridging ligand μ-η 1 ,η 2 -SbSe 4 to the lanthanide(III) center depending on the Ln 3+ ions and the mixed ethylene polyamines, indicating the effect of lanthanide contraction on the structures of the lanthanide(III) selenidoantimonates. The lanthanide selenidoantimonates exhibit semiconducting properties with E g between 2.08 and 2.51 eV. - Graphical Abstract: Two structural types of lanthanide(III) selenidoantimonates are formed in both en-dien and en-trien mixed polyamines across lanthanide series, indicating the lanthanide contraction effect on the structures of the lanthanide(III) selenidoantimonates. Highlights: → Two structural types of lanthanide selenidoantimonates are prepared across the lanthanide series in both Ln/Sb/Se/(en+dien) and Ln/Sb/Se/(en+trien) systems. → The [SbSe 4 ] 3- anion acts as a mono-SbSe 4 , a η 2 -SbSe 4 or a μ-η 1 ,η 2 -SbSe 4 ligand to the Ln 3+ ions. → The soft base ligand [SbSe 4 ] 3- can be controlled to coordinate to the Ln 3+ ions with en+dien and en+trien as co-ligands.

  9. Synthesis and characterisation of iron, cobalt and gallium complexes wit the redox-active amide ligand systems pyridinocarboxiamidobenzene and hydroxy phenyl oxamide; Synthese und Charakterisierung von Eisen-, Cobalt- und Galliumkomplexen mit den redoxaktiven Amidligandsystemen Pyridincarboxamidobenzol und Hydroxyphenyloxamid

    Energy Technology Data Exchange (ETDEWEB)

    Beckmann, U.

    2001-07-01

    The interactions of the redox-active ligand systems piridinocarboxamidobenzene and hydroxy phenyl oxamide with the metals iron, cobalt and gallium were investigated. It was found that metal complexes with ligands of the pyridinocarboxamidobenzene and hydroxy phenyl oxamide type can be redox-active in the sense of a ligand-centered reaction. This may provide a better understanding of natural catalysis mechanisms and redox processes. [German] In dieser Arbeit wurde die Wechselwirkung der redoxaktiven Ligandsysteme Pyridincarboxamidobenzol und Hydroxyphenyloxamid mit den Metallen Eisen, Cobalt und Gallium untersucht. Es konnte gezeigt werden, dass Metallkomplexe mit Liganden vom Typ Pyridincarboxamidobenzol und Hydroxyphenyloxamid auch im Sinne einer ligandzentrierten Reaktion redoxaktiv sein koennen. Dies kann dazu beitragen, Katalysemechanismen und Redoxprozesse in der Natur besser zu verstehen. (orig.)

  10. Clinical experience of laboratory follow-up with noninvasive prenatal testing using cell-free DNA and positive microdeletion results in 349 cases.

    Science.gov (United States)

    Schwartz, S; Kohan, M; Pasion, R; Papenhausen, P R; Platt, L D

    2018-02-01

    Screening via noninvasive prenatal testing (NIPT) involving the analysis of cell-free DNA (cfDNA) from plasma has become readily available to screen for chromosomal and DNA aberrations through maternal blood. This report reviews a laboratory's experience with follow-up of positive NIPT screens for microdeletions. Patients that were screened positive by NIPT for a microdeletion involving 1p, 4p, 5p, 15q, or 22q who underwent diagnostic studies by either chorionic villus sampling or amniocentesis were evaluated. The overall positive predictive value for 349 patients was 9.2%. When a microdeletion was confirmed, 39.3% of the cases had additional abnormal microarray findings. Unrelated abnormal microarray findings were detected in 11.8% of the patients in whom the screen positive microdeletion was not confirmed. Stretches of homozygosity in the microdeletion were frequently associated with a false positive cfDNA microdeletion result. Overall, this report reveals that while cfDNA analysis will screen for microdeletions, the positive predictive value is low; in our series it is 9.2%. Therefore, the patient should be counseled accordingly. Confirmatory diagnostic microarray studies are imperative because of the high percentage of false positives and the frequent additional abnormalities not delineated by cfDNA analysis. © 2018 John Wiley & Sons, Ltd.

  11. Effects of heme-PrP complex on cell-free conversion and peroxidase-linked immunodetection of prions in blood-based assays.

    Science.gov (United States)

    Soutyrine, Andrei; Yogasingam, Nishandan; Huang, Hongsheng; Mitchell, Gordon

    2015-08-01

    Prion protein (PrP) binding to natural and synthetic porphyrins has been previously demonstrated but the effects of endogenous heme interactions with PrP remain uncertain. This study investigated implications of this interaction in blood-based peroxidase-linked prion immunodetection and seeded conversion of cellular prion (PrP(C)) into disease associated form (PrP(Sc)). Heme binding to recombinant PrP(C) enhanced intrinsic peroxidase activity (POD) by 2.5-fold and POD inherent to denatured blood accounted for over 84% of luminol-based substrate oxidation in a prion immunodetection assay. An immuno-capture assay showed that 75-98% of blood POD was attributable to binding of PrP(C) with endogenous heme. Additionally, 10 μM heme inhibited (PPrP(C) to PrP(Sc) through the protein misfolding cycling amplification assay. We conclude that the observed effects can interfere with cell-free conversion and peroxidase-linked immunodetection of prions in blood-based assays. These results indicate that heme-PrP interactions could modulate intrinsic POD and protect PrP(C) from conversion into PrP(Sc). Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

  12. Low-Dose Ionizing Radiation Affects Mesenchymal Stem Cells via Extracellular Oxidized Cell-Free DNA: A Possible Mediator of Bystander Effect and Adaptive Response

    Directory of Open Access Journals (Sweden)

    V. A. Sergeeva

    2017-01-01

    Full Text Available We have hypothesized that the adaptive response to low doses of ionizing radiation (IR is mediated by oxidized cell-free DNA (cfDNA fragments. Here, we summarize our experimental evidence for this model. Studies involving measurements of ROS, expression of the NOX (superoxide radical production, induction of apoptosis and DNA double-strand breaks, antiapoptotic gene expression and cell cycle inhibition confirm this hypothesis. We have demonstrated that treatment of mesenchymal stem cells (MSCs with low doses of IR (10 cGy leads to cell death of part of cell population and release of oxidized cfDNA. cfDNA has the ability to penetrate into the cytoplasm of other cells. Oxidized cfDNA, like low doses of IR, induces oxidative stress, ROS production, ROS-induced oxidative modifications of nuclear DNA, DNA breaks, arrest of the cell cycle, activation of DNA reparation and antioxidant response, and inhibition of apoptosis. The MSCs pretreated with low dose of irradiation or oxidized cfDNA were equally effective in induction of adaptive response to challenge further dose of radiation. Our studies suggest that oxidized cfDNA is a signaling molecule in the stress signaling that mediates radiation-induced bystander effects and that it is an important component of the development of radioadaptive responses to low doses of IR.

  13. The usage and current approaches of cell free fetal DNA (cffDNA as a prenatal diagnostic method in fetal aneuploidy screening

    Directory of Open Access Journals (Sweden)

    Hülya Erbaba

    2015-12-01

    Full Text Available Prenatal diagnosis of invasive and noninvasive tests can be done in a way (NIPT, but because of the invasive methods have risks of infection and abortion, diagnosing non-invasive procedure increasing day by day. One of the widespread cell free fetal DNA in maternal blood test (cffDNA that is increasing in clinical use has been drawing attention. The incidence of aneuploidy chromosomal anomaly of the kind in which all live births; Trisomy 21 (Down Syndrome 1/800, trisomy 13 (Patau syndrome 1 /10,000, trisomy 18 (Edwards syndrome is a form of 1/6000. Because of the high mortality and morbidity, it is vital that congenital anomalies should be diagnosed in prenatal period. Aneuploidy testing for high-risk pregnant women after the 10th week of pregnancy in terms of the blood sample is taken and free fetal DNA in maternal plasma is based on the measurement of the relative amount. Knowledge of the current criteria for use by healthcare professionals in the field test will allow the exclusion of maternal and fetal risks. In this study, it is aimed to demonstrate current international approaches related to the positive and negative sides of non-invasive that is one of the prenatal diagnostic methods of cffDNA test. J Clin Exp Invest 2015; 6 (4: 414-417

  14. Anti-adherence potential of Enterococcus durans cells and its cell-free supernatant on plastic and stainless steel against foodborne pathogens.

    Science.gov (United States)

    Amel, Ait Meddour; Farida, Bendali; Djamila, Sadoun

    2015-07-01

    It is demonstrated that numerous bacteria are able to attach to surfaces of equipment used for food handling or processing. In this study, a strain of Enterococcus durans, originally isolated from a milking machine surface, was firstly studied for its biofilm formation potential on plastic and stainless steel supports. The strain was found to be a biofilm producer either at 25, 30 or 37 °C on polystyrene microtitre plates, with a best adherence level observed at 25 °C. En. durans showed a strong adhesion to stainless steel AISI-304. Antibacterial and anti-adherence activities of En. durans were tested against four foodborne pathogens (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853 and Listeria innocua CLIP 74915) which were shown as biofilm producers on both plastic and stainless steel. En. durans cells and cell-free culture supernatant showed a significant (P < 0.05) inhibition potential of the pathogens either on solid media or in broth co-cultures. Characterization of the antibacterial substances indicated their proteinaceous nature which assigned them most probably to bacteriocins group.

  15. Impact of Cell-Free Fetal DNA Screening on Patients’ Choice of Invasive Procedures after a Positive California Prenatal Screen Result

    Directory of Open Access Journals (Sweden)

    Forum T. Shah

    2014-07-01

    Full Text Available Until recently, maternal serum analyte levels paired with sonographic fetal nuchal translucency measurement was the most accurate prenatal screen available for Trisomies 18 and 21, (91% and 94% detection and false positive rates of 0.31% and 4.5% respectively. Women with positive California Prenatal Screening Program (CPSP results have the option of diagnostic testing to determine definitively if the fetus has a chromosomal abnormality. Cell-free fetal (cff- DNA screening for Trisomies 13, 18, and 21 was first offered in 2012, allowing women with positive screens to choose additional screening before diagnostic testing. Cff-DNA sensitivity rates are as high as 99.9% and 99.1%, with false positive rates of 0.4% and 0.1%, for Trisomies 18 and 21, respectively. A retrospective chart review was performed in 2012 on 500 CPSP referrals at the University of California, San Diego Thornton Hospital. Data were collected prior to and after the introduction of cff-DNA. There was a significant increase in the number of participants who chose to pursue additional testing and a decrease in the number of invasive procedures performed after cff-DNA screening was available. We conclude that as fetal aneuploidy screening improves, the number of invasive procedures will continue to decrease.

  16. Plasma cell-free DNA and its DNA integrity as biomarker to distinguish prostate cancer from benign prostatic hyperplasia in patients with increased serum prostate-specific antigen.

    Science.gov (United States)

    Feng, Jiang; Gang, Feng; Li, Xiao; Jin, Tang; Houbao, Huang; Yu, Cao; Guorong, Li

    2013-08-01

    To investigate whether plasma cell-free DNA (cfDNA) or its integrity could differentiate prostate cancer from benign prostate hyperplasia (BPH) in patients with serum prostate-specific antigen (PSA) ≥ 4 ng/ml. Ninety-six patients with prostate cancer and 112 patients with BPH were enrolled. cfDNA levels in plasma before prostate biopsy were quantified by real-time PCR amplification of ALU gene (product size of 115 bp), and quantitative ratio of ALU (247 bp) to ALU (115 bp) reflected the integrity of cfDNA. In patients with serum PSA ≥ 4 ng/ml, there were significant differences in plasma cfDNA or its integrity between the patients with prostate cancer (19.74 ± 4.43, 0.34 ± 0.05) and patients with BPH (7.36 ± 1.58, 0.19 ± 0.03; P Prostate cancer could be differentiated with a sensitivity of 73.2 % and a specificity of 72.7 % by cfDNA (AUC = 0.864). The integrity of cfDNA had a sensitivity of 81.7 % and a specificity of 78.8 % for the distinguishing prostate cancer from BPH (AUC = 0.910). cfDNA and its integrity could be applied to differentiate prostate cancer from BPH in patients with serum PSA ≥ 4 ng/ml.

  17. Quantification of cell-free DNA in blood plasma and DNA damage degree in lymphocytes to evaluate dysregulation of apoptosis in schizophrenia patients.

    Science.gov (United States)

    Ershova, E S; Jestkova, E M; Chestkov, I V; Porokhovnik, L N; Izevskaya, V L; Kutsev, S I; Veiko, N N; Shmarina, G; Dolgikh, O; Kostyuk, S V

    2017-04-01

    Oxidative DNA damage has been proposed as one of the causes of schizophrenia (SZ), and post mortem data indicate a dysregulation of apoptosis in SZ patients. To evaluate apoptosis in vivo we quantified the concentration of plasma cell-free DNA (cfDNA index, determined using fluorescence), the levels of 8-oxodG in cfDNA (immunoassay) and lymphocytes (FL1-8-oxodG index, flow cytometry) of male patients with acute psychotic disorders: paranoid SZ (total N = 58), schizophreniform (N = 11) and alcohol-induced (N = 14) psychotic disorder, and 30 healthy males. CfDNA in SZ (N = 58) does not change compared with controls. In SZ patients. Elevated levels of 8-oxodG were found in cfDNA (N = 58) and lymphocytes (n = 45). The main sources of cfDNA are dying cells with oxidized DNA. Thus, the cfDNA/FL1-8-oxodG ratio shows the level of apoptosis in damaged cells. Two subgroups were identified among the SZ patients (n = 45). For SZ-1 (31%) and SZ-2 (69%) median values of cfDNA/FL1-8-oxodG index are related as 1:6 (p DNA in the patient's body tissues and may be a contributing cause of acute psychotic disorder. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Comparison of the quantification of KRAS mutations by digital PCR and E-ice-COLD-PCR in circulating-cell-free DNA from metastatic colorectal cancer patients.

    Science.gov (United States)

    Sefrioui, David; Mauger, Florence; Leclere, Laurence; Beaussire, Ludivine; Di Fiore, Frédéric; Deleuze, Jean-François; Sarafan-Vasseur, Nasrin; Tost, Jörg

    2017-02-01

    Circulating cell-free DNA (ccfDNA) bears great promise as biomarker for personalized medicine, but ccfDNA is present only at low levels in the plasma or serum of cancer patients. E-ice-COLD-PCR is a recently developed enrichment method to detect and identify mutations present at low-abundance in clinical samples. However, recent studies have shown the importance to accurately quantify low-abundance mutations as clinically important decisions will depend on certain mutation thresholds. The possibility for an enrichment method to accurately quantify the mutation levels remains a point of concern and might limit its clinical applicability. In the present study, we compared the quantification of KRAS mutations in ccfDNA from metastatic colorectal cancer patients by E-ice-COLD-PCR with two digital PCR approaches. For the quantification of mutations by E-ice-COLD-PCR, cell lines with known mutations diluted into WT genomic DNA were used for calibration. E-ice-COLD-PCR and the two digital PCR approaches showed the same range of the mutation level and were concordant for mutation levels below the clinical relevant threshold. E-ice-COLD-PCR can accurately detect and quantify low-abundant mutations in ccfDNA and has a shorter time to results making it compatible with the requirements of analyses in a clinical setting without the loss of quantitative accuracy. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  19. The diagnostic value of circulating cell free DNA quantification in non-small cell lung cancer: A systematic review with meta-analysis.

    Science.gov (United States)

    Jiang, Tao; Zhai, Changyun; Su, Chunxia; Ren, Shengxiang; Zhou, Caicun

    2016-10-01

    The aim of the current study was to assess the diagnostic value of circulating cell free DNA (cfDNA) quantification in discriminating non-small cell lung cancer (NSCLC) from healthy individuals. An electronic search was conducted on PubMed, EMBASE, Web of Science, and Cochrane Library. Eligible studies regarding to examine the diagnostic value of cfDNA in the detection of NSCLC were extracted and analyzed. We identified 15 eligible studies with a total of 2125 patients. The pooled results for quantification of cfDNA in lung cancer screening in the included studies were as follows: sensitivity, 81% (95% confidence interval (CI), 76%-84%); specificity, 85% (95% CI, 77%-91%); diagnostic odds ratio, 23.87 (95% CI, 13.37-42.61); and areas under the summary receiver operating characteristic curves were 0.89 (95% CI, 0.86-0.92). Subgroup analyses according to the time of sample collection, sample materials, test method, reference gene and cutoff value did not improve sensitivity, but specificity could be significantly improved when we only included the studies using cfDNA sample before surgery or antitumor treatment and real-time PCR to detect cfDNA and human β-actin as a reference gene. Quantification of cfDNA was a promising and effective biomarker for discriminating NSCLC from healthy individuals. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  20. eTumorType, An Algorithm of Discriminating Cancer Types for Circulating Tumor Cells or Cell-free DNAs in Blood

    Directory of Open Access Journals (Sweden)

    Jinfeng Zou

    2017-04-01

    Full Text Available With the technology development on detecting circulating tumor cells (CTCs and cell-free DNAs (cfDNAs in blood, serum, and plasma, non-invasive diagnosis of cancer becomes promising. A few studies reported good correlations between signals from tumor tissues and CTCs or cfDNAs, making it possible to detect cancers using CTCs and cfDNAs. However, the detection cannot tell which cancer types the person has. To meet these challenges, we developed an algorithm, eTumorType, to identify cancer types based on copy number variations (CNVs of the cancer founding clone. eTumorType integrates cancer hallmark concepts and a few computational techniques such as stochastic gradient boosting, voting, centroid, and leading patterns. eTumorType has been trained and validated on a large dataset including 18 common cancer types and 5327 tumor samples. eTumorType produced high accuracies (0.86–0.96 and high recall rates (0.79–0.92 for predicting colon, brain, prostate, and kidney cancers. In addition, relatively high accuracies (0.78–0.92 and recall rates (0.58–0.95 have also been achieved for predicting ovarian, breast luminal, lung, endometrial, stomach, head and neck, leukemia, and skin cancers. These results suggest that eTumorType could be used for non-invasive diagnosis to determine cancer types based on CNVs of CTCs and cfDNAs.

  1. Antioxidative response of the three macrophytes Ceratophyllum demersum, Egeria densa, and Hydrilla verticillata to a time dependent exposure of cell-free crude extracts containing three microcystins from cyanobacterial blooms of Lake Amatitlán, Guatemala.

    Science.gov (United States)

    Romero-Oliva, Claudia Suseth; Contardo-Jara, Valeska; Pflugmacher, Stephan

    2015-06-01

    Microcystins (MCs) produced by cyanobacteria in natural environments are a potential risk to the integrity of ecosystems. In this study, the effects of cyanobacterial cell-free crude extracts from a Microcystis aeruginosa bloom containing three MC-congeners MC-LR, -RR, and -YR at environmental relevant concentrations of 49.3±2.9, 49.8±5.9, and 6.9±3.8μg/L, respectively, were evaluated on Ceratophyllum demersum (L.), Egeria densa (Planch.), and Hydrilla verticillata (L.f.). Effects on photosynthetic pigments (total chlorophyll (chl), chl a, chl b, and carotenoids), enzymatic defense led by catalase (CAT), peroxidase (POD) and glutathione reductase (GR), and biotransformation enzyme glutathione S-transferase (GST) were measured after 1, 4, and 8h and after 1, 3, 7, and 14 days of exposure. Results show that in all exposed macrophytes, photosynthetic pigments were negatively affected. While chl a and total chl decreased with increasing exposure time, a parallel increase in chl b was observed after 8h. Concomitant increase of ∼5, 16, and 34% of antioxidant carotenoid concentration in exposed C. demersum, E. densa, and H. verticillata, respectively, was also displayed. Enzymatic antioxidant defense systems in all exposed macrophytes were initiated within the first hour of exposure. In exposed E. densa, highest values of CAT and GR activities were observed after 4 and 8h, respectively, while in exposed H. verticillata highest value of POD activity was observed after 8h. An early induction with a significant increase of biotransformation enzyme GST was observed in E. densa after 4h and in C. demersum and H. verticillata after 8h. These results are the first to show rapid induction of stress and further possible MC biotransformation (based on the activation of GST enzymatic activity included in MC metabolization during the biotransformation mechanism) in macrophytes exposed to crude extract containing a mixture of MCs. Copyright © 2015 Elsevier B.V. All rights

  2. [Femicides in ethnic and racialized groups: syntheses].

    Science.gov (United States)

    Meneghel, Stela Nazareth; Lerma, Betty Ruth Lozano

    2017-01-01

    The text entitled "Femicides in ethnic and racialized groups: syntheses" presents some of the discussions that took place during a seminar on this topic in Buenaventura. Buenaventura is the main Colombian port on the Pacific, a region rich in minerals and a corridor for the movement of goods, which makes it a strategic territory and a center for disputes. At the seminar, the social and political determinants of femicide were discussed, understanding it as a tactic of waging war against women. The forum provided a space for academic discussion, but also for grievances over inter-personal violence, the manifestation of feelings and the elaboration of pain and grief through the medium of art. We believe that the dissemination of this experience to the Brazilian public, in a country with ethnic, social and racial vulnerability similar to that in Colombia, will be of value to social and health workers. The scope of this paper is therefore to provide the opinion of its authors on the determinants of femicides and on actions to tackle them, in addition to a synthesis of the discussions and debates that permeated the event.

  3. Mechanochemically Driven Syntheses of Boride Nanomaterials

    Science.gov (United States)

    Blair, Richard G.

    Solid state metathesis reactions have proven to be a viable route to the production of unfunctionalized nanomaterials. However, current implementations of this approach are limited to self-propagating reactions. We have been investigating mechanically driven metathesis reactions. The use of high-energy ball mills allows control of crystallite sizes without the use of a capping group. Reinforcement materials with crystallite sizes on the order of 5-30 nm can be produced in such a manner. Borides are of particular interest due to their strength, high melting point, and electrical conductivity. The ultimate goal of this work is to prepare oxide and capping group-free nanoparticles suitable for incorporation in thermoelectric, polymer, and ceramic composites. Ultimately this work will facilitate the production of improved thermoelectric materials that will provide robust, deployable, power generation modules to supplement or replace fuel cell, Stirling, and battery-derived power sources. It will also result in scalable, bulk syntheses of tough, refractory, conductive nanomaterials for polymer composites with improved electrical properties, ceramic composites with enhanced fracture toughness, and composites with enhanced neutron reflectance and/or absorbance.

  4. Planning chemical syntheses with deep neural networks and symbolic AI

    Science.gov (United States)

    Segler, Marwin H. S.; Preuss, Mike; Waller, Mark P.

    2018-03-01

    To plan the syntheses of small organic molecules, chemists use retrosynthesis, a problem-solving technique in which target molecules are recursively transformed into increasingly simpler precursors. Computer-aided retrosynthesis would be a valuable tool but at present it is slow and provides results of unsatisfactory quality. Here we use Monte Carlo tree search and symbolic artificial intelligence (AI) to discover retrosynthetic routes. We combined Monte Carlo tree search with an expansion policy network that guides the search, and a filter network to pre-select the most promising retrosynthetic steps. These deep neural networks were trained on essentially all reactions ever published in organic chemistry. Our system solves for almost twice as many molecules, thirty times faster than the traditional computer-aided search method, which is based on extracted rules and hand-designed heuristics. In a double-blind AB test, chemists on average considered our computer-generated routes to be equivalent to reported literature routes.

  5. Study of photoconductor polymers synthesized by plasma

    International Nuclear Information System (INIS)

    Enriquez P, M.A.

    2007-01-01

    In this work the photoconductivity in poly thiophene (PTh), poly pyrrole (PPy) and doped poly pyrrole with iodine (PPy/I) is studied, whose structures depend of the intensity of the electric field applied during the synthesis by plasma. The conjugated organic polymers possess double alternated bonds in its chemical structure that its allow the one movement of π electrons through the polymeric chains. The plasma is produced by means of splendor discharges to 13.5 MHz, resistive coupling, at one pressure that oscillates in the interval from 2 to 3x10 -1 mbar, 180 min and powers of 10, 24, 40, 60 , 80 and 100 W. Its were used heteroaromatic polymers like PTh and PPy/I, due to their potential applications in optoelectronics. The influence of the iodine is evaluated as dopant in PPy and it is compared with their similar one without doping in the light absorption/emission processes. The polymers synthesized by plasma can ramify or to intersect due to the energy applied during the synthesis. However, if the polymer intersects, the aromaticity can continue through the polymeric chains. The absorptions obtained by infrared spectroscopy, suggest that the polymer conserves the aromatic structure of the monomer fundamentally with substitutions that indicate inter crossing and partial fragmentation. The structure of most of the polymers spreads to be amorphous because they don't possess any classification. However, the PPy/I and PTh synthesized by this technique present crystalline segments whose intensity diminishes with the power of the discharge. In PTh, the average crystallinity diminishes from 19.8% to 9.9%, and in PPy/I of 15.9% to 13.3% in the interval of 10 to 100 W of power. In this work, however, its were crystalline arrangements in all the studied powers. The classification of the polymeric structure favors the formation of trajectories of transfer of electric loads among the chains, that which influences in the global electric conductivity of the material. In UV

  6. Comparison of real-time reverse transcriptase polymerase chain reaction of peripheral blood mononuclear cells, serum and cell-free body cavity effusion for the diagnosis of feline infectious peritonitis.

    Science.gov (United States)

    Doenges, Stephanie J; Weber, Karin; Dorsch, Roswitha; Fux, Robert; Hartmann, Katrin

    2017-04-01

    Objectives Diagnosis of feline infectious peritonitis (FIP) remains challenging, especially in cats without effusions. The objective of this study was to evaluate the sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction (RT-PCR) detecting feline coronavirus (FCoV) RNA in peripheral blood mononuclear cells (PBMCs) and serum in comparison with the same real-time RT-PCR in cell-free body cavity effusion. Methods This prospective case-control study included 92 cats. Forty-three cats had a definitive diagnosis of FIP, established either by histopathological examination (n = 28) or by positive immunofluorescence staining of FCoV antigen in macrophages of effusions (n = 11), or by both methods (n = 4). Forty-nine control cats had other diseases but similar clinical signs. Real-time RT-PCR was performed on PBMCs of 37 cats (21 cats with FIP, 16 controls), on serum of 51 cats (26 cats with FIP, 25 controls) and on cell-free body cavity effusion of 69 cats (36 cats with FIP, 33 controls). Sensitivity, specificity, positive and negative predictive value, including 95% confidence intervals (CI), were calculated. Results Real-time RT-PCR of PBMCs, serum and cell-free body cavity effusion showed a specificity of 100% (95% CI 79.4-100% in PBMCs, 86.3-100% in serum, 89.4-100% in cell-free body cavity effusion) and a sensitivity of 28.6% (95% CI 11.3-52.2%) in PBMCs, 15.4% (95% CI 4.4-34.9%) in serum and 88.9% (95% CI 73.9-96.9%) in cell-free body cavity effusion to diagnose FIP. Conclusions and relevance Although it is known that RT-PCR can often provide false-positive results in healthy cats, this real-time RT-PCR was shown to be a specific tool for the diagnosis of FIP when applied in a clinical setting. Sensitivity in cell-free body cavity effusion was high but low in PBMCs and serum. PBMC samples showed a higher sensitivity than serum samples, and are therefore a better choice if no effusion is present.

  7. An Algorithm Measuring Donor Cell-Free DNA in Plasma of Cellular and Solid Organ Transplant Recipients That Does Not Require Donor or Recipient Genotyping

    Directory of Open Access Journals (Sweden)

    Paul MK Gordon

    2016-09-01

    Full Text Available Cell-free DNA (cfDNA has significant potential in the diagnosis and monitoring of clinical conditions but accurately and easily distinguishing the relative proportion of DNA molecules in a mixture derived from two different sources (i.e. donor and recipient tissues after transplantation is challenging. In human cellular transplantation there is currently no useable method to detect in vivo engraftment and blood-based non-invasive tests for allograft rejection in solid organ transplantation are either non-specific (e.g. creatinine in kidney transplantation, liver enzymes in hepatic transplantation or absent (i.e. heart transplantation. Elevated levels of donor cfDNA have been shown to correlate with solid organ rejection but complex methodology limits implementation of this promising biomarker. We describe a cost-effective method to quantify donor cfDNA in recipient plasma using a panel of high-frequency single nucleotide polymorphisms, next-generation (semiconductor sequencing and a novel mixture model algorithm. In vitro, our method accurately and rapidly determined donor/recipient DNA admixture. For in vivo testing, donor cfDNA was serially quantified in an infant with a urea cycle disorder after receiving six daily infusions of donor liver cells. Donor cfDNA isolated from 1-2 ml of recipient plasma was detected as late as 24 weeks after infusion suggesting engraftment. The percentage of circulating donor cfDNA was also assessed in pediatric and adult heart transplant recipients undergoing routine endomyocardial biopsy with levels observed to be stable over time and generally measuring <1% in cases without moderate or severe cellular rejection. Unlike existing non-invasive methods used to define the proportion of donor cfDNA in solid organ transplant patients, our assay does not require sex mismatch, donor genotyping or whole-genome sequencing and potentially has broad application to detect cellular engraftment or allograft injury after

  8. Cell-Free DNA-Based Non-invasive Prenatal Screening for Common Aneuploidies in a Canadian Province: A Cost-Effectiveness Analysis.

    Science.gov (United States)

    Nshimyumukiza, Léon; Beaumont, Jean-Alexandre; Duplantie, Julie; Langlois, Sylvie; Little, Julian; Audibert, François; McCabe, Christopher; Gekas, Jean; Giguère, Yves; Gagné, Christian; Reinharz, Daniel; Rousseau, François

    2018-01-01

    Yearly, 450 000 pregnant Canadians are eligible for voluntary prenatal screening for trisomy 21. Different screening strategies select approximately 4% of women for invasive fetal chromosome testing. Non-invasive prenatal testing (NIPT) using maternal blood cell-free DNA could reduce those invasive procedures but is expensive. This study evaluated the cost-effectiveness of NIPT strategies compared with conventional strategies. This study used a decision analytic model to estimate the cost-effectiveness of 13 prenatal screening strategies for fetal aneuploidies: six frequently used strategies, universal NIPT, and six strategies incorporating NIPT as a second-tier test. The study considered a virtual cohort of pregnant women of similar size and age as women in Quebec. Model data were obtained from published sources and government databases. The study predicted the number of chromosomal anomalies detected (trisomies 21, 13, and 18), invasive procedures and euploid fetal losses, direct costs, and incremental cost-effectiveness ratios. Of the 13 strategies compared, eight identified fewer cases at a higher cost than at least one of the remaining five strategies. Integrated serum screening with conditional NIPT had the lowest cost, and the cost per case detected was $63 139, with a 90% reduction of invasive procedures. The number of cases identified was improved with four other screening strategies, but with increasing of incremental costs per case (from $61 623 to $1 553 615). Results remained robust, except when NIPT costs and risk cut-offs varied. NIPT as a second-tier test for high-risk women is likely to be cost-effective as compared with screening algorithms not involving NIPT. Copyright © 2018 The Society of Obstetricians and Gynaecologists of Canada/La Société des obstétriciens et gynécologues du Canada. Published by Elsevier Inc. All rights reserved.

  9. Highly sensitive detection of ESR1 mutations in cell-free DNA from patients with metastatic breast cancer using molecular barcode sequencing.

    Science.gov (United States)

    Masunaga, Nanae; Kagara, Naofumi; Motooka, Daisuke; Nakamura, Shota; Miyake, Tomohiro; Tanei, Tomonori; Naoi, Yasuto; Shimoda, Masafumi; Shimazu, Kenzo; Kim, Seung Jin; Noguchi, Shinzaburo

    2018-01-01

    We aimed to develop a highly sensitive method to detect ESR1 mutations in cell-free DNA (cfDNA) using next-generation sequencing with molecular barcode (MB-NGS) targeting the hotspot segment (c.1600-1713). The sensitivity of MB-NGS was tested using serially diluted ESR1 mutant DNA and then cfDNA samples from 34 patients with metastatic breast cancer were analyzed with MB-NGS. The results of MB-NGS were validated in comparison with conventional NGS and droplet digital PCR (ddPCR). MB-NGS showed a higher sensitivity (0.1%) than NGS without barcode (1%) by reducing background errors. Of the cfDNA samples from 34 patients with metastatic breast cancer, NGS without barcode revealed seven mutations in six patients (17.6%) and MB-NGS revealed six additional mutations including three mutations not reported in the COSMIC database of breast cancer, resulting in total 13 ESR1 mutations in ten patients (29.4%). Regarding the three hotspot mutations, all the patients with mutations detected by MB-NGS had identical mutations detected by droplet digital PCR (ddPCR), and mutant allele frequency correlated very well between both (r = 0.850, p < 0.01). Moreover, all the patients without these mutations by MB-NGS were found to have no mutations by ddPCR. In conclusion, MB-NGS could successfully detect ESR1 mutations in cfDNA with a higher sensitivity of 0.1% than conventional NGS and was considered as clinically useful as ddPCR.

  10. Cell-free fetal DNA versus maternal serum screening for trisomy 21 in pregnant women with and without assisted reproduction technology: a prospective interventional study.

    Science.gov (United States)

    Costa, Jean-Marc; Letourneau, Alexandra; Favre, Romain; Bidat, Laurent; Belaisch-Allart, Joelle; Jouannic, Jean-Marie; Quarello, Edwin; Senat, Marie-Victoire; Broussin, Bernard; Tsatsaris, Vassilis; Demain, Adèle; Kleinfinger, Pascale; Lohmann, Laurence; Agostini, Hélène; Bouyer, Jean; Benachi, Alexandra

    2018-03-01

    PurposeCell-free DNA (cfDNA) as a primary screening test has been available for years but few studies have addressed this option in a prospective manner. The question is of interest after reports that maternal serum screening (MSS) is less accurate for pregnancies resulting from assisted reproduction technologies (ART) than for spontaneous pregnancies (SP).MethodsA prospective interventional study was designed to address the performances of cfDNA compared with MSS in pregnancies with or without ART. Each patient was offered both MSS and cfDNA testing. The primary analysis cohort ultimately included 794 patients with a spontaneous pregnancy (SP) (n = 472) or pregnancy obtained after ART (n = 322).ResultsOverall, the false-positive rate and positive predictive value were 6.6% and 8.8% for MSS but 0% and 100% for cfDNA. MSS false-positive rate and positive predictive values were clearly poorer in the ART group (11.7% and 2.6%) than in the SP group (3.2% and 21.1%). The global rates of invasive procedures were 1.9% (15/794) with cfDNA but 8.4% (65/794) if MSS alone was proposed.ConclusioncfDNA achieved better performance than MSS in both spontaneous and ART pregnancies, thus decreasing the number of invasive procedures. Our findings suggest that cfDNA should be considered for primary screening, especially in pregnancies obtained after ART.GENETICS in MEDICINE advance online publication, 1 March 2018; doi:10.1038/gim.2018.4.

  11. The price of performance: a cost and performance analysis of the implementation of cell-free fetal DNA testing for Down syndrome in Ontario, Canada.

    Science.gov (United States)

    Okun, N; Teitelbaum, M; Huang, T; Dewa, C S; Hoch, J S

    2014-04-01

    To examine the cost and performance implications of introducing cell-free fetal DNA (cffDNA) testing within modeled scenarios in a publicly funded Canadian provincial Down syndrome (DS) prenatal screening program. Two clinical algorithms were created: the first to represent the current screening program and the second to represent one that incorporates cffDNA testing. From these algorithms, eight distinct scenarios were modeled to examine: (1) the current program (no cffDNA), (2) the current program with first trimester screening (FTS) as the nuchal translucency-based primary screen (no cffDNA), (3) a program substituting current screening with primary cffDNA, (4) contingent cffDNA with current FTS performance, (5) contingent cffDNA at a fixed price to result in overall cost neutrality,(6) contingent cffDNA with an improved detection rate (DR) of FTS, (7) contingent cffDNA with higher uptake of FTS, and (8) contingent cffDNA with optimized FTS (higher uptake and improved DR). This modeling study demonstrates that introducing contingent cffDNA testing improves performance by increasing the number of cases of DS detected prenatally, and reducing the number of amniocenteses performed and concomitant iatrogenic pregnancy loss of pregnancies not affected by DS. Costs are modestly increased, although the cost per case of DS detected is decreased with contingent cffDNA testing. Contingent models of cffDNA testing can improve overall screening performance while maintaining the provision of an 11- to 13-week scan. Costs are modestly increased, but cost per prenatally detected case of DS is decreased. © 2013 John Wiley & Sons, Ltd.

  12. Prognostic role of APC and RASSF1A promoter methylation status in cell free circulating DNA of operable gastric cancer patients.

    Science.gov (United States)

    Balgkouranidou, I; Matthaios, D; Karayiannakis, A; Bolanaki, H; Michailidis, P; Xenidis, N; Amarantidis, K; Chelis, L; Trypsianis, G; Chatzaki, E; Lianidou, E S; Kakolyris, S

    2015-08-01

    Gastric carcinogenesis is a multistep process including not only genetic mutations but also epigenetic alterations. The best known and more frequent epigenetic alteration is DNA methylation affecting tumor suppressor genes that may be involved in various carcinogenetic pathways. The aim of the present study was to investigate the methylation status of APC promoter 1A and RASSF1A promoter in cell free DNA of operable gastric cancer patients. Using methylation specific PCR, we examined the methylation status of APC promoter 1A and RASSF1A promoter in 73 blood samples obtained from patients with gastric cancer. APC and RASSF1A promoters were found to be methylated in 61 (83.6%) and 50 (68.5%) of the 73 gastric cancer samples examined, but in none of the healthy control samples (p APC promoter and elevated CEA (p = 0.033) as well as CA-19.9 (p = 0.032) levels, was noticed. The Kaplan-Meier estimates of survival, significantly favored patients with a non-methylated APC promoter status (p = 0.008). No other significant correlations between APC and RASSF1A methylation status and different tumor variables examined was observed. Serum RASSF1A and APC promoter hypermethylation is a frequent epigenetic event in patients with early operable gastric cancer. The observed correlations between APC promoter methylation status and survival as well as between a hypermethylated RASSF1A promoter and nodal positivity may be indicative of a prognostic role for those genes in early operable gastric cancer. Additional studies, in a larger cohort of patients are required to further explore whether these findings could serve as potential molecular biomarkers of survival and/or response to specific treatments. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Follicle-stimulating hormone (FSH) unmasks specific high affinity FSH-binding sites in cell-free membrane preparations of porcine granulosa cells

    Energy Technology Data Exchange (ETDEWEB)

    Ford, K.A.; LaBarbera, A.R.

    1988-11-01

    The purpose of these studies was to determine whether changes in FSH receptors correlated with FSH-induced attenuation of FSH-responsive adenylyl cyclase in immature porcine granulosa cells. Cells were incubated with FSH (1-1000 ng/ml) for up to 24 h, treated with acidified medium (pH 3.5) to remove FSH bound to cells, and incubated with (125I)iodo-porcine FSH to quantify FSH-binding sites. FSH increased binding of FSH in a time-, temperature-, and FSH concentration-dependent manner. FSH (200 ng/ml) increased binding approximately 4-fold within 16 h. Analysis of equilibrium saturation binding data indicated that the increase in binding sites reflected a 2.3-fold increase in receptor number and a 5.4-fold increase in apparent affinity. The increase in binding did not appear to be due to 1) a decrease in receptor turnover, since the basal rate of turnover appeared to be very slow; 2) an increase in receptor synthesis, since agents that inhibit protein synthesis and glycosylation did not block the increase in binding; or 3) an increase in intracellular receptors, since agents that inhibit cytoskeletal components had no effect. Agents that increase intracellular cAMP did not affect FSH binding. The increase in binding appeared to result from unmasking of cryptic FSH-binding sites, since FSH increased binding in cell-free membrane preparations to the same extent as in cells. Unmasking of cryptic sites was hormone specific, and the sites bound FSH specifically. Unmasking of sites was reversible in a time- and temperature-dependent manner after removal of bound FSH. The similarity between the FSH dose-response relationships for unmasking of FSH-binding sites and attenuation of FSH-responsive cAMP production suggests that the two processes are functionally linked.

  14. eTumorType, An Algorithm of Discriminating Cancer Types for Circulating Tumor Cells or Cell-free DNAs in Blood.

    Science.gov (United States)

    Zou, Jinfeng; Wang, Edwin

    2017-04-01

    With the technology development on detecting circulating tumor cells (CTCs) and cell-free DNAs (cfDNAs) in blood, serum, and plasma, non-invasive diagnosis of cancer becomes promising. A few studies reported good correlations between signals from tumor tissues and CTCs or cfDNAs, making it possible to detect cancers using CTCs and cfDNAs. However, the detection cannot tell which cancer types the person has. To meet these challenges, we developed an algorithm, eTumorType, to identify cancer types based on copy number variations (CNVs) of the cancer founding clone. eTumorType integrates cancer hallmark concepts and a few computational techniques such as stochastic gradient boosting, voting, centroid, and leading patterns. eTumorType has been trained and validated on a large dataset including 18 common cancer types and 5327 tumor samples. eTumorType produced high accuracies (0.86-0.96) and high recall rates (0.79-0.92) for predicting colon, brain, prostate, and kidney cancers. In addition, relatively high accuracies (0.78-0.92) and recall rates (0.58-0.95) have also been achieved for predicting ovarian, breast luminal, lung, endometrial, stomach, head and neck, leukemia, and skin cancers. These results suggest that eTumorType could be used for non-invasive diagnosis to determine cancer types based on CNVs of CTCs and cfDNAs. Copyright © 2017 Beijing Institute of Genomics, Chinese Academy of Sciences and Genetics Society of China. Production and hosting by Elsevier B.V. All rights reserved.

  15. A "Hybrid" Approach for Synthesizing Optimal Controllers of Hybrid Systems

    DEFF Research Database (Denmark)

    Zhao, Hengjun; Zhan, Naijun; Kapur, Deepak

    2012-01-01

    to discretization manageable and within bounds. A major advantage of our approach is not only that it avoids errors due to numerical computation, but it also gives a better optimal controller. In order to illustrate our approach, we use the real industrial example of an oil pump provided by the German company HYDAC...

  16. Zooplankton as a compound mineralising and synthesizing system: Phosphorus excretion

    NARCIS (Netherlands)

    Gulati, R.D.; Martinez, C.P.; Siewertsen, K.

    1995-01-01

    Data on phosphate excretion rates of zooplankton are based on measurements using the pelagic crustacean zoo-plankton of Lake Vechten and laboratory-cultured Daphnia galeata. In case of Daphnia sp we measured the effects of feeding on P-rich algae and P-poor algae (Scenedesmus) as food on the

  17. Synthesizing Soft Systems Methodology and Human Performance Technology

    Science.gov (United States)

    Scott, Glen; Winiecki, Donald J.

    2012-01-01

    Human performance technology (HPT), like other concepts, models, and frameworks that we use to describe the world in which we live and the way we organize ourselves to accomplish valuable activities, is built from paradigms that were fresh and relevant at the time it was conceived and from the fields of study from which it grew. However, when the…

  18. Syntheses and electronic structures of decamethylmetallocenes

    International Nuclear Information System (INIS)

    Robbins, J.L.

    1981-04-01

    The synthesis of decamethylmanganocene [(eta-C 5 (CH 3 ) 5 ) 2 Mn or (Me 5 Cp) 2 Mn)] is described. Magnetic susceptibility and electron paramagnetic resonance (EPR) studies show that (Me 5 Cp) 2 Mn is a low-spin, 17-electron compound with an orbitally degenerate, 2 E/sub 2g/ [e/sub 2g/ 3 a/sub 1g/ 2 ] ground state. An x-ray crystallographic study of (Me 5 Cp) 2 Mn shows that it is a monomeric, D/sub 5d/ decamethylmetallocene with metal to ring carbon distances that are about 0.3 A shorter than those determined for high-spin manganocenes. The syntheses of new (Me 5 Cp) 2 M (M = Mg,V,Cr,Co, and Ni) and [(Me 5 Cp) 2 M]PF 6 (M = Cr,Co, and Ni) compounds are described. In addition, a preparative route to a novel, dicationic decamethylmetallocene, [(Me 5 Cp) 2 Ni](PF 6 ) 2 is reported. Infrared, nuclear magnetic resonance, magnetic susceptibility, and/or x-ray crystallographic studies indicate that all the above compounds are D/sub 5d/ or D/sub 5h/ decamethylmetallocenes with low-spin electronic configurations. Cyclic voltammetry studies verify the reversibility and the one-electron nature of the (Me 5 Cp) 2 M → [(Me 5 Cp) 2 M] + (M = Cr,Mn,Fe,Co,Ni), [(Me 5 Cp) 2 Mn] - → (Me 5 Cp) 2 Mn and [(Me 5 Cp) 2 Ni] + → [Me 5 Cp) 2 Ni] 2+ redox reactions. These studies reveal that the neutral decamethylmetallocenes are much more easily oxidized than their metallocene counterparts. This result attests to the electron-donating properties of the ten substituent methyl groups. Proton and carbon-13 NMR data are reported for the diamagnetic Mg(II), Mn(I), Fe(II), Co(III), and Ni(IV) decamethylmetallocenes and for [(Me 5 Cp) 2 V(CO) 2 ] + . The uv-visible absorption spectra of the 15-, 18- and 20- electron decamethylmetallocenes are also reported

  19. Modification of Lime Mortars with Synthesized Aluminosilicates

    Science.gov (United States)

    Loganina, Valentina I.; Sadovnikova, Marija E.; Jezierski, Walery; Małaszkiewicz, Dorota

    2017-10-01

    The increasing attention for restoration of buildings of historical and architectural importance has increased the interest for lime-based binders, which could be applied for manufacturing repair mortars and plasters compatible with historical heritage. Different additives, admixtures or fibers may be incorporated to improve mechanical and thermal features of such materials. In this study synthesized aluminosilicates (SA) were applied as an additive for lime mortar. The technology of synthesis consisted in the deposition of aluminosilicates from a sodium liquid glass by the aluminum sulphate Al2(SO4)3. The goal of this investigation was developing a new method of aluminosilicates synthesis from a sodium liquid glass and using this new material as a component for a lime mortar. Aluminosilicates were precipitated from the solution of aluminum sulphate Al2(SO)3 and sodium silicate. SA were then used as an additive to calcareous compositions and their influence was tested. Mortars were prepared with commercial air lime and siliceous river sand. Air lime binder was replaced by 5 and 10 wt.% of SA. Calcareous composition specimens were formed at water/lime ratio 1.0. The following analyses were made: grain size distribution of SA, X-ray diffraction analysis (XRD), sorption properties, plastic strength and compressive strength of lime mortars. XRD pattern of the SA shows the presence of thenardite, gibbsite and amorphous phase represented by aggregate of nano-size cristobalite-like crystallites. Application of SA leads to increase of compressive strength after 90 days of hardening by 28% and 53% at SA content 5 and 10% respectively comparing to specimens without this additive. Contents of chemically bound lime in the reference specimens after 28 days of hardening in air-dry conditions was 46.5%, while in specimens modified with SA contained 50.0-55.3% of bound lime depending on filtrate pH. This testifies to high activity of calcareous composition. The new blended lime

  20. Syntheses and electronic structures of decamethylmetallocenes

    Energy Technology Data Exchange (ETDEWEB)

    Robbins, J.L.

    1981-04-01

    The synthesis of decamethylmanganocene ((eta-C/sub 5/(CH/sub 3/)/sub 5/)/sub 2/Mn or (Me/sub 5/Cp)/sub 2/Mn)) is described. Magnetic susceptibility and electron paramagnetic resonance (EPR) studies show that (Me/sub 5/Cp)/sub 2/Mn is a low-spin, 17-electron compound with an orbitally degenerate, /sup 2/E/sub 2g/ (e/sub 2g//sup 3/ a/sub 1g//sup 2/) ground state. An x-ray crystallographic study of (Me/sub 5/Cp)/sub 2/Mn shows that it is a monomeric, D/sub 5d/ decamethylmetallocene with metal to ring carbon distances that are about 0.3 A shorter than those determined for high-spin manganocenes. The syntheses of new (Me/sub 5/Cp)/sub 2/M (M = Mg,V,Cr,Co, and Ni) and ((Me/sub 5/Cp)/sub 2/M)PF/sub 6/ (M = Cr,Co, and Ni) compounds are described. In addition, a preparative route to a novel, dicationic decamethylmetallocene, ((Me/sub 5/Cp)/sub 2/Ni)(PF/sub 6/)/sub 2/ is reported. Infrared, nuclear magnetic resonance, magnetic susceptibility, and/or x-ray crystallographic studies indicate that all the above compounds are D/sub 5d/ or D/sub 5h/ decamethylmetallocenes with low-spin electronic configurations. Cyclic voltammetry studies verify the reversibility and the one-electron nature of the (Me/sub 5/Cp)/sub 2/M ..-->.. ((Me/sub 5/Cp)/sub 2/M)/sup +/ (M = Cr,Mn,Fe,Co,Ni), ((Me/sub 5/Cp)/sub 2/Mn)/sup -/ ..-->.. (Me/sub 5/Cp)/sub 2/Mn and ((Me/sub 5/Cp)/sub 2/Ni)/sup +/ ..-->.. (Me/sub 5/Cp)/sub 2/Ni)/sup 2 +/ redox reactions. These studies reveal that the neutral decamethylmetallocenes are much more easily oxidized than their metallocene counterparts. This result attests to the electron-donating properties of the ten substituent methyl groups. Proton and carbon-13 NMR data are reported for the diamagnetic Mg(II), Mn(I), Fe(II), Co(III), and Ni(IV) decamethylmetallocenes and for ((Me/sub 5/Cp)/sub 2/V(CO)/sub 2/)/sup +/. The uv-visible absorption spectra of the 15-, 18- and 20- electron decamethylmetallocenes are also reported.

  1. Antibacterial and Cytotoxicity Studies of Silver Nanoparticles Synthesized by Endophytic Fusarium solani Isolated from Withania somnifera (L.

    Directory of Open Access Journals (Sweden)

    Smitha Vijayan

    2016-11-01

    Full Text Available The present study establish extracellular production of silver nanoparticles (AgNP using Fusarium solani, from medicinal plant Withania somnifera (L. (ashwagandha and it’s antibacterial and cytotoxicity effects. Biological- AgNP (Bio- AgNP were synthesized by using fungal cell free extract and characterized by SEM, TEM, UV spectroscopy, XRD, FTIR and AFM analysis.  Antibacterial properties were assayed by well diffusion and cytotoxicity by RBC lysis test and MTT assay respectively. X- ray diffraction and microscopic analysis revealed the well dispersed and crystalline nature of spherical nanoparticles with a calculated size ranging from 10 - 50 nm. The Bio-AgNP exhibited significant antibacterial properties in a range of 50-100 µgml-1 against the selected clinical pathogens Escherichia coli,Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus and Klebsiella pneumoniae. The observed hemolysis of 3.906 % at 50 µg ml-1   suggested the safe therapeutic application of Bio - AgNP. MTT assay revealed that at the suggeseted concentration 69 % of cells are viable. These outcomes are extremely encouraging to utilize Bio-AgNP as a medication. Exploiting the endophytic organisms from therapeutic plants for improvement of nanomaterial is a uninvestigated and relatively novel territory. This may improve the likelihood in future to push the limit ahead in nanomedicine.

  2. Synthesizing and salvaging NAD: lessons learned from Chlamydomonas reinhardtii.

    Directory of Open Access Journals (Sweden)

    Huawen Lin

    2010-09-01

    Full Text Available The essential coenzyme nicotinamide adenine dinucleotide (NAD+ plays important roles in metabolic reactions and cell regulation in all organisms. Bacteria, fungi, plants, and animals use different pathways to synthesize NAD+. Our molecular and genetic data demonstrate that in the unicellular green alga Chlamydomonas NAD+ is synthesized from aspartate (de novo synthesis, as in plants, or nicotinamide, as in mammals (salvage synthesis. The de novo pathway requires five different enzymes: L-aspartate oxidase (ASO, quinolinate synthetase (QS, quinolate phosphoribosyltransferase (QPT, nicotinate/nicotinamide mononucleotide adenylyltransferase (NMNAT, and NAD+ synthetase (NS. Sequence similarity searches, gene isolation and sequencing of mutant loci indicate that mutations in each enzyme result in a nicotinamide-requiring mutant phenotype in the previously isolated nic mutants. We rescued the mutant phenotype by the introduction of BAC DNA (nic2-1 and nic13-1 or plasmids with cloned genes (nic1-1 and nic15-1 into the mutants. NMNAT, which is also in the de novo pathway, and nicotinamide phosphoribosyltransferase (NAMPT constitute the nicotinamide-dependent salvage pathway. A mutation in NAMPT (npt1-1 has no obvious growth defect and is not nicotinamide-dependent. However, double mutant strains with the npt1-1 mutation and any of the nic mutations are inviable. When the de novo pathway is inactive, the salvage pathway is essential to Chlamydomonas for the synthesis of NAD+. A homolog of the human SIRT6-like gene, SRT2, is upregulated in the NS mutant, which shows a longer vegetative life span than wild-type cells. Our results suggest that Chlamydomonas is an excellent model system to study NAD+ metabolism and cell longevity.

  3. Antiaromatic bisindeno-[n]thienoacenes with small singlet biradical characters: Syntheses, structures and chain length dependent physical properties

    KAUST Repository

    Shi, Xueliang; Burrezo, Paula Mayorga; Lee, Sangsu; Zhang, Wenhua; Zheng, Bin; Dai, Gaole; Chang, Jingjing; Lõ pez Navarrete, Juan Teodomiro; Huang, Kuo-Wei; Kim, Dongho; Casado, Juan; Chi, Chunyan

    2014-01-01

    showing promising applications for organic electronics, photonics and spintronics. In this work, we designed and synthesized a new type of hybrid system, the so-called bisindeno-[n]thienoacenes (n = 1-4), by annulation of quinoidal fused α

  4. New aids for the non-invasive prenatal diagnosis of achondroplasia: dysmorphic features, charts of fetal size and molecular confirmation using cell-free fetal DNA in maternal plasma.

    Science.gov (United States)

    Chitty, L S; Griffin, D R; Meaney, C; Barrett, A; Khalil, A; Pajkrt, E; Cole, T J

    2011-03-01

    To improve the prenatal diagnosis of achondroplasia by constructing charts of fetal size, defining frequency of sonographic features and exploring the role of non-invasive molecular diagnosis based on cell-free fetal deoxyribonucleic acid (DNA) in maternal plasma. Data on fetuses with a confirmed diagnosis of achondroplasia were obtained from our databases, records reviewed, sonographic features and measurements determined and charts of fetal size constructed using the LMS (lambda-mu-sigma) method and compared with charts used in normal pregnancies. Cases referred to our regional genetics laboratory for molecular diagnosis using cell-free fetal DNA were identified and results reviewed. Twenty-six cases were scanned in our unit. Fetal size charts showed that femur length was usually on or below the 3(rd) centile by 25 weeks' gestation, and always below the 3(rd) by 30 weeks. Head circumference was above the 50(th) centile, increasing to above the 95(th) when compared with normal for the majority of fetuses. The abdominal circumference was also increased but to a lesser extent. Commonly reported sonographic features were bowing of the femora, frontal bossing, short fingers, a small chest and polyhydramnios. Analysis of cell-free fetal DNA in six pregnancies confirmed the presence of the c.1138G > A mutation in the FGRF3 gene in four cases with achondroplasia, but not the two subsequently found to be growth restricted. These data should improve the accuracy of diagnosis of achondroplasia based on sonographic findings, and have implications for targeted molecular confirmation that can reliably and safely be carried out using cell-free fetal DNA. Copyright © 2011 ISUOG. Published by John Wiley & Sons, Ltd.

  5. Syntheses and absorption-structure relationships of some new ...

    Indian Academy of Sciences (India)

    New biheterocyclic compound was synthesized as starting material to prepare new photosensitizers mono-, tri-, substituted tri-, azadimethine and mixed cyanine dyes. Absorption-structure relationship of the synthesized cyanine dyes were determined by studying their electronic spectral behaviour in ethanol. The structure of ...

  6. Model-based analysis of costs and outcomes of non-invasive prenatal testing for Down's syndrome using cell free fetal DNA in the UK National Health Service.

    Directory of Open Access Journals (Sweden)

    Stephen Morris

    Full Text Available Non-invasive prenatal testing (NIPT for Down's syndrome (DS using cell free fetal DNA in maternal blood has the potential to dramatically alter the way prenatal screening and diagnosis is delivered. Before NIPT can be implemented into routine practice, information is required on its costs and benefits. We investigated the costs and outcomes of NIPT for DS as contingent testing and as first-line testing compared with the current DS screening programme in the UK National Health Service.We used a pre-existing model to evaluate the costs and outcomes associated with NIPT compared with the current DS screening programme. The analysis was based on a hypothetical screening population of 10,000 pregnant women. Model inputs were taken from published sources. The main outcome measures were number of DS cases detected, number of procedure-related miscarriages and total cost.At a screening risk cut-off of 1∶150 NIPT as contingent testing detects slightly fewer DS cases, has fewer procedure-related miscarriages, and costs the same as current DS screening (around UK£280,000 at a cost of £500 per NIPT. As first-line testing NIPT detects more DS cases, has fewer procedure-related miscarriages, and is more expensive than current screening at a cost of £50 per NIPT. When NIPT uptake increases, NIPT detects more DS cases with a small increase in procedure-related miscarriages and costs.NIPT is currently available in the private sector in the UK at a price of £400-£900. If the NHS cost was at the lower end of this range then at a screening risk cut-off of 1∶150 NIPT as contingent testing would be cost neutral or cost saving compared with current DS screening. As first-line testing NIPT is likely to produce more favourable outcomes but at greater cost. Further research is needed to evaluate NIPT under real world conditions.

  7. Cell-free spent media obtained from Bifidobacterium bifidum and Bifidobacterium crudilactis grown in media supplemented with 3’-sialyllactose exert virulence modulation on intestinal pathogens

    Directory of Open Access Journals (Sweden)

    Bondue Pauline

    2016-09-01

    Full Text Available Complex oligosaccharides from human milk (HMO possess an antimicrobial activity and can promote the growth of bifidobacteria such as Bifidobacterium bifidum and Bifidobacterium longum subsp. infantis. In addition, fermentation of carbohydrates by bifidobacteria can result in the production of metabolites presenting an antivirulence effect on several pathogenic bacteria. Whey is rich in complex bovine milk oligosaccharides (BMO structurally similar to HMO and B. crudilactis, a species of bovine origin, is able to metabolize some of those complex carbohydrates. This study focused on the ability of B. bifidum and B. crudilactis to grow in a culture medium supplemented in 3’-sialyllactose (3’SL as sole source of carbon, a main BMO encountered in cow milk. Next, the effects of cell-free spent media (CFSM were tested against virulence expression of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium. Both strains were able to grow in presence of 3’SL, but B. crudilactis showed the best growth (7.92 ± 0.3 log cfu/ml compared to B. bifidum (6.84 ± 0.9 log cfu/ml. Then, CFSM were tested for their effects on virulence gene expression by ler and hilA promoter activity of luminescent mutants of E. coli and S. Typhimurium, respectively, and on wild type strains of E. coli O157:H7 and S. Typhimurium using RT-qPCR. All CFSM resulted in significant under expression of the ler and hilA genes for the luminescent mutants and ler (ratios of -15.4 and -8.1 respectively and qseA (ratios of -2.1 and -3.1 for the wild type strain of E. coli O157:H7. The 3’SL, a major BMO, combined with some bifidobacteria strains of bovine or human origin could therefore be an interesting synbiotic to maintain or restore the intestinal health of young children. These effects observed in vitro will be further investigated regarding the exact nature of the active molecules.

  8. Implications Enzymatic Degradation of the Endothelial Glycocalyx on the Microvascular Hemodynamics and the Arteriolar Red Cell Free Layer of the Rat Cremaster Muscle

    Directory of Open Access Journals (Sweden)

    Ozlem Yalcin

    2018-03-01

    Full Text Available The endothelial glycocalyx is a complex network of glycoproteins, proteoglycans, and glycosaminoglycans; it lines the vascular endothelial cells facing the lumen of blood vessels forming the endothelial glycocalyx layer (EGL. This study aims to investigate the microvascular hemodynamics implications of the EGL by quantifying changes in blood flow hydrodynamics post-enzymatic degradation of the glycocalyx layer. High-speed intravital microscopy videos of small arteries (around 35 μm of the rat cremaster muscle were recorded at various time points after enzymatic degradation of the EGL. The thickness of the cell free layer (CFL, blood flow velocity profiles, and volumetric flow rates were quantified. Hydrodynamic effects of the presence of the EGL were observed in the differences between the thickness of CFL in microvessels with an intact EGL and glass tubes of similar diameters. Maximal changes in the thickness of CFL were observed 40 min post-enzymatic degradation of the EGL. Analysis of the frequency distribution of the thickness of CFL allows for estimation of the thickness of the endothelial surface layer (ESL, the plasma layer, and the glycocalyx. Peak flow, maximum velocity, and mean velocity were found to statistically increase by 24, 27, and 25%, respectively, after enzymatic degradation of the glycocalyx. The change in peak-to-peak maximum velocity and mean velocity were found to statistically increase by 39 and 32%, respectively, after 40 min post-enzymatic degradation of the EGL. The bluntness of blood flow velocity profiles was found to be reduced post-degradation of the EGL, as the exclusion volume occupied by the EGL increased the effective volume impermeable to RBCs in microvessels. This study presents the effects of the EGL on microvascular hemodynamics. Enzymatic degradation of the EGL resulted in a decrease in the thickness of CFL, an increase in blood velocity, blood flow, and decrease of the bluntness of the blood flow

  9. The value of fluorimetry (Qubit) and spectrophotometry (NanoDrop) in the quantification of cell-free DNA (cfDNA) in malignant melanoma and prostate cancer patients.

    Science.gov (United States)

    Ponti, Giovanni; Maccaferri, Monia; Manfredini, Marco; Kaleci, Shaniko; Mandrioli, Mauro; Pellacani, Giovanni; Ozben, Tomris; Depenni, Roberta; Bianchi, Giampaolo; Pirola, Giacomo Maria; Tomasi, Aldo

    2018-04-01

    Circulating cell-free tumor DNA (cfDNA) is of crucial interest in oncology. cfDNA constitutes a potential prognostic and therapeutic marker for different solid tumors and can be used in the diagnostic and therapeutic management of cancer patients for which nowadays there are no valid laboratory markers. In the present study, the quality and quantity of the cfDNA were assessed by different quantification procedures, in order to identify the potential applications of these techniques in the preliminary cfDNA quantification. Qubit with single (ss) and double strand (ds) DNA assay kits, NanoDrop and quantitative Real Time PCR (qPCR), were adopted to assess the cfDNA in the blood samples of 18 melanoma patients, 67 prostate cancer patients and 15 healthy controls. The quantification by NanoDrop (average value 8.48ng/μl, 95% confidence limit (CL)=7.23-9.73), Qubit ssDNA (average value 23.08ng/μl, CL=19.88-26.28), dsDNA (average value 4.32ng/μl, CL=3.52-5.12) assay kits and qPCR (average value 0.39ng/μl, CL=0.31-0.47) revealed differences among the four procedures. Qubit 2.0 ss-DNA kit gave higher cfDNA concentration values for all the samples analyzed. In detail, Qubit ssDNA assay revealed higher sensitivity in the quantification of small amounts of pure ss-DNA and ds-DNA, while NanoDrop allowed the assessment of the purity of cfDNA samples. The NanoDrop and Qubit 2.0 measurements were analyzed in order to define their correlation with qPCR cfDNA assessment, showing good correlation values with the qPCR that should be considered the "gold standard". In our proposal, the sequential combination of NanoDrop and Qubit ssDNA methods should be adopted for a cost-effective preliminary assessment of total circulating cfDNA in melanoma and prostate cancer patients, and only discordant values should undergo qPCR assessment. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. Implications Enzymatic Degradation of the Endothelial Glycocalyx on the Microvascular Hemodynamics and the Arteriolar Red Cell Free Layer of the Rat Cremaster Muscle.

    Science.gov (United States)

    Yalcin, Ozlem; Jani, Vivek P; Johnson, Paul C; Cabrales, Pedro

    2018-01-01

    The endothelial glycocalyx is a complex network of glycoproteins, proteoglycans, and glycosaminoglycans; it lines the vascular endothelial cells facing the lumen of blood vessels forming the endothelial glycocalyx layer (EGL). This study aims to investigate the microvascular hemodynamics implications of the EGL by quantifying changes in blood flow hydrodynamics post-enzymatic degradation of the glycocalyx layer. High-speed intravital microscopy videos of small arteries (around 35 μm) of the rat cremaster muscle were recorded at various time points after enzymatic degradation of the EGL. The thickness of the cell free layer (CFL), blood flow velocity profiles, and volumetric flow rates were quantified. Hydrodynamic effects of the presence of the EGL were observed in the differences between the thickness of CFL in microvessels with an intact EGL and glass tubes of similar diameters. Maximal changes in the thickness of CFL were observed 40 min post-enzymatic degradation of the EGL. Analysis of the frequency distribution of the thickness of CFL allows for estimation of the thickness of the endothelial surface layer (ESL), the plasma layer, and the glycocalyx. Peak flow, maximum velocity, and mean velocity were found to statistically increase by 24, 27, and 25%, respectively, after enzymatic degradation of the glycocalyx. The change in peak-to-peak maximum velocity and mean velocity were found to statistically increase by 39 and 32%, respectively, after 40 min post-enzymatic degradation of the EGL. The bluntness of blood flow velocity profiles was found to be reduced post-degradation of the EGL, as the exclusion volume occupied by the EGL increased the effective volume impermeable to RBCs in microvessels. This study presents the effects of the EGL on microvascular hemodynamics. Enzymatic degradation of the EGL resulted in a decrease in the thickness of CFL, an increase in blood velocity, blood flow, and decrease of the bluntness of the blood flow velocity profile in

  11. Carbon-14 labelled nitrogen heterocycles; the syntheses of three phosphodiesterase inhibitors

    International Nuclear Information System (INIS)

    Lawrie, K.W.M.; Novelli, C.E.A.; Saunders, David

    1995-01-01

    The syntheses of three heterocyclic phosphodiesterase inhibitors are described from a common radiolabelled precursor, namely 2-propoxybenzo[cyano- 14 C] nitrile. Conversion of the nitrile to the corresponding methyl ketone or amidine allows elaboration of the heterocycles radiolabelled within the ring systems. (Author)

  12. Carbon-14 labelled nitrogen heterocycles; the syntheses of three phosphodiesterase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Lawrie, K.W.M.; Novelli, C.E.A.; Saunders, David [SmithKline Beecham Pharmaceuticals Research and Development, Harlow (United Kingdom). Synthetic Isotope Chemistry Dept.; Coates, W.J. [SmithKline Beecham Pharmaceuticals Research and Development, Welwyn (United Kingdom)

    1995-09-01

    The syntheses of three heterocyclic phosphodiesterase inhibitors are described from a common radiolabelled precursor, namely 2-propoxybenzo[cyano-{sup 14}C] nitrile. Conversion of the nitrile to the corresponding methyl ketone or amidine allows elaboration of the heterocycles radiolabelled within the ring systems. (Author).

  13. Organic thin film transistors with polymer brush gate dielectrics synthesized by atom transfer radical polymerization

    DEFF Research Database (Denmark)

    Pinto, J.C.; Whiting, G.L.; Khodabakhsh, S.

    2008-01-01

    , synthesized by atom transfer radical polymerization (ATRP), were used to fabricate low voltage OFETs with both evaporated pentacene and solution deposited poly(3-hexylthiophene). The semiconductor-dielectric interfaces in these systems were studied with a variety of methods including scanning force microscopy...

  14. A fractional-N frequency synthesizer for WCDMA/Bluetooth/ZigBee applications

    Science.gov (United States)

    Chunyuan, Zhou; Guolin, Li; Chun, Zhang; Baoyong, Chi; Dongmei, Li; Zhihua, Wang

    2009-07-01

    A triple-mode fractional-N frequency synthesizer with a noise-filter voltage controlled oscillator (VCO) for WCDMA/Bluetooth/ZigBee applications has been implemented in 0.18-μm RF-CMOS technology. The proposed synthesizer achieves a good phase noise lower than -80 dBc/Hz in band and -115 dBc/Hz@1 MHz for the three modes, and only draws 21 mA from a 1.8 V supply. It has a high hardware sharing and a small size, only 1.5 × 1.4 mm2. The system architecture, circuit design, and measured results are also presented.

  15. A fractional-N frequency synthesizer for wireless sensor network nodes

    International Nuclear Information System (INIS)

    Ma Xiao; Du Zhankun; Liu Chang; Liu Ke; Yan Yuepeng; Ye Tianchun

    2014-01-01

    This paper presents a fractional-N frequency synthesizer for wireless sensor network (WSN) nodes. The proposed frequency synthesizer adopts a phase locked loop (PLL) based structure, which employs an LC voltage-controlled oscillator (VCO) with small VCO gain (K VCO ) and frequency step (f step ) variations, a charge pump (CP) with current changing in proportion with the division ratio and a 20-bit ΔΣ modulator, etc. To realize constant K VCO and f step , a novel capacitor sub-bands grouping method is proposed. The VCO sub-groups' sizes are arranged according to the maximal allowed K VCO variation of the system. Besides, a current mode logic divide-by-2 circuit with inside-loop buffers ensures the synthesizer generates I/Q quadrature signals robustly. This synthesizer is implemented in a 0.13 μm CMOS process. Measurement results show that the frequency synthesizer has a frequency span from 2.07 to 3.11 GHz and the typical phase noise is −86.34 dBc/Hz at 100 kHz offset and −114.17 dBc/Hz at 1 MHz offset with a loop bandwidth of about 200 kHz, which meet the WSN nodes' requirements. (semiconductor integrated circuits)

  16. Bias Voltage-Dependent Impedance Spectroscopy Analysis of Hydrothermally Synthesized ZnS Nanoparticles

    Science.gov (United States)

    Dey, Arka; Dhar, Joydeep; Sil, Sayantan; Jana, Rajkumar; Ray, Partha Pratim

    2018-04-01

    In this report, bias voltage-dependent dielectric and electron transport properties of ZnS nanoparticles were discussed. ZnS nanoparticles were synthesized by introducing a modified hydrothermal process. The powder XRD pattern indicates the phase purity, and field emission scanning electron microscope image demonstrates the morphology of the synthesized sample. The optical band gap energy (E g = 4.2 eV) from UV measurement explores semiconductor behavior of the synthesized material. The electrical properties were performed at room temperature using complex impedance spectroscopy (CIS) technique as a function of frequency (40 Hz-10 MHz) under different forward dc bias voltages (0-1 V). The CIS analysis demonstrates the contribution of bulk resistance in conduction mechanism and its dependency on forward dc bias voltages. The imaginary part of the impedance versus frequency curve exhibits the existence of relaxation peak which shifts with increasing dc forward bias voltages. The dc bias voltage-dependent ac and dc conductivity of the synthesized ZnS was studied on thin film structure. A possible hopping mechanism for electrical transport processes in the system was investigated. Finally, it is worth to mention that this analysis of bias voltage-dependent dielectric and transport properties of as-synthesized ZnS showed excellent properties for emerging energy applications.

  17. A study on the effect of chemically synthesized magnetite nanoparticles on earthworm: Eudrilus eugeniae

    Science.gov (United States)

    Samrot, Antony V.; Justin, C.; Padmanaban, S.; Burman, Ujjala

    2017-02-01

    Most look into the benefits of the nanoparticles, but keeping aside the benefits; this study focuses on the impacts of nanoparticles on living systems. Improper disposal of nanoparticles into the environment is a subject of pollution or nano-pollution which in turn affects the flora and fauna in the ecosystem, particularly soil ecosystem. Thus, this study was done to understand the impacts of chemically synthesized magnetite nanoparticles on earthworm— Eudrilus eugeniae, a soil-dependent organism which acquires food and nutrition from decaying matters. The chemically synthesized magnetite nanoparticles were characterized by UV-visible spectrophotometry, Fourier transform infrared spectroscopy and field emission scanning electron microscopy. Earthworms were allowed to interact with different concentrations of synthesized nanoparticles and the effect of the nanoparticles was analysed by studying the phenotypic changes followed by histology and inductively coupled plasma optical emission spectrometry analyses.

  18. Synthesized view comparison method for no-reference 3D image quality assessment

    Science.gov (United States)

    Luo, Fangzhou; Lin, Chaoyi; Gu, Xiaodong; Ma, Xiaojun

    2018-04-01

    We develop a no-reference image quality assessment metric to evaluate the quality of synthesized view rendered from the Multi-view Video plus Depth (MVD) format. Our metric is named Synthesized View Comparison (SVC), which is designed for real-time quality monitoring at the receiver side in a 3D-TV system. The metric utilizes the virtual views in the middle which are warped from left and right views by Depth-image-based rendering algorithm (DIBR), and compares the difference between the virtual views rendered from different cameras by Structural SIMilarity (SSIM), a popular 2D full-reference image quality assessment metric. The experimental results indicate that our no-reference quality assessment metric for the synthesized images has competitive prediction performance compared with some classic full-reference image quality assessment metrics.

  19. Implementation of Different Variants of Table-Based Frequency Synthesizers with Quadrature Output in VHDL

    Directory of Open Access Journals (Sweden)

    Daniel Kekrt

    2012-01-01

    Full Text Available This article describes the modelling and implementation of two different variants of direct frequency synthesizer, and evaluation of the performance of the finished design, in terms of memory and speed efficiency. The frequency synthesizer requirement comes from our complex radio transmission system design. The research activity has been focused on finding an optimal balance between simplicity, speed and memory consumption. The modelling was done in MATLAB environment in floating-point and fixed-point arithmetic, and the actual design was implemented and synthesized using the Xilinx ISE suite. The output has been connected to our customized radio front-end built on the Texas Instruments TRF2443 chip. The front-end output signal has been captured and compared with simulation results.

  20. Assessment voice synthesizers for reading in digital books

    Directory of Open Access Journals (Sweden)

    Sérvulo Fernandes da Silva Neto

    2013-07-01

    Full Text Available The digital accessibility shows ways to information access in digital media that assist people with different types of disabilities to a better interaction with the computer independent of its limitations. Of these tools are composed by voice synthesizers, that supposedly simplifying their access to any recorded knowledge through digital technologies. However such tools have emerged originally in countries foreign language. Which brings us to the following research problem: the voice synthesizers are appropriate for reading digital books in the Portuguese language? The objective of this study was to analyze and classify different software tools voice synthesizers in combination with software digital book readers to support accessibility to e-books in Portuguese. Through literature review were identified applications software voice synthesizers, composing the sample analyzed in this work. We used a simplified version of the method of Multiple Criteria Decision Support - MMDA, to assess these. In the research 12 were considered readers of e-books and 11 software voice synthesizer, tested with six formats of e-books (E-pub, PDF, HTML, DOC, TXT, and Mobi. In accordance with the results, the software Virtual Vision achieved the highest score. Relative to formats, it was found that the PDF has measured a better score when summed the results of the three synthesizers. In the studied universe contacted that many synthesizers simply cannot be used because they did not support the Portuguese language.

  1. Analysis on nonlinear optical properties of Cd (Zn) Se quantum dots synthesized using three different stabilizing agents

    Science.gov (United States)

    J, Joy Sebastian Prakash; G, Vinitha; Ramachandran, Murugesan; Rajamanickam, Karunanithi

    2017-10-01

    Three different stabilizing agents, namely, L-cysteine, Thioglycolic acid and cysteamine hydrochloride were used to synthesize Cd(Zn)Se quantum dots (QDs). It was characterized using UV-vis spectroscopy, x-ray diffraction (XRD) and transmission electron microscopy (TEM). The non-linear optical properties (non-linear absorption and non-linear refraction) of synthesized Cd(Zn)Se quantum dots were studied with z-scan technique using diode pumped continuous wavelaser system at a wavelength of 532 nm. Our (organic) synthesized quantum dots showed optical properties similar to the inorganic materials reported elsewhere.

  2. Detection of a high-molecular-weight LHRH precursor by cell-free translation of mRNA from human, rat, and mouse hypothalamus

    International Nuclear Information System (INIS)

    Curtis, A.; Szelke, M.; Fink, G.

    1986-01-01

    Large precursors have also been predicted using the immunoprecipitation technique which relies upon the identification of large immunoreactive molecules following in vitro translation of mRNA. The mRNA is presumed to represent the largest form of a nascent precursor polypeptide molecule irrespective of the number of biosynthetic cleavage steps which are necessary to liberate the active peptide. However, as has been shown for somatostatin, nonprotein modifications may be made which apparently increase molecular weight, such as glycosylation or phosphorylation of the molecule. The authors employed the immunoprecipitation technique to confirm earlier chromatographic studies that the hypothalamic decapeptide, luteinizing hormone releasing hormone (LHRH) is also synthesized by way of a large precursor form. The authors' finding show that the translation of hypothalamic mRNA produces a primary translation product with an apparent molecule weight of 28,000 which contains an amino acid sequence immunologically similar to that of biologically active LHRH. The procedure involved the incorporation of a radioactive amino acid into polypeptides synthesized by in vitro translation of hypothalamic messenger RNA. The resulting complex protein mixture was immunoprecipitated with a specific anti-LHRH serum, and the immunoprecipitate was identified by polyacrylamide gel electrophoresis and autoradiography

  3. Synthesizing and characterization of bilayer ferro fluid

    International Nuclear Information System (INIS)

    Akhavan, M.; Ghominezhad, M.

    1997-01-01

    Adsorption role of the sodium dodecyl sulfate (SDS) as the second layer surfactant in a double layer surfactant ferro fluid is investigated. Preparation of the solid phase is based on the growth of the magnetic oxide by dehydration of the salt solution of FeCl sub 2 and FeCl sub 3 in an identical molar ratio. The particle size was determined through the magnetic measurements by VSM to be about 8-10 nm. The data show that in a certain thermal interval, a local maxima appears in the magnetic oxide concentration which is a function of the stirring time and the SDS concentration. Increasing temperature, causes surface oxidation which decreases the magnetization, similar to what happens in the monolayer ferro fluids. In addition, in the double layer systems, the solubility of the layers into each other varies. This has a more profound effect on the saturation magnetization. (author)

  4. Impact of 50% Synthesized Iso-Paraffins (SIP) on Middle Distillate Fuel Filtration and Coalescence

    Science.gov (United States)

    2014-10-30

    Paraffins DEFINITIONS Coalescence - the ability to shed water Conventional Material Source - crude oil , natural gas liquid condensates...Impact of 50% Synthesized Iso-Paraffins (SIP) on Middle Distillate Fuel Filtration and Coalescence NF&LCFT REPORT 441/15-003 30 October 2014...heavy oil , shale oil , and oil sands Effluent - stream leaving a system Influent - stream entering a system Turnover - time required to flow the

  5. Two-Volt Josephson Arbitrary Waveform Synthesizer Using Wilkinson Dividers

    Science.gov (United States)

    Flowers-Jacobs, Nathan E.; Fox, Anna E.; Dresselhaus, Paul D.; Schwall, Robert E.; Benz, Samuel P.

    2016-01-01

    The root-mean-square (rms) output voltage of the NIST Josephson arbitrary waveform synthesizer (JAWS) has been doubled from 1 V to a record 2 V by combining two new 1 V chips on a cryocooler. This higher voltage will improve calibrations of ac thermal voltage converters and precision voltage measurements that require state-of-the-art quantum accuracy, stability, and signal-to-noise ratio. We achieved this increase in output voltage by using four on-chip Wilkinson dividers and eight inner-outer dc blocks, which enable biasing of eight Josephson junction (JJ) arrays with high-speed inputs from only four high-speed pulse generator channels. This approach halves the number of pulse generator channels required in future JAWS systems. We also implemented on-chip superconducting interconnects between JJ arrays, which reduces systematic errors and enables a new modular chip package. Finally, we demonstrate a new technique for measuring and visualizing the operating current range that reduces the measurement time by almost two orders of magnitude and reveals the relationship between distortion in the output spectrum and output pulse sequence errors. PMID:27453676

  6. Understanding betrayals in marriage: a synthesized model of forgiveness.

    Science.gov (United States)

    Gordon, K C; Baucom, D H

    1998-01-01

    Forgiveness is an issue that is problematic for many couples, particularly those in marital therapy. However, little attention has been paid to this construct in the psychological literature. The purpose of this article is to describe a synthesized model of forgiveness using constructs from multiple theories, including forgiveness, trauma recovery, cognitive-behavioral, family systems, and insight-oriented theories. Forgiveness is conceptualized as a process consisting of three stages, each of which has cognitive, behavioral, and affective components. Furthermore, these stages seem to parallel a person's natural response to traumatic stress. First, there is a response to the initial impact; second, there is an attempt to give the event some kind of meaning, or put it into context; and finally, the person begins to move forward and readjust. Forgiveness is conceptualized as attaining: (a) a realistic, nondistorted, balanced view of the relationship; (b) a release from being controlled by negative affect toward the participating partner; and (c) a lessened desire to punish the participating partner. Implications for marital therapy also are discussed.

  7. Study of as-synthesized and calcined hydrocalumites as possible ...

    Indian Academy of Sciences (India)

    Administrator

    ments. Finally, these solids were tested as antacids by using a synthetic gastric juice. Results showed that calcined samples were able to neutralize the synthetic gastric juice in more extension as an as-synthesized ..... D 2010 Appl. Clay Sci.

  8. Syntheses, Protonation Constants and Antimicrobial Activity of 2 ...

    African Journals Online (AJOL)

    carboxaldehyde and N-alkylimidazole-2-methanol derivatives [alkyl = benzyl, methyl, ethyl, propyl, butyl, heptyl, octyl and decyl] have been synthesized and the protonation constants determined. The antimicrobial properties of the compounds were tested ...

  9. Characterization of combustion synthesized zirconia powder by UV

    Indian Academy of Sciences (India)

    . The surface acidbase properties of these samples were also investigated by indicator titration method. The catalytic activity was probed with transfer hydrogenation reaction in liquid phase. It was found that combustion synthesized zirconia did ...

  10. Bilateral Manipulandum to Synthesize Ground Referenced and Interlimb Viscoelastic Loads

    National Research Council Canada - National Science Library

    Gallasch, E

    2001-01-01

    .... The mechatronics consists of two angular voice coil actuators (+/- 40 Nm) with embedded rotary (+/- 20 degrees) and torque sensors driven by voltage controlled current sources, DSP software routines to synthesize isotonic...

  11. Exploitation of rare earth catalysts in polymer syntheses

    Institute of Scientific and Technical Information of China (English)

    Shen Zhiquan

    2006-01-01

    The studies over forty years on rare earth catalysts in polymer syntheses of diene,alkyne,alkylene oxide,thiirane, carbon dioxide copolymerization, lactide,caprolactone,cyclic carbonate and so forth in China have been reviewed.

  12. Multiblock copolymers synthesized in aqueous dispersions using multifunctional RAFT agents

    NARCIS (Netherlands)

    Bussels, R.; Bergman-Göttgens, C.M.; Meuldijk, J.; Koning, C.E.

    2005-01-01

    Triblock copolymers were synthesized in aqueous dispersions in two polymerization steps using a low molar mass difunctional dithiocarbamate-based RAFT agent, and in merely one polymerization step using a macromolecular difunctional dithiocarbamate-based RAFT agent. Segmented block copolymers

  13. Sol–gel synthesized mesoporous anatase titanium dioxide ...

    Indian Academy of Sciences (India)

    for dye sensitized solar cell (DSSC) applications. R GOVINDARAJ1,∗, M ... DSSC than rutile phase. In this work, we have synthesized hierarchically structured ... Hydrolysis and polycondensation reaction mechanism of sol–gel process. 2.

  14. GaN Nanowires Synthesized by Electroless Etching Method

    KAUST Repository

    Najar, Adel; Anjum, Dalaver H.; Ng, Tien Khee; Ooi, Boon S.; Ben Slimane, Ahmed

    2012-01-01

    Ultra-long Gallium Nitride Nanowires is synthesized via metal-electroless etching method. The morphologies and optical properties of GaN NWs show a single crystal GaN with hexagonal Wurtzite structure and high luminescence properties.

  15. Entwicklung eines kontinuierlichen Verfahrens zur enzymkatalysierten Synthese eines strukturierten Triglycerides

    OpenAIRE

    Stadler, Hans-Gerhard

    2005-01-01

    Ausgangspunkt für die in der vorliegenden Arbeit durchgeführten Entwicklung eines kontinuierlichen Verfahrens zur Synthese des strukturierten Triglycerides 1,3-Oleyl-2-palmitoylglycerin war die von Schmid [Schmid 1999] entwickelte lipase-katalysierte Zwei-Schritt-Synthese für strukturierte Triglyceride vom Typ ABA [European Patent Application; EP 0 882 797 A2]. In der ersten Reaktionsstufe dieser zweistufigen Umesterung wird in einer Folgereaktion das Substrat Tripalmitin mit Hilfe ein...

  16. Proteins synthesized in tobacco mosaic virus infected protoplasts

    International Nuclear Information System (INIS)

    Huber, R.

    1979-01-01

    The author deals with research on the multiplication of tobacco mosaic virus (TMV) in leaf cell protoplasts. An attempt is made to answer three questions: (1) Which proteins are synthesized in TMV infected protoplasts as a result of TMV multiplication. (2) Which of the synthesized proteins are made under the direction of the TMV genome and, if any, which of the proteins are host specific. (3) In which functions are these proteins involved. (Auth.)

  17. Design of a synthesizer for magnetic resonance equipment using FPGA

    International Nuclear Information System (INIS)

    Sonora A

    2006-01-01

    This paper exposes the design of a direct digital synthesizer in FPGA. This desing can generate a sine wave output up to 4MHZ with 3,33 mHz of precision. The frequency is set by 32bit word of phase increment in 350ns. The desing was made for Magnetic Resonance scanners and uses a 97% of logic resources of device. Functions for the synthesizer control are implemented in the same chip

  18. SYNTHESIZER CONTROLLED BEAM TRANSFER FROM THE AGS TO RHIC

    Int