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Sample records for cell-free metabolic platform

  1. Controlling cell-free metabolism through physiochemical perturbations.

    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

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

    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

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

    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.

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

    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.

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

    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.

  6. MicrobesFlux: a web platform for drafting metabolic models from the KEGG database

    Feng Xueyang

    2012-08-01

    Full Text Available Abstract Background Concurrent with the efforts currently underway in mapping microbial genomes using high-throughput sequencing methods, systems biologists are building metabolic models to characterize and predict cell metabolisms. One of the key steps in building a metabolic model is using multiple databases to collect and assemble essential information about genome-annotations and the architecture of the metabolic network for a specific organism. To speed up metabolic model development for a large number of microorganisms, we need a user-friendly platform to construct metabolic networks and to perform constraint-based flux balance analysis based on genome databases and experimental results. Results We have developed a semi-automatic, web-based platform (MicrobesFlux for generating and reconstructing metabolic models for annotated microorganisms. MicrobesFlux is able to automatically download the metabolic network (including enzymatic reactions and metabolites of ~1,200 species from the KEGG database (Kyoto Encyclopedia of Genes and Genomes and then convert it to a metabolic model draft. The platform also provides diverse customized tools, such as gene knockouts and the introduction of heterologous pathways, for users to reconstruct the model network. The reconstructed metabolic network can be formulated to a constraint-based flux model to predict and analyze the carbon fluxes in microbial metabolisms. The simulation results can be exported in the SBML format (The Systems Biology Markup Language. Furthermore, we also demonstrated the platform functionalities by developing an FBA model (including 229 reactions for a recent annotated bioethanol producer, Thermoanaerobacter sp. strain X514, to predict its biomass growth and ethanol production. Conclusion MicrobesFlux is an installation-free and open-source platform that enables biologists without prior programming knowledge to develop metabolic models for annotated microorganisms in the KEGG

  7. Evolutionary programming as a platform for in silico metabolic engineering

    Patil, Kiran Raosaheb; Rocha, Isabel; Förster, Jochen

    2005-01-01

    , and it is therefore interesting to develop new faster algorithms. Results In this study we report an evolutionary programming based method to rapidly identify gene deletion strategies for optimization of a desired phenotypic objective function. We illustrate the proposed method for two important design parameters...... of close to optimal solutions. The identified metabolic engineering strategies suggest that non-intuitive genetic modifications span several different pathways and may be necessary for solving challenging metabolic engineering problems....

  8. Evolutionary programming as a platform for in silico metabolic engineering

    Förster Jochen

    2005-12-01

    Full Text Available Abstract Background Through genetic engineering it is possible to introduce targeted genetic changes and hereby engineer the metabolism of microbial cells with the objective to obtain desirable phenotypes. However, owing to the complexity of metabolic networks, both in terms of structure and regulation, it is often difficult to predict the effects of genetic modifications on the resulting phenotype. Recently genome-scale metabolic models have been compiled for several different microorganisms where structural and stoichiometric complexity is inherently accounted for. New algorithms are being developed by using genome-scale metabolic models that enable identification of gene knockout strategies for obtaining improved phenotypes. However, the problem of finding optimal gene deletion strategy is combinatorial and consequently the computational time increases exponentially with the size of the problem, and it is therefore interesting to develop new faster algorithms. Results In this study we report an evolutionary programming based method to rapidly identify gene deletion strategies for optimization of a desired phenotypic objective function. We illustrate the proposed method for two important design parameters in industrial fermentations, one linear and other non-linear, by using a genome-scale model of the yeast Saccharomyces cerevisiae. Potential metabolic engineering targets for improved production of succinic acid, glycerol and vanillin are identified and underlying flux changes for the predicted mutants are discussed. Conclusion We show that evolutionary programming enables solving large gene knockout problems in relatively short computational time. The proposed algorithm also allows the optimization of non-linear objective functions or incorporation of non-linear constraints and additionally provides a family of close to optimal solutions. The identified metabolic engineering strategies suggest that non-intuitive genetic modifications span

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

    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. Genome-scale metabolic models as platforms for strain design and biological discovery.

    Mienda, Bashir Sajo

    2017-07-01

    Genome-scale metabolic models (GEMs) have been developed and used in guiding systems' metabolic engineering strategies for strain design and development. This strategy has been used in fermentative production of bio-based industrial chemicals and fuels from alternative carbon sources. However, computer-aided hypotheses building using established algorithms and software platforms for biological discovery can be integrated into the pipeline for strain design strategy to create superior strains of microorganisms for targeted biosynthetic goals. Here, I described an integrated workflow strategy using GEMs for strain design and biological discovery. Specific case studies of strain design and biological discovery using Escherichia coli genome-scale model are presented and discussed. The integrated workflow presented herein, when applied carefully would help guide future design strategies for high-performance microbial strains that have existing and forthcoming genome-scale metabolic models.

  11. Systems Biology as an Integrated Platform for Bioinformatics, Systems Synthetic Biology, and Systems Metabolic Engineering

    Bor-Sen Chen

    2013-10-01

    Full Text Available Systems biology aims at achieving a system-level understanding of living organisms and applying this knowledge to various fields such as synthetic biology, metabolic engineering, and medicine. System-level understanding of living organisms can be derived from insight into: (i system structure and the mechanism of biological networks such as gene regulation, protein interactions, signaling, and metabolic pathways; (ii system dynamics of biological networks, which provides an understanding of stability, robustness, and transduction ability through system identification, and through system analysis methods; (iii system control methods at different levels of biological networks, which provide an understanding of systematic mechanisms to robustly control system states, minimize malfunctions, and provide potential therapeutic targets in disease treatment; (iv systematic design methods for the modification and construction of biological networks with desired behaviors, which provide system design principles and system simulations for synthetic biology designs and systems metabolic engineering. This review describes current developments in systems biology, systems synthetic biology, and systems metabolic engineering for engineering and biology researchers. We also discuss challenges and future prospects for systems biology and the concept of systems biology as an integrated platform for bioinformatics, systems synthetic biology, and systems metabolic engineering.

  12. Systems Biology as an Integrated Platform for Bioinformatics, Systems Synthetic Biology, and Systems Metabolic Engineering

    Chen, Bor-Sen; Wu, Chia-Chou

    2013-01-01

    Systems biology aims at achieving a system-level understanding of living organisms and applying this knowledge to various fields such as synthetic biology, metabolic engineering, and medicine. System-level understanding of living organisms can be derived from insight into: (i) system structure and the mechanism of biological networks such as gene regulation, protein interactions, signaling, and metabolic pathways; (ii) system dynamics of biological networks, which provides an understanding of stability, robustness, and transduction ability through system identification, and through system analysis methods; (iii) system control methods at different levels of biological networks, which provide an understanding of systematic mechanisms to robustly control system states, minimize malfunctions, and provide potential therapeutic targets in disease treatment; (iv) systematic design methods for the modification and construction of biological networks with desired behaviors, which provide system design principles and system simulations for synthetic biology designs and systems metabolic engineering. This review describes current developments in systems biology, systems synthetic biology, and systems metabolic engineering for engineering and biology researchers. We also discuss challenges and future prospects for systems biology and the concept of systems biology as an integrated platform for bioinformatics, systems synthetic biology, and systems metabolic engineering. PMID:24709875

  13. Platforms.

    Josko, Deborah

    2014-01-01

    The advent of DNA sequencing technologies and the various applications that can be performed will have a dramatic effect on medicine and healthcare in the near future. There are several DNA sequencing platforms available on the market for research and clinical use. Based on the medical laboratory scientist or researcher's needs and taking into consideration laboratory space and budget, one can chose which platform will be beneficial to their institution and their patient population. Although some of the instrument costs seem high, diagnosing a patient quickly and accurately will save hospitals money with fewer hospital stays and targeted treatment based on an individual's genetic make-up. By determining the type of disease an individual has, based on the mutations present or having the ability to prescribe the appropriate antimicrobials based on the knowledge of the organism's resistance patterns, the clinician will be better able to treat and diagnose a patient which ultimately will improve patient outcomes and prognosis.

  14. Extremely Thermophilic Microorganisms as Metabolic Engineering Platforms for Production of Fuels and Industrial Chemicals

    Benjamin M Zeldes

    2015-11-01

    Full Text Available Enzymes from extremely thermophilic microorganisms have been of technological interest for some time because of their ability to catalyze reactions of industrial significance at elevated temperatures. Thermophilic enzymes are now routinely produced in recombinant mesophilic hosts for use as discrete biocatalysts. Genome and metagenome sequence data for extreme thermophiles provide useful information for putative biocatalysts for a wide range of biotransformations, albeit involving at most a few enzymatic steps. However, in the past several years, unprecedented progress has been made in establishing molecular genetics tools for extreme thermophiles to the point that the use of these microorganisms as metabolic engineering platforms has become possible. While in its early days, complex metabolic pathways have been altered or engineered into recombinant extreme thermophiles, such that the production of fuels and chemicals at elevated temperatures has become possible. Not only does this expand the thermal range for industrial biotechnology, it also potentially provides biodiverse options for specific biotransformations unique to these microorganisms. The list of extreme thermophiles growing optimally between 70 and 100°C with genetic toolkits currently available includes archaea and bacteria, aerobes and anaerobes, coming from genera such as Caldicellulosiruptor, Sulfolobus, Thermotoga, Thermococcus and Pyrococcus. These organisms exhibit unusual and potentially useful native metabolic capabilities, including cellulose degradation, metal solubilization, and RuBisCO-free carbon fixation. Those looking to design a thermal bioprocess now have a host of potential candidates to choose from, each with its own advantages and challenges that will influence its appropriateness for specific applications. Here, the issues and opportunities for extremely thermophilic metabolic engineering platforms are considered with an eye towards potential technological

  15. Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals

    Zeldes, Benjamin M.; Keller, Matthew W.; Loder, Andrew J.; Straub, Christopher T.; Adams, Michael W. W.; Kelly, Robert M.

    2015-01-01

    Enzymes from extremely thermophilic microorganisms have been of technological interest for some time because of their ability to catalyze reactions of industrial significance at elevated temperatures. Thermophilic enzymes are now routinely produced in recombinant mesophilic hosts for use as discrete biocatalysts. Genome and metagenome sequence data for extreme thermophiles provide useful information for putative biocatalysts for a wide range of biotransformations, albeit involving at most a few enzymatic steps. However, in the past several years, unprecedented progress has been made in establishing molecular genetics tools for extreme thermophiles to the point that the use of these microorganisms as metabolic engineering platforms has become possible. While in its early days, complex metabolic pathways have been altered or engineered into recombinant extreme thermophiles, such that the production of fuels and chemicals at elevated temperatures has become possible. Not only does this expand the thermal range for industrial biotechnology, it also potentially provides biodiverse options for specific biotransformations unique to these microorganisms. The list of extreme thermophiles growing optimally between 70 and 100°C with genetic toolkits currently available includes archaea and bacteria, aerobes and anaerobes, coming from genera such as Caldicellulosiruptor, Sulfolobus, Thermotoga, Thermococcus, and Pyrococcus. These organisms exhibit unusual and potentially useful native metabolic capabilities, including cellulose degradation, metal solubilization, and RuBisCO-free carbon fixation. Those looking to design a thermal bioprocess now have a host of potential candidates to choose from, each with its own advantages and challenges that will influence its appropriateness for specific applications. Here, the issues and opportunities for extremely thermophilic metabolic engineering platforms are considered with an eye toward potential technological advantages for high

  16. OptFlux: an open-source software platform for in silico metabolic engineering.

    Rocha, Isabel; Maia, Paulo; Evangelista, Pedro; Vilaça, Paulo; Soares, Simão; Pinto, José P; Nielsen, Jens; Patil, Kiran R; Ferreira, Eugénio C; Rocha, Miguel

    2010-04-19

    Over the last few years a number of methods have been proposed for the phenotype simulation of microorganisms under different environmental and genetic conditions. These have been used as the basis to support the discovery of successful genetic modifications of the microbial metabolism to address industrial goals. However, the use of these methods has been restricted to bioinformaticians or other expert researchers. The main aim of this work is, therefore, to provide a user-friendly computational tool for Metabolic Engineering applications. OptFlux is an open-source and modular software aimed at being the reference computational application in the field. It is the first tool to incorporate strain optimization tasks, i.e., the identification of Metabolic Engineering targets, using Evolutionary Algorithms/Simulated Annealing metaheuristics or the previously proposed OptKnock algorithm. It also allows the use of stoichiometric metabolic models for (i) phenotype simulation of both wild-type and mutant organisms, using the methods of Flux Balance Analysis, Minimization of Metabolic Adjustment or Regulatory on/off Minimization of Metabolic flux changes, (ii) Metabolic Flux Analysis, computing the admissible flux space given a set of measured fluxes, and (iii) pathway analysis through the calculation of Elementary Flux Modes. OptFlux also contemplates several methods for model simplification and other pre-processing operations aimed at reducing the search space for optimization algorithms. The software supports importing/exporting to several flat file formats and it is compatible with the SBML standard. OptFlux has a visualization module that allows the analysis of the model structure that is compatible with the layout information of Cell Designer, allowing the superimposition of simulation results with the model graph. The OptFlux software is freely available, together with documentation and other resources, thus bridging the gap from research in strain optimization

  17. Metabolic engineering of Escherichia coli: a sustainable industrial platform for bio-based chemical production.

    Chen, Xianzhong; Zhou, Li; Tian, Kangming; Kumar, Ashwani; Singh, Suren; Prior, Bernard A; Wang, Zhengxiang

    2013-12-01

    In order to decrease carbon emissions and negative environmental impacts of various pollutants, more bulk and/or fine chemicals are produced by bioprocesses, replacing the traditional energy and fossil based intensive route. The Gram-negative rod-shaped bacterium, Escherichia coli has been studied extensively on a fundamental and applied level and has become a predominant host microorganism for industrial applications. Furthermore, metabolic engineering of E. coli for the enhanced biochemical production has been significantly promoted by the integrated use of recent developments in systems biology, synthetic biology and evolutionary engineering. In this review, we focus on recent efforts devoted to the use of genetically engineered E. coli as a sustainable platform for the production of industrially important biochemicals such as biofuels, organic acids, amino acids, sugar alcohols and biopolymers. In addition, representative secondary metabolites produced by E. coli will be systematically discussed and the successful strategies for strain improvements will be highlighted. Moreover, this review presents guidelines for future developments in the bio-based chemical production using E. coli as an industrial platform. Copyright © 2013 Elsevier Inc. All rights reserved.

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

    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

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

    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

  20. Metabolic cost of running is greater on a treadmill with a stiffer running platform.

    Smith, James A H; McKerrow, Alexander D; Kohn, Tertius A

    2017-08-01

    Exercise testing on motorised treadmills provides valuable information about running performance and metabolism; however, the impact of treadmill type on these tests has not been investigated. This study compared the energy demand of running on two laboratory treadmills: an HP Cosmos (C) and a Quinton (Q) model, with the latter having a 4.5 times stiffer running platform. Twelve experienced runners ran identical bouts on these treadmills at a range of four submaximal velocities (reported data is for the velocity that approximated 75-81% VO 2max ). The stiffer treadmill elicited higher oxygen consumption (C: 46.7 ± 3.8; Q: 50.1 ± 4.3 ml·kg -1 · min -1 ), energy expenditure (C: 16.0 ± 2.5; Q: 17.7 ± 2.9 kcal · min -1 ), carbohydrate oxidation (C: 9.6 ± 3.1; Q: 13.0 ± 3.9 kcal · min -1 ), heart rate (C: 155 ± 16; Q: 163 ± 16 beats · min -1 ) and rating of perceived exertion (C: 13.8 ± 1.2; Q: 14.7 ± 1.2), but lower fat oxidation (C: 6.4 ± 2.3; Q: 4.6 ± 2.5 kcal · min -1 ) (all analysis of variance treadmill comparisons P running depending on the running platform stiffness.

  1. Camelina sativa: An ideal platform for the metabolic engineering and field production of industrial lipids.

    Bansal, Sunil; Durrett, Timothy P

    2016-01-01

    Triacylglycerols (TAG) containing modified fatty acids with functionality beyond those found in commercially grown oil seed crops can be used as feedstocks for biofuels and bio-based materials. Over the years, advances have been made in transgenically engineering the production of various modified fatty acids in the model plant Arabidopsis thaliana. However, the inability to produce large quantities of transgenic seed has limited the functional testing of the modified oil. In contrast, the emerging oil seed crop Camelina sativa possesses important agronomic traits that recommend it as an ideal production platform for biofuels and industrial feedstocks. Camelina possesses low water and fertilizer requirements and is capable of yields comparable to other oil seed crops, particularly under stress conditions. Importantly, its relatively short growing season enables it to be grown as part of a double cropping system. In addition to these valuable agronomic features, Camelina is amenable to rapid metabolic engineering. The development of a simple and effective transformation method, combined with the availability of abundant transcriptomic and genomic data, has allowed the generation of transgenic Camelina lines capable of synthesizing high levels of unusual lipids. In some cases these levels have surpassed what was achieved in Arabidopsis. Further, the ability to use Camelina as a crop production system has allowed for the large scale growth of transgenic oil seed crops, enabling subsequent physical property testing. The application of new techniques such as genome editing will further increase the suitability of Camelina as an ideal platform for the production of biofuels and bio-materials. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  2. Prenatal Cell-Free DNA Screening

    ... 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 ...

  3. Dedicated Industrial Oilseed Crops as Metabolic Engineering Platforms for Sustainable Industrial Feedstock Production.

    Zhu, Li-Hua; Krens, Frans; Smith, Mark A; Li, Xueyuan; Qi, Weicong; van Loo, Eibertus N; Iven, Tim; Feussner, Ivo; Nazarenus, Tara J; Huai, Dongxin; Taylor, David C; Zhou, Xue-Rong; Green, Allan G; Shockey, Jay; Klasson, K Thomas; Mullen, Robert T; Huang, Bangquan; Dyer, John M; Cahoon, Edgar B

    2016-02-26

    Feedstocks for industrial applications ranging from polymers to lubricants are largely derived from petroleum, a non-renewable resource. Vegetable oils with fatty acid structures and storage forms tailored for specific industrial uses offer renewable and potentially sustainable sources of petrochemical-type functionalities. A wide array of industrial vegetable oils can be generated through biotechnology, but will likely require non-commodity oilseed platforms dedicated to specialty oil production for commercial acceptance. Here we show the feasibility of three Brassicaceae oilseeds crambe, camelina, and carinata, none of which are widely cultivated for food use, as hosts for complex metabolic engineering of wax esters for lubricant applications. Lines producing wax esters >20% of total seed oil were generated for each crop and further improved for high temperature oxidative stability by down-regulation of fatty acid polyunsaturation. Field cultivation of optimized wax ester-producing crambe demonstrated commercial utility of these engineered crops and a path for sustainable production of other industrial oils in dedicated specialty oilseeds.

  4. Arraying proteins by cell-free synthesis.

    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.

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

    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

  6. IMGMD: A platform for the integration and standardisation of In silico Microbial Genome-scale Metabolic Models.

    Ye, Chao; Xu, Nan; Dong, Chuan; Ye, Yuannong; Zou, Xuan; Chen, Xiulai; Guo, Fengbiao; Liu, Liming

    2017-04-07

    Genome-scale metabolic models (GSMMs) constitute a platform that combines genome sequences and detailed biochemical information to quantify microbial physiology at the system level. To improve the unity, integrity, correctness, and format of data in published GSMMs, a consensus IMGMD database was built in the LAMP (Linux + Apache + MySQL + PHP) system by integrating and standardizing 328 GSMMs constructed for 139 microorganisms. The IMGMD database can help microbial researchers download manually curated GSMMs, rapidly reconstruct standard GSMMs, design pathways, and identify metabolic targets for strategies on strain improvement. Moreover, the IMGMD database facilitates the integration of wet-lab and in silico data to gain an additional insight into microbial physiology. The IMGMD database is freely available, without any registration requirements, at http://imgmd.jiangnan.edu.cn/database.

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

    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.

  8. Translation in cell-free systems

    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

  9. Growth platform-dependent and -independent phenotypic and metabolic responses of Arabidopsis and its halophytic relative, Eutrema salsugineum, to salt stress.

    Kazachkova, Yana; Batushansky, Albert; Cisneros, Aroldo; Tel-Zur, Noemi; Fait, Aaron; Barak, Simon

    2013-07-01

    Comparative studies of the stress-tolerant Arabidopsis (Arabidopsis thaliana) halophytic relative, Eutrema salsugineum, have proven a fruitful approach to understanding natural stress tolerance. Here, we performed comparative phenotyping of Arabidopsis and E. salsugineum vegetative development under control and salt-stress conditions, and then compared the metabolic responses of the two species on different growth platforms in a defined leaf developmental stage. Our results reveal both growth platform-dependent and -independent phenotypes and metabolic responses. Leaf emergence was affected in a similar way in both species grown in vitro but the effects observed in Arabidopsis occurred at higher salt concentrations in E. salsugineum. No differences in leaf emergence were observed on soil. A new effect of a salt-mediated reduction in E. salsugineum leaf area was unmasked. On soil, leaf area reduction in E. salsugineum was mainly due to a fall in cell number, whereas both cell number and cell size contributed to the decrease in Arabidopsis leaf area. Common growth platform-independent leaf metabolic signatures such as high raffinose and malate, and low fumarate contents that could reflect core stress tolerance mechanisms, as well as growth platform-dependent metabolic responses were identified. In particular, the in vitro growth platform led to repression of accumulation of many metabolites including sugars, sugar phosphates, and amino acids in E. salsugineum compared with the soil system where these same metabolites accumulated to higher levels in E. salsugineum than in Arabidopsis. The observation that E. salsugineum maintains salt tolerance despite growth platform-specific phenotypes and metabolic responses suggests a considerable degree of phenotypic and metabolic adaptive plasticity in this extremophile.

  10. Metabolic engineering of Saccharomyces cerevisiae: a key cell factory platform for future biorefineries.

    Hong, Kuk-Ki; Nielsen, Jens

    2012-08-01

    Metabolic engineering is the enabling science of development of efficient cell factories for the production of fuels, chemicals, pharmaceuticals, and food ingredients through microbial fermentations. The yeast Saccharomyces cerevisiae is a key cell factory already used for the production of a wide range of industrial products, and here we review ongoing work, particularly in industry, on using this organism for the production of butanol, which can be used as biofuel, and isoprenoids, which can find a wide range of applications including as pharmaceuticals and as biodiesel. We also look into how engineering of yeast can lead to improved uptake of sugars that are present in biomass hydrolyzates, and hereby allow for utilization of biomass as feedstock in the production of fuels and chemicals employing S. cerevisiae. Finally, we discuss the perspectives of how technologies from systems biology and synthetic biology can be used to advance metabolic engineering of yeast.

  11. OptFlux: an open-source software platform for in silico metabolic engineering

    Rocha, I.; Maia, P.; Evangelista, P.

    2010-01-01

    to address industrial goals. However, the use of these methods has been restricted to bioinformaticians or other expert researchers. The main aim of this work is, therefore, to provide a user-friendly computational tool for Metabolic Engineering applications. Results: OptFlux is an open-source and modular...... available a number of useful tools. Its open-source nature invites contributions by all those interested in making their methods available for the community. Given its plug-in based architecture it can be extended with new functionalities. Currently, several plug-ins are being developed, including network...

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

    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.

  13. [The Design Requirements for an E-Health Management Platform: Addressing the Needs of Adolescent Girls at High Risk of Metabolic Syndrome].

    Su, Mei-Chen; Chen, Wen-Chin; Liu, Chieh-Yu; Jou, Hei-Jen; Hsiao, Ya-Chu; Tsao, Lee-Ing

    2015-10-01

    The rising prevalence of metabolic syndrome among adolescents has not been effectively addressed by current campus-based health promotions. Using the Internet in these promotions may help health professionals achieve better healthcare management. The purpose of the present study was to explore the design requirements of an e-health management platform from the subjective perspective of adolescent girls who were at a high risk of metabolic syndrome. The findings may provide a reference for designing nursing interventions that more effectively promote healthly lifestyle habits to adolescents. This qualitative study employed a snowball approach and used a semi-structured interview guide to collect data. A total of 20 Taiwanese adolescent females who were at a high risk of metabolic syndrome, aged 16-20 years, able to speak Mandarin or Taiwanese, and willing to participate and to have their sessions tape-recorded were enrolled as participants and engaged individually in in-depth interviews. The constant comparative method was used to inductively analyze the interview data. Five main themes related to the e-health management platform emerged from the data. These themes included: an attractive and user-friendly website interface, access to reliable information and resources, provision of tailored health information, access to peer support, and self-monitoring and learning tools. The findings highlight the key design needs of an e-Health management platform from the perspective of adolescent girls who are at a high risk of metabolic syndrome. The identified themes may be addressed in future revisions / developments of these platforms in order to better address the needs of this vulnerable population and to effectively reduce the incidence of metabolic syndrome. The authors hope that the results of the present study may be used to provide better healthcare and support for adolescent girls with metabolic syndrome.

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

    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

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

    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.

  16. Nonhomologous DNA End Joining in Cell-Free Extracts

    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.

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

    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.

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

    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.

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

    Hodgman, C Eric; Jewett, Michael C

    2012-05-01

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

  20. Development of gold-immobilized P450 platform for exploring the effect of oligomer formation on P450-mediated metabolism for in vitro to in vivo drug metabolism predictions

    Kabulski, Jarod L.

    The cytochrome P450 (P450) enzyme family is responsible for the biotransformation of a wide range of endogenous and xenobiotic compounds, as well as being the major metabolic enzyme in first pass drug metabolism. In vivo drug metabolism for P450 enzymes is predicted using in vitro data obtained from a reconstituted expressed P450 system, but these systems have not always been proven to accurately represent in vivo enzyme kinetics, due to interactions caused by oligomer formation. These in vitro systems use soluble P450 enzymes prone to oligomer formation and studies have shown that increased states of protein aggregation directly affect the P450 enzyme kinetics. We have developed an immobilized enzyme system that isolates the enzyme and can be used to elucidate the effect of P450 aggregation on metabolism kinetics. The long term goal of my research is to develop a tool that will help improve the assessment of pharmaceuticals by better predicting in vivo kinetics in an in vitro system. The central hypothesis of this research is that P450-mediated kinetics measured in vitro is dependent on oligomer formation and that the accurate prediction of in vivo P450-mediated kinetics requires elucidation of the effect of oligomer formation. The rationale is that the development of a P450 bound to a Au platform can be used to control the aggregation of enzymes and bonding to Au may also permit replacement of the natural redox partners with an electrode capable of supplying a constant flow of electrons. This dissertation explains the details of the enzyme attachment, monitoring substrate binding, and metabolism using physiological and electrochemical methods, determination of enzyme kinetics, and the development of an immobilized-P450 enzyme bioreactor. This work provides alternative approaches to studying P450-mediated kinetics, a platform for controlling enzyme aggregation, electrochemically-driven P450 metabolism, and for investigating the effect of protein

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

    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.

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

    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.

  3. A Lab-on-a-disc platform for trapping of cells, monitoring of cell behaviour and evaluation of redox metabolism

    Amato, Letizia; Kuldeep, Kuldeep; Esmail Tehrani, Sheida

    2015-01-01

    In this work, we demonstrate an integrated electrochemical system on a centrifugal microfluidic platform for cell studies by combining electrochemical impedance spectroscopy and amperometry, and comparison of different cleaning protocols for gold electrodes on plastic substrate.......In this work, we demonstrate an integrated electrochemical system on a centrifugal microfluidic platform for cell studies by combining electrochemical impedance spectroscopy and amperometry, and comparison of different cleaning protocols for gold electrodes on plastic substrate....

  4. Cell-Free, De Nova Synthesis of Poliovirus

    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.

  5. Metabolism

    ... Are More Common in People With Type 1 Diabetes Metabolic Syndrome Your Child's Weight Healthy Eating Endocrine System Blood Test: Basic Metabolic Panel (BMP) Activity: Endocrine System Growth Disorders Diabetes Center Thyroid Disorders Your Endocrine System Movie: Endocrine ...

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

    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.

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

    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

  8. Coping with complexity: machine learning optimization of cell-free protein synthesis.

    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.

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

    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. Metabolism

    ... lin), which signals cells to increase their anabolic activities. Metabolism is a complicated chemical process, so it's not ... how those enzymes or hormones work. When the metabolism of body chemicals is ... Hyperthyroidism (pronounced: hi-per-THIGH-roy-dih-zum). Hyperthyroidism ...

  11. Ultra high resolution SFC-MS as a high throughput platform for metabolic phenotyping: application to metabolic profiling of rat and dog bile.

    Jones, Michael D; Rainville, Paul D; Isaac, Giorgis; Wilson, Ian D; Smith, Norman W; Plumb, Robert S

    2014-09-01

    Ultra high resolution SFC-MS (on sub-2μm particles) coupled to mass spectrometry has been evaluated for the metabolic profiling of rat and dog bile. The selectivity of the SFC separation differed from that seen in previous reversed-phase UPLC-MS studies on bile, with the order of elution for analytes such as e.g., the bile acids showing many differences. The chromatography system showed excellent stability, reproducibility and robustness with relative standard deviation of less than 1% for retention time obtained over the course of the analysis. SFC showed excellent chromatographic performance with chromatographic peak widths in the order of 3s at the base of the peak. The use of supercritical fluid carbon dioxide as a mobile phase solvent also reduced the overall consumption of organic solvent by a factor of 3 and also reduced the overall analysis time by a factor of 30% compared to reversed-phase gradient LC. SFC-MS appear complementary to RPLC for the metabolic profiling of complex samples such as bile. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    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.

  13. Reconstituted AIM2 inflammasome in cell-free system.

    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.

  14. Recent advances in the metabolic engineering of microorganisms for the production of 3-hydroxypropionic acid as C3 platform chemical.

    Valdehuesa, Kris Niño G; Liu, Huaiwei; Nisola, Grace M; Chung, Wook-Jin; Lee, Seung Hwan; Park, Si Jae

    2013-04-01

    Development of sustainable technologies for the production of 3-hydroxypropionic acid (3HP) as a platform chemical has recently been gaining much attention owing to its versatility in applications for the synthesis of other specialty chemicals. Several proposed biological synthesis routes and strategies for producing 3HP from glucose and glycerol are reviewed presently. Ten proposed routes for 3HP production from glucose are described and one of which was recently constructed successfully in Escherichia coli with malonyl-Coenzyme A as a precursor. This resulted in a yield still far from the required level for industrial application. On the other hand, strategies employing engineered E. coli and Klebsiella pneumoniae capable of producing 3HP from glycerol are also evaluated. The titers produced by these recombinant strains reached around 3 %. At its current state, it is evident that a bulk of engineering works is yet to be done to acquire a biosynthesis route for 3HP that is acceptable for industrial-scale production.

  15. Metabolic Profiling and Classification of Propolis Samples from Southern Brazil: An NMR-Based Platform Coupled with Machine Learning.

    Maraschin, Marcelo; Somensi-Zeggio, Amélia; Oliveira, Simone K; Kuhnen, Shirley; Tomazzoli, Maíra M; Raguzzoni, Josiane C; Zeri, Ana C M; Carreira, Rafael; Correia, Sara; Costa, Christopher; Rocha, Miguel

    2016-01-22

    The chemical composition of propolis is affected by environmental factors and harvest season, making it difficult to standardize its extracts for medicinal usage. By detecting a typical chemical profile associated with propolis from a specific production region or season, certain types of propolis may be used to obtain a specific pharmacological activity. In this study, propolis from three agroecological regions (plain, plateau, and highlands) from southern Brazil, collected over the four seasons of 2010, were investigated through a novel NMR-based metabolomics data analysis workflow. Chemometrics and machine learning algorithms (PLS-DA and RF), including methods to estimate variable importance in classification, were used in this study. The machine learning and feature selection methods permitted construction of models for propolis sample classification with high accuracy (>75%, reaching ∼90% in the best case), better discriminating samples regarding their collection seasons comparatively to the harvest regions. PLS-DA and RF allowed the identification of biomarkers for sample discrimination, expanding the set of discriminating features and adding relevant information for the identification of the class-determining metabolites. The NMR-based metabolomics analytical platform, coupled to bioinformatic tools, allowed characterization and classification of Brazilian propolis samples regarding the metabolite signature of important compounds, i.e., chemical fingerprint, harvest seasons, and production regions.

  16. MpTheory Java library: a multi-platform Java library for systems biology based on the Metabolic P theory.

    Marchetti, Luca; Manca, Vincenzo

    2015-04-15

    MpTheory Java library is an open-source project collecting a set of objects and algorithms for modeling observed dynamics by means of the Metabolic P (MP) theory, that is, a mathematical theory introduced in 2004 for modeling biological dynamics. By means of the library, it is possible to model biological systems both at continuous and at discrete time. Moreover, the library comprises a set of regression algorithms for inferring MP models starting from time series of observations. To enhance the modeling experience, beside a pure Java usage, the library can be directly used within the most popular computing environments, such as MATLAB, GNU Octave, Mathematica and R. The library is open-source and licensed under the GNU Lesser General Public License (LGPL) Version 3.0. Source code, binaries and complete documentation are available at http://mptheory.scienze.univr.it. luca.marchetti@univr.it, marchetti@cosbi.eu Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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

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

    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.

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

    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.

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

    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.

  1. Cell-Free DNA and Active Rejection in Kidney Allografts.

    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.

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

    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.

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

    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 ...

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

    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.

  5. Metabolic engineering of a glycerol-oxidative pathway in Lactobacillus panis PM1 for utilization of bioethanol thin stillage: potential to produce platform chemicals from glycerol.

    Kang, Tae Sun; Korber, Darren R; Tanaka, Takuji

    2014-12-01

    Lactobacillus panis PM1 has the ability to produce 1,3-propanediol (1,3-PDO) from thin stillage (TS), which is the major waste material after bioethanol production, and is therefore of significance. However, the fact that L. panis PM1 cannot use glycerol as a sole carbon source presents a considerable problem in terms of utilization of this strain in a wide range of industrial applications. Accordingly, L. panis PM1 was genetically engineered to directly utilize TS as a fermentable substrate for the production of valuable platform chemicals without the need for exogenous nutrient supplementation (e.g., sugars and nitrogen sources). An artificial glycerol-oxidative pathway, comprised of glycerol facilitator, glycerol kinase, glycerol 3-phosphate dehydrogenase, triosephosphate isomerase, and NADPH-dependent aldehyde reductase genes of Escherichia coli, was introduced into L. panis PM1 in order to directly utilize glycerol for the production of energy for growth and value-added chemicals. A pH 6.5 culture converted glycerol to mainly lactic acid (85.43 mM), whereas a significant amount of 1,3-propanediol (59.96 mM) was formed at pH 7.5. Regardless of the pH, ethanol (82.16 to 83.22 mM) was produced from TS fermentations, confirming that the artificial pathway metabolized glycerol for energy production and converted it into lactic acid or 1,3-PDO and ethanol in a pH-dependent manner. This study demonstrates the cost-effective conversion of TS to value-added chemicals by the engineered PM1 strain cultured under industrial conditions. Thus, application of this strain or these research findings can contribute to reduced costs of bioethanol production. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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

    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.

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

    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...

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

    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.

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

    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

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

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

    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.

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

    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.

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

    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.

  14. Payment Platform

    Hjelholt, Morten; Damsgaard, Jan

    2012-01-01

    thoroughly and substitute current payment standards in the decades to come. This paper portrays how digital payment platforms evolve in socio-technical niches and how various technological platforms aim for institutional attention in their attempt to challenge earlier platforms and standards. The paper...... applies a co-evolutionary multilevel perspective to model the interplay and processes between technology and society wherein digital payment platforms potentially will substitute other payment platforms just like the credit card negated the check. On this basis this paper formulate a multilevel conceptual...

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

    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

    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.

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

    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.

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

    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.

  20. Platform Constellations

    Staykova, Kalina Stefanova; Damsgaard, Jan

    2016-01-01

    This research paper presents an initial attempt to introduce and explain the emergence of new phenomenon, which we refer to as platform constellations. Functioning as highly modular systems, the platform constellations are collections of highly connected platforms which co-exist in parallel and a......’ acquisition and users’ engagement rates as well as unlock new sources of value creation and diversify revenue streams....

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

    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.

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

    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

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

    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...

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

    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.

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

    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

  6. Cell-free placental DNA beyond Down syndrome: Lessons learned from fetal RHD genotyping

    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

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

    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...

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

    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.

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

    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.

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

    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.

  11. 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.

    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.

  12. Synthetic biology platform technologies for antimicrobial applications.

    Braff, Dana; Shis, David; Collins, James J

    2016-10-01

    The growing prevalence of antibiotic resistance calls for new approaches in the development of antimicrobial therapeutics. Likewise, improved diagnostic measures are essential in guiding the application of targeted therapies and preventing the evolution of therapeutic resistance. Discovery platforms are also needed to form new treatment strategies and identify novel antimicrobial agents. By applying engineering principles to molecular biology, synthetic biologists have developed platforms that improve upon, supplement, and will perhaps supplant traditional broad-spectrum antibiotics. Efforts in engineering bacteriophages and synthetic probiotics demonstrate targeted antimicrobial approaches that can be fine-tuned using synthetic biology-derived principles. Further, the development of paper-based, cell-free expression systems holds promise in promoting the clinical translation of molecular biology tools for diagnostic purposes. In this review, we highlight emerging synthetic biology platform technologies that are geared toward the generation of new antimicrobial therapies, diagnostics, and discovery channels. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. A Multi-scale Computational Platform to Mechanistically Assess the Effect of Genetic Variation on Drug Responses in Human Erythrocyte Metabolism.

    Mih, Nathan; Brunk, Elizabeth; Bordbar, Aarash; Palsson, Bernhard O

    2016-07-01

    Progress in systems medicine brings promise to addressing patient heterogeneity and individualized therapies. Recently, genome-scale models of metabolism have been shown to provide insight into the mechanistic link between drug therapies and systems-level off-target effects while being expanded to explicitly include the three-dimensional structure of proteins. The integration of these molecular-level details, such as the physical, structural, and dynamical properties of proteins, notably expands the computational description of biochemical network-level properties and the possibility of understanding and predicting whole cell phenotypes. In this study, we present a multi-scale modeling framework that describes biological processes which range in scale from atomistic details to an entire metabolic network. Using this approach, we can understand how genetic variation, which impacts the structure and reactivity of a protein, influences both native and drug-induced metabolic states. As a proof-of-concept, we study three enzymes (catechol-O-methyltransferase, glucose-6-phosphate dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase) and their respective genetic variants which have clinically relevant associations. Using all-atom molecular dynamic simulations enables the sampling of long timescale conformational dynamics of the proteins (and their mutant variants) in complex with their respective native metabolites or drug molecules. We find that changes in a protein's structure due to a mutation influences protein binding affinity to metabolites and/or drug molecules, and inflicts large-scale changes in metabolism.

  14. A Multi-scale Computational Platform to Mechanistically Assess the Effect of Genetic Variation on Drug Responses in Human Erythrocyte Metabolism.

    Nathan Mih

    2016-07-01

    Full Text Available Progress in systems medicine brings promise to addressing patient heterogeneity and individualized therapies. Recently, genome-scale models of metabolism have been shown to provide insight into the mechanistic link between drug therapies and systems-level off-target effects while being expanded to explicitly include the three-dimensional structure of proteins. The integration of these molecular-level details, such as the physical, structural, and dynamical properties of proteins, notably expands the computational description of biochemical network-level properties and the possibility of understanding and predicting whole cell phenotypes. In this study, we present a multi-scale modeling framework that describes biological processes which range in scale from atomistic details to an entire metabolic network. Using this approach, we can understand how genetic variation, which impacts the structure and reactivity of a protein, influences both native and drug-induced metabolic states. As a proof-of-concept, we study three enzymes (catechol-O-methyltransferase, glucose-6-phosphate dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase and their respective genetic variants which have clinically relevant associations. Using all-atom molecular dynamic simulations enables the sampling of long timescale conformational dynamics of the proteins (and their mutant variants in complex with their respective native metabolites or drug molecules. We find that changes in a protein's structure due to a mutation influences protein binding affinity to metabolites and/or drug molecules, and inflicts large-scale changes in metabolism.

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

    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

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

    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.

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

    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.

  19. Percutaneous Mitral Valve Repair in Mitral Regurgitation Reduces Cell-Free Hemoglobin and Improves Endothelial Function.

    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.

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

    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.

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

    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.

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

    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.

  3. Stability of cell-free DNA from maternal plasma isolated following a single centrifugation step.

    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.

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

    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.

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

    Annika Müller-Lucks

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

  6. Metabolic profiling of potential lung cancer biomarkers using bronchoalveolar lavage fluid and the integrated direct infusion/ gas chromatography mass spectrometry platform.

    Callejón-Leblic, Belén; García-Barrera, Tamara; Grávalos-Guzmán, Jesús; Pereira-Vega, Antonio; Gómez-Ariza, José Luis

    2016-08-11

    Lung cancer is one of the ten most common causes of death worldwide, so that the search for early diagnosis biomarkers is a very challenging task. Bronchoalveolar lavage fluid (BALF) provides information on cellular and biochemical epithelial surface of the lower respiratory tract constituents and no previous metabolomic studies have been performed with BALF samples from patients with lung cancer. Therefore, this fluid has been explored looking for new contributions in lung cancer metabolism. In this way, two complementary metabolomics techniques based on direct infusion high resolution mass spectrometry (DI-ESI-QTOF-MS) and gas chromatography mass spectrometry (GC-MS) have been applied to compare statistically differences between lung cancer (LC) and control (C) BALF samples, using partial least square discriminant analysis (PLS-DA) in order to find and identify potential biomarkers of the disease. A total of 42 altered metabolites were found in BALF from LC. The metabolic pathway analysis showed that glutamate and glutamine metabolism pathway was mainly altered by this disease. In addition, we assessed the biomarker specificity and sensitivity according to the area under the receiver operator characteristic (ROC) curves, indicating that glycerol and phosphoric acid were potential sensitive and specific biomarkers for lung cancer diagnosis and prognosis. The search for early diagnosis of lung cancer is a very challenging task because of the high mortality associated to this disease and its critical linkage to the initiation of treatment. Bronchoalveolar lavage fluid provides information on cellular and biochemical epithelial surface of the lower respiratory tract constituents and no previous metabolomic studies have been performed with BALF samples from patients with lung cancer. Since BALF is in close interaction with lung tissue it is a more representative sample of lung status than other peripheral biofluids as blood or urine studied in previous works

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

    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. Platform computing

    2002-01-01

    "Platform Computing releases first grid-enabled workload management solution for IBM eServer Intel and UNIX high performance computing clusters. This Out-of-the-box solution maximizes the performance and capability of applications on IBM HPC clusters" (1/2 page) .

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

    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.

  10. A multi-platform metabolomics approach demonstrates changes in energy metabolism and the transsulfuration pathway in Chironomus tepperi following exposure to zinc

    Long, Sara M.; Tull, Dedreia L.; Jeppe, Katherine J.; De Souza, David P.; Dayalan, Saravanan; Pettigrove, Vincent J.; McConville, Malcolm J.; Hoffmann, Ary A.

    2015-01-01

    Highlights: • An integrated metabolomics approach was applied to examine zinc exposure in midges. • Changes in carbohydrate and energy metabolism were observed using GC–MS. • Transsulfuration pathway is affected by zinc exposure. • Heavy metals other than zinc affect the transsulfuration pathways differently. - Abstract: Measuring biological responses in resident biota is a commonly used approach to monitoring polluted habitats. The challenge is to choose sensitive and, ideally, stressor-specific endpoints that reflect the responses of the ecosystem. Metabolomics is a potentially useful approach for identifying sensitive and consistent responses since it provides a holistic view to understanding the effects of exposure to chemicals upon the physiological functioning of organisms. In this study, we exposed the aquatic non-biting midge, Chironomus tepperi, to two concentrations of zinc chloride and measured global changes in polar metabolite levels using an untargeted gas chromatography–mass spectrometry (GC–MS) analysis and a targeted liquid chromatography–mass spectrometry (LC–MS) analysis of amine-containing metabolites. These data were correlated with changes in the expression of a number of target genes. Zinc exposure resulted in a reduction in levels of intermediates in carbohydrate metabolism (i.e., glucose 6-phosphate, fructose 6-phosphate and disaccharides) and an increase in a number of TCA cycle intermediates. Zinc exposure also resulted in decreases in concentrations of the amine containing metabolites, lanthionine, methionine and cystathionine, and an increase in metallothionein gene expression. Methionine and cystathionine are intermediates in the transsulfuration pathway which is involved in the conversion of methionine to cysteine. These responses provide an understanding of the pathways affected by zinc toxicity, and how these effects are different to other heavy metals such as cadmium and copper. The use of complementary

  11. A multi-platform metabolomics approach demonstrates changes in energy metabolism and the transsulfuration pathway in Chironomus tepperi following exposure to zinc

    Long, Sara M., E-mail: hoskins@unimelb.edu.au [Centre for Aquatic Pollution, Identification and Management (CAPIM), School of BioSciences, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Road, Parkville, 3052 (Australia); Tull, Dedreia L., E-mail: dedreia@unimelb.edu.au [Metabolomics Australia, Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road, Parkville, 3052 (Australia); Jeppe, Katherine J., E-mail: k.jeppe@unimelb.edu.au [Centre for Aquatic Pollution, Identification and Management (CAPIM), School of BioSciences, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Road, Parkville, 3052 (Australia); Centre for Aquatic Pollution, Identification and Management (CAPIM), School of BioSciences, The University of Melbourne, 3010 (Australia); De Souza, David P., E-mail: desouzad@unimelb.edu.au [Metabolomics Australia, Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road, Parkville, 3052 (Australia); Dayalan, Saravanan, E-mail: sdayalan@unimelb.edu.au [Metabolomics Australia, Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road, Parkville, 3052 (Australia); Pettigrove, Vincent J., E-mail: vpet@unimelb.edu.au [Centre for Aquatic Pollution, Identification and Management (CAPIM), School of BioSciences, The University of Melbourne, 3010 (Australia); McConville, Malcolm J., E-mail: malcolmm@unimelb.edu.au [Metabolomics Australia, Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road, Parkville, 3052 (Australia); Hoffmann, Ary A., E-mail: ary@unimelb.edu.au [Centre for Aquatic Pollution, Identification and Management (CAPIM), School of BioSciences, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Road, Parkville, 3052 (Australia); School of BioSciences, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Road, Parkville, 3052 (Australia)

    2015-05-15

    Highlights: • An integrated metabolomics approach was applied to examine zinc exposure in midges. • Changes in carbohydrate and energy metabolism were observed using GC–MS. • Transsulfuration pathway is affected by zinc exposure. • Heavy metals other than zinc affect the transsulfuration pathways differently. - Abstract: Measuring biological responses in resident biota is a commonly used approach to monitoring polluted habitats. The challenge is to choose sensitive and, ideally, stressor-specific endpoints that reflect the responses of the ecosystem. Metabolomics is a potentially useful approach for identifying sensitive and consistent responses since it provides a holistic view to understanding the effects of exposure to chemicals upon the physiological functioning of organisms. In this study, we exposed the aquatic non-biting midge, Chironomus tepperi, to two concentrations of zinc chloride and measured global changes in polar metabolite levels using an untargeted gas chromatography–mass spectrometry (GC–MS) analysis and a targeted liquid chromatography–mass spectrometry (LC–MS) analysis of amine-containing metabolites. These data were correlated with changes in the expression of a number of target genes. Zinc exposure resulted in a reduction in levels of intermediates in carbohydrate metabolism (i.e., glucose 6-phosphate, fructose 6-phosphate and disaccharides) and an increase in a number of TCA cycle intermediates. Zinc exposure also resulted in decreases in concentrations of the amine containing metabolites, lanthionine, methionine and cystathionine, and an increase in metallothionein gene expression. Methionine and cystathionine are intermediates in the transsulfuration pathway which is involved in the conversion of methionine to cysteine. These responses provide an understanding of the pathways affected by zinc toxicity, and how these effects are different to other heavy metals such as cadmium and copper. The use of complementary

  12. [Orange Platform].

    Toba, Kenji

    2017-07-01

    The Organized Registration for the Assessment of dementia on Nationwide General consortium toward Effective treatment in Japan (ORANGE platform) is a recently established nationwide clinical registry for dementia. This platform consists of multiple registries of patients with dementia stratified by the following clinical stages: preclinical, mild cognitive impairment, early-stage, and advanced-stage dementia. Patients will be examined in a super-longitudinal fashion, and their lifestyle, social background, genetic risk factors, and required care process will be assessed. This project is also notable because the care registry includes information on the successful, comprehensive management of patients with dementia. Therefore, this multicenter prospective cohort study will contribute participants to all clinical trials for Alzheimer's disease as well as improve the understanding of individuals with dementia.

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

    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.

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

    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

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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

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

    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.

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

    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.

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

    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.

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

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

    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

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

    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.

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

    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.

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

    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

    Circulating DNA can be detected and quantified in the blood of cancer patients and used for detection of tumor-specific genetic alterations. The clinical utility has been intensively investigated for the past 10 years. The majority of reports focus on analyzing the clinical potential of tumor-specific mutations, whereas the use of total cell-free DNA (cfDNA) quantification is somehow controversial and sparsely described in the literature, but holds important clinical information in itself. The purpose of the present report was to present a systematic review and meta-analysis of the prognostic value of total cfDNA in patients with metastatic colorectal cancer (mCRC) treated with chemotherapy. In addition, we report on the overall performance of cfDNA as source for KRAS mutation detection. A systematic literature search of PubMed and Embase was performed by two independent investigators. Eligibility criteria were (a) total cfDNA analysis, (b) mCRC, and (c) prognostic value during palliative treatment. The preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines were followed, and meta-analysis applied on both aggregate data extraction and individual patients' data. Ten eligible cohorts were identified, including a total of 1,076 patients. Seven studies used quantitative polymerase chain reaction methods, two BEAMing [beads, emulsification, amplification, and magnetics] technology, and one study digital droplet polymerase chain reaction. The baseline levels of cfDNA was similar in the presented studies, and all studies reported a clear prognostic value in favor of patients with lowest levels of baseline cfDNA. A meta-analysis revealed a combined estimate of favorable overall survival hazard ratio (HR) in patients with levels below the median cfDNA (HR = 2.39, 95% confidence interval 2.03-2.82, p  meta-analysis. Reliable prognostic markers could help to guide patients and treating physicians regarding the relevance and choice of

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

    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.

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

    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.

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

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

    2017-01-01

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

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

    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...

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

    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.

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

    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...

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

    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.

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

    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...

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

    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

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

    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

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

    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

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

    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...

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

    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...

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

    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

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

    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...

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

    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

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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

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

    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.

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

    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.

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

    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

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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

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

    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.

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

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

    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

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

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

    2015-03-01

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

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

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

    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

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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...

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

    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...

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

    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)

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

    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.

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

    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)

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

    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.

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

    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.

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

    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.

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

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

    2009-10-01

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

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

    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.

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

    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

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

    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

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

    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

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

    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.

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

    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.

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

    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

  4. Product Platform Performance

    Munk, Lone

    The aim of this research is to improve understanding of platform-based product development by studying platform performance in relation to internal effects in companies. Platform-based product development makes it possible to deliver product variety and at the same time reduce the needed resources...... engaging in platform-based product development. Similarly platform assessment criteria lack empirical verification regarding relevance and sufficiency. The thesis focuses on • the process of identifying and estimating internal effects, • verification of performance of product platforms, (i...... experienced representatives from the different life systems phase systems of the platform products. The effects are estimated and modeled within different scenarios, taking into account financial and real option aspects. The model illustrates and supports estimation and quantification of internal platform...

  5. Mobile platform security

    Asokan, N; Dmitrienko, Alexandra

    2013-01-01

    Recently, mobile security has garnered considerable interest in both the research community and industry due to the popularity of smartphones. The current smartphone platforms are open systems that allow application development, also for malicious parties. To protect the mobile device, its user, and other mobile ecosystem stakeholders such as network operators, application execution is controlled by a platform security architecture. This book explores how such mobile platform security architectures work. We present a generic model for mobile platform security architectures: the model illustrat

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

    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

  7. Data Platforms and Cities

    Blok, Anders; Courmont, Antoine; Hoyng, Rolien

    2017-01-01

    This section offers a series of joint reflections on (open) data platform from a variety of cases, from cycling, traffic and mapping to activism, environment and data brokering. Data platforms play a key role in contemporary urban governance. Linked to open data initiatives, such platforms are of...

  8. Dynamic Gaming Platform (DGP)

    2009-04-01

    GAMING PLATFORM (DGP) Lockheed Martin Corporation...YYYY) APR 09 2. REPORT TYPE Final 3. DATES COVERED (From - To) Jul 07 – Mar 09 4. TITLE AND SUBTITLE DYNAMIC GAMING PLATFORM (DGP) 5a...CMU Carnegie Mellon University DGP Dynamic Gaming Platform GA Genetic Algorithm IARPA Intelligence Advanced Research Projects Activity LM ATL Lockheed Martin Advanced Technology Laboratories PAINT ProActive INTelligence

  9. ITS Platform North Denmark

    Lahrmann, Harry; Agerholm, Niels; Juhl, Jens

    2012-01-01

    This paper presents the project entitled “ITS Platform North Denmark” which is used as a test platform for Intelligent Transportation System (ITS) solutions. The platform consists of a newly developed GNSS/GPRS On Board Unit (OBU) to be installed in 500 cars, a backend server and a specially...

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

    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...

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

    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.

  12. Continuous Platform Development

    Nielsen, Ole Fiil

    low risks and investments but also with relatively fuzzy results. When looking for new platform projects, it is important to make sure that the company and market is ready for the introduction of platforms, and to make sure that people from marketing and sales, product development, and downstream......, but continuous product family evolution challenges this strategy. The concept of continuous platform development is based on the fact that platform development should not be a one-time experience but rather an ongoing process of developing new platforms and updating existing ones, so that product family...

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

    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

  14. Lab-on-a-disc platform for screening of genetically modified E. coli cells via cell-free electrochemical detection of p-Coumaric acid

    Sanger, Kuldeep; Zor, Kinga; Jendresen, Christian Bille

    2017-01-01

    filtration and electrochemical detection units, the sample filtration was performed by rotating the disc using a programmable closed-loop stepper motor. The electrodes, patterned on plastic substrate, were connected through a printed circuit board to the slip ring using a robust magnetic clamping system...

  15. Cross-Platform Technologies

    Maria Cristina ENACHE

    2017-04-01

    Full Text Available Cross-platform - a concept becoming increasingly used in recent years especially in the development of mobile apps, but this consistently over time and in the development of conventional desktop applications. The notion of cross-platform software (multi-platform or platform-independent refers to a software application that can run on more than one operating system or computing architecture. Thus, a cross-platform application can operate independent of software or hardware platform on which it is execute. As a generic definition presents a wide range of meanings for purposes of this paper we individualize this definition as follows: we will reduce the horizon of meaning and we use functionally following definition: a cross-platform application is a software application that can run on more than one operating system (desktop or mobile identical or in a similar way.

  16. ADMS Evaluation Platform

    2018-01-23

    Deploying an ADMS or looking to optimize its value? NREL offers a low-cost, low-risk evaluation platform for assessing ADMS performance. The National Renewable Energy Laboratory (NREL) has developed a vendor-neutral advanced distribution management system (ADMS) evaluation platform and is expanding its capabilities. The platform uses actual grid-scale hardware, large-scale distribution system models, and advanced visualization to simulate realworld conditions for the most accurate ADMS evaluation and experimentation.

  17. Platform development supportedby gaming

    Mikkola, Juliana Hsuan; Hansen, Poul H. Kyvsgård

    2007-01-01

    The challenge of implementing industrial platforms in practice can be described as a configuration problem caused by high number of variables, which often have contradictory influences on the total performance of the firm. Consequently, the specific platform decisions become extremely complex......, possibly increasing the strategic risks for the firm. This paper reports preliminary findings on platform management process at LEGO, a Danish toy company.  Specifically, we report the process of applying games combined with simulations and workshops in the platform development. We also propose a framework...

  18. Omnidirectional holonomic platforms

    Pin, F.G.; Killough, S.M.

    1994-01-01

    This paper presents the concepts for a new family of wheeled platforms which feature full omnidirectionality with simultaneous and independently controlled rotational and translational motion capabilities. The authors first present the orthogonal-wheels concept and the two major wheel assemblies on which these platforms are based. They then describe how a combination of these assemblies with appropriate control can be used to generate an omnidirectional capability for mobile robot platforms. The design and control of two prototype platforms are then presented and their respective characteristics with respect to rotational and translational motion control are discussed

  19. Platform decommissioning costs

    Rodger, David

    1998-01-01

    There are over 6500 platforms worldwide contributing to the offshore oil and gas production industry. In the North Sea there are around 500 platforms in place. There are many factors to be considered in planning for platform decommissioning and the evaluation of options for removal and disposal. The environmental impact, technical feasibility, safety and cost factors all have to be considered. This presentation considers what information is available about the overall decommissioning costs for the North Sea and the costs of different removal and disposal options for individual platforms. 2 figs., 1 tab

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

    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.

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

    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.

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

    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.).

  3. A robust robotic high-throughput antibody purification platform.

    Schmidt, Peter M; Abdo, Michael; Butcher, Rebecca E; Yap, Min-Yin; Scotney, Pierre D; Ramunno, Melanie L; Martin-Roussety, Genevieve; Owczarek, Catherine; Hardy, Matthew P; Chen, Chao-Guang; Fabri, Louis J

    2016-07-15

    Monoclonal antibodies (mAbs) have become the fastest growing segment in the drug market with annual sales of more than 40 billion US$ in 2013. The selection of lead candidate molecules involves the generation of large repertoires of antibodies from which to choose a final therapeutic candidate. Improvements in the ability to rapidly produce and purify many antibodies in sufficient quantities reduces the lead time for selection which ultimately impacts on the speed with which an antibody may transition through the research stage and into product development. Miniaturization and automation of chromatography using micro columns (RoboColumns(®) from Atoll GmbH) coupled to an automated liquid handling instrument (ALH; Freedom EVO(®) from Tecan) has been a successful approach to establish high throughput process development platforms. Recent advances in transient gene expression (TGE) using the high-titre Expi293F™ system have enabled recombinant mAb titres of greater than 500mg/L. These relatively high protein titres reduce the volume required to generate several milligrams of individual antibodies for initial biochemical and biological downstream assays, making TGE in the Expi293F™ system ideally suited to high throughput chromatography on an ALH. The present publication describes a novel platform for purifying Expi293F™-expressed recombinant mAbs directly from cell-free culture supernatant on a Perkin Elmer JANUS-VariSpan ALH equipped with a plate shuttle device. The purification platform allows automated 2-step purification (Protein A-desalting/size exclusion chromatography) of several hundred mAbs per week. The new robotic method can purify mAbs with high recovery (>90%) at sub-milligram level with yields of up to 2mg from 4mL of cell-free culture supernatant. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Groundwater Assessment Platform

    Podgorski, Joel; Berg, Michael

    2018-01-01

    The Groundwater Assessment Platform is a free, interactive online GIS platform for the mapping, sharing and statistical modeling of groundwater quality data. The modeling allows users to take advantage of publicly available global datasets of various environmental parameters to produce prediction maps of their contaminant of interest.

  5. EURESCOM Services Platform

    Nieuwenhuis, Lambertus Johannes Maria; van Halteren, Aart

    1999-01-01

    This paper presents the results of the EURESCOM Project 715. In February 1999, a large team of researchers from six European public network operators completed a two year period of cooperative experiments on a TINA-based environment, called the EURESCOM Services Platform (ESP). This platform

  6. Product Platform Modeling

    Pedersen, Rasmus

    for customisation of products. In many companies these changes in the business environment have created a controversy between the need for a wide variety of products offered to the marketplace and a desire to reduce variation within the company in order to increase efficiency. Many companies use the concept...... other. These groups can be varied and combined to form different product variants without increasing the internal variety in the company. Based on the Theory of Domains, the concept of encapsulation in the organ domain is introduced, and organs are formulated as platform elements. Included......This PhD thesis has the title Product Platform Modelling. The thesis is about product platforms and visual product platform modelling. Product platforms have gained an increasing attention in industry and academia in the past decade. The reasons are many, yet the increasing globalisation...

  7. Product Platform Replacements

    Sköld, Martin; Karlsson, Christer

    2012-01-01

    . To shed light on this unexplored and growing managerial concern, the purpose of this explorative study is to identify operational challenges to management when product platforms are replaced. Design/methodology/approach – The study uses a longitudinal field-study approach. Two companies, Gamma and Omega...... replacement was chosen in each company. Findings – The study shows that platform replacements primarily challenge managers' existing knowledge about platform architectures. A distinction can be made between “width” and “height” in platform replacements, and it is crucial that managers observe this in order...... to challenge their existing knowledge about platform architectures. Issues on technologies, architectures, components and processes as well as on segments, applications and functions are identified. Practical implications – Practical implications are summarized and discussed in relation to a framework...

  8. Methanol Metabolism in Yeasts : Regulation of the Synthesis of Catabolic Enzymes

    Egli, Th.; Dijken, J.P. van; Veenhuis, M.; Harder, W.; Fiechter, A.

    1980-01-01

    The regulation of the synthesis of four dissimilatory enzymes involved in methanol metabolism, namely alcohol oxidase, formaldehyde dehydrogenase, formate dehydrogenase and catalase was investigated in the yeasts Hansenula polymorpha and Kloeckera sp. 2201. Enzyme profiles in cell-free extracts of

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

    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.

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

    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.

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

    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

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

    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.

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

    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.

  14. The vacuum platform

    McNab, A.

    2017-10-01

    This paper describes GridPP’s Vacuum Platform for managing virtual machines (VMs), which has been used to run production workloads for WLCG and other HEP experiments. The platform provides a uniform interface between VMs and the sites they run at, whether the site is organised as an Infrastructure-as-a-Service cloud system such as OpenStack, or an Infrastructure-as-a-Client system such as Vac. The paper describes our experience in using this platform, in developing and operating VM lifecycle managers Vac and Vcycle, and in interacting with VMs provided by LHCb, ATLAS, ALICE, CMS, and the GridPP DIRAC service to run production workloads.

  15. The universal modular platform

    North, R.B.

    1995-01-01

    A new and patented design for offshore wellhead platforms has been developed to meet a 'fast track' requirement for increased offshore production, from field locations not yet identified. The new design uses modular construction to allow for radical changes in the water depth of the final location and assembly line efficiency in fabrication. By utilizing high strength steels and structural support from the well conductors the new design accommodates all planned production requirements on a support structure significantly lighter and less expensive than the conventional design it replaces. Twenty two platforms based on the new design were ready for installation within 18 months of the project start. Installation of the new platforms began in 1992 for drilling support and 1993 for production support. The new design has become the Company standard for all future production platforms. Large saving and construction costs have been realized through its light weight, flexibility in both positioning and water depth, and its modular construction

  16. Identification of platform levels

    Mortensen, Niels Henrik

    2005-01-01

    reduction, ability to launch a wider product portfolio without increasing resources and reduction of complexity within the whole company. To support the multiple product development process, platform based product development has in many companies such as Philips, VW, Ford etc. proven to be a very effective...... product development in one step and therefore the objective of this paper is to identify levels of platform based product development. The structure of this paper is as follows. First the applied terminology for platforms will be briefly explained and then characteristics between single and multi product...... development will be examined. Based on the identification of the above characteristics five platform levels are described. The research presented in this paper is a result of MSc, Ph.D projects at the Technical University of Denmark and consultancy projects within the organisation of Institute of Product...

  17. Paper based electronics platform

    Nassar, Joanna Mohammad; Sevilla, Galo Andres Torres; Hussain, Muhammad Mustafa

    2017-01-01

    A flexible and non-functionalized low cost paper-based electronic system platform fabricated from common paper, such as paper based sensors, and methods of producing paper based sensors, and methods of sensing using the paper based sensors

  18. USA Hire Testing Platform

    Office of Personnel Management — The USA Hire Testing Platform delivers tests used in hiring for positions in the Federal Government. To safeguard the integrity of the hiring processes and ensure...

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

    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.

  20. National Community Solar Platform

    Rupert, Bart [Clean Energy Collective, Louisville, CO (United States)

    2016-06-30

    This project was created to provide a National Community Solar Platform (NCSP) portal known as Community Solar Hub, that is available to any entity or individual who wants to develop community solar. This has been done by providing a comprehensive portal to make CEC’s solutions, and other proven community solar solutions, externally available for everyone to access – making the process easy through proven platforms to protect subscribers, developers and utilities. The successful completion of this project provides these tools via a web platform and integration APIs, a wide spectrum of community solar projects included in the platform, multiple groups of customers (utilities, EPCs, and advocates) using the platform to develop community solar, and open access to anyone interested in community solar. CEC’s Incubator project includes web-based informational resources, integrated systems for project information and billing systems, and engagement with customers and users by community solar experts. The combined effort externalizes much of Clean Energy Collective’s industry-leading expertise, allowing third parties to develop community solar without duplicating expensive start-up efforts. The availability of this platform creates community solar projects that are cheaper to build and cheaper to participate in, furthering the goals of DOE’s SunShot Initiative. Final SF 425 Final SF 428 Final DOE F 2050.11 Final Report Narrative

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

    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.)

  2. Mini-review: In vitro Metabolic Engineering for Biomanufacturing of High-value Products

    Weihua Guo

    Full Text Available With the breakthroughs in biomolecular engineering and synthetic biology, many valuable biologically active compound and commodity chemicals have been successfully manufactured using cell-based approaches in the past decade. However, because of the high complexity of cell metabolism, the identification and optimization of rate-limiting metabolic pathways for improving the product yield is often difficult, which represents a significant and unavoidable barrier of traditional in vivo metabolic engineering. Recently, some in vitro engineering approaches were proposed as alternative strategies to solve this problem. In brief, by reconstituting a biosynthetic pathway in a cell-free environment with the supplement of cofactors and substrates, the performance of each biosynthetic pathway could be evaluated and optimized systematically. Several value-added products, including chemicals, nutraceuticals, and drug precursors, have been biosynthesized as proof-of-concept demonstrations of in vitro metabolic engineering. This mini-review summarizes the recent progresses on the emerging topic of in vitro metabolic engineering and comments on the potential application of cell-free technology to speed up the “design-build-test” cycles of biomanufacturing. Keywords: Cell-free, Biosynthesis, Metabolic pathways, Design-build-test cycle

  3. Cell-free spent media obtained from Bifidobacterium bifidum and Bifidobacterium crudilactis grown in media supplemented with 3’-sialyllactose exert virulence modulation on intestinal pathogens

    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.

  4. The Platformization of the Web: Making Web Data Platform Ready

    Helmond, A.

    2015-01-01

    In this article, I inquire into Facebook’s development as a platform by situating it within the transformation of social network sites into social media platforms. I explore this shift with a historical perspective on, what I refer to as, platformization, or the rise of the platform as the dominant

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

    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. Transactional Network Platform: Applications

    Katipamula, Srinivas; Lutes, Robert G.; Ngo, Hung; Underhill, Ronald M.

    2013-10-31

    In FY13, Pacific Northwest National Laboratory (PNNL) with funding from the Department of Energy’s (DOE’s) Building Technologies Office (BTO) designed, prototyped and tested a transactional network platform to support energy, operational and financial transactions between any networked entities (equipment, organizations, buildings, grid, etc.). Initially, in FY13, the concept demonstrated transactions between packaged rooftop air conditioners and heat pump units (RTUs) and the electric grid using applications or "agents" that reside on the platform, on the equipment, on a local building controller or in the Cloud. The transactional network project is a multi-lab effort with Oakridge National Laboratory (ORNL) and Lawrence Berkeley National Laboratory (LBNL) also contributing to the effort. PNNL coordinated the project and also was responsible for the development of the transactional network (TN) platform and three different applications associated with RTUs. This document describes two applications or "agents" in details, and also summarizes the platform. The TN platform details are described in another companion document.

  7. Platform-based production development

    Bossen, Jacob; Brunoe, Thomas Ditlev; Nielsen, Kjeld

    2015-01-01

    Platforms as a means for applying modular thinking in product development is relatively well studied, but platforms in the production system has until now not been given much attention. With the emerging concept of platform-based co-development the importance of production platforms is though...

  8. In vitro flow cytometry-based screening platform for cellulase engineering

    Körfer, Georgette; Pitzler, Christian; Vojcic, Ljubica; Martinez, Ronny; Schwaneberg, Ulrich

    2016-01-01

    Ultrahigh throughput screening (uHTS) plays an essential role in directed evolution for tailoring biocatalysts for industrial applications. Flow cytometry-based uHTS provides an efficient coverage of the generated protein sequence space by analysis of up to 107 events per hour. Cell-free enzyme production overcomes the challenge of diversity loss during the transformation of mutant libraries into expression hosts, enables directed evolution of toxic enzymes, and holds the promise to efficiently design enzymes of human or animal origin. The developed uHTS cell-free compartmentalization platform (InVitroFlow) is the first report in which a flow cytometry-based screened system has been combined with compartmentalized cell-free expression for directed cellulase enzyme evolution. InVitroFlow was validated by screening of a random cellulase mutant library employing a novel screening system (based on the substrate fluorescein-di-β-D-cellobioside), and yielded significantly improved cellulase variants (e.g. CelA2-H288F-M1 (N273D/H288F/N468S) with 13.3-fold increased specific activity (220.60 U/mg) compared to CelA2 wildtype: 16.57 U/mg). PMID:27184298

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

    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.

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

    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...

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

    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....

  12. Reusable platform concepts

    Gudmestad, O.T.; Sparby, B.K.; Stead, B.L.

    1993-01-01

    There is an increasing need to reduce costs of offshore production facilities in order to make development of offshore fields profitable. For small fields with short production time there is in particular a need to investigate ways to reduce costs. The idea of platform reuse is for such fields particularly attractive. This paper will review reusable platform concepts and will discuss their range of application. Particular emphasis will be placed on technical limitations. Traditional concepts as jackups and floating production facilities will be discussed by major attention will be given to newly developed ideas for reuse of steel jackets and concrete structures. It will be shown how the operator for several fields can obtain considerable savings by applying such reusable platform concepts

  13. Wireless sensor platform

    Joshi, Pooran C.; Killough, Stephen M.; Kuruganti, Phani Teja

    2017-08-08

    A wireless sensor platform and methods of manufacture are provided. The platform involves providing a plurality of wireless sensors, where each of the sensors is fabricated on flexible substrates using printing techniques and low temperature curing. Each of the sensors can include planar sensor elements and planar antennas defined using the printing and curing. Further, each of the sensors can include a communications system configured to encode the data from the sensors into a spread spectrum code sequence that is transmitted to a central computer(s) for use in monitoring an area associated with the sensors.

  14. Windows Azure Platform

    Redkar, Tejaswi

    2010-01-01

    The Azure Services Platform is a brand-new cloud-computing technology from Microsoft. It is composed of four core components-Windows Azure, .NET Services, SQL Services, and Live Services-each with a unique role in the functioning of your cloud service. It is the goal of this book to show you how to use these components, both separately and together, to build flawless cloud services. At its heart Windows Azure Platform is a down-to-earth, code-centric book. This book aims to show you precisely how the components are employed and to demonstrate the techniques and best practices you need to know

  15. Windows Azure Platform

    Redkar, Tejaswi

    2011-01-01

    The Windows Azure Platform has rapidly established itself as one of the most sophisticated cloud computing platforms available. With Microsoft working to continually update their product and keep it at the cutting edge, the future looks bright - if you have the skills to harness it. In particular, new features such as remote desktop access, dynamic content caching and secure content delivery using SSL make the latest version of Azure a more powerful solution than ever before. It's widely agreed that cloud computing has produced a paradigm shift in traditional architectural concepts by providin

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

    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.

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

    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.

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

    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.

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

    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...

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

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

    2018-01-04

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

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

    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.

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

    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.

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

    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.

  10. The Creative Platform

    Byrge, Christian; Hansen, Søren

    whether you consider thirdgrade teaching, human-resource development, or radical new thinking in product development in a company. The Creative Platform was developed at Aalborg University through a series of research-and-development activities in collaboration with educational institutions and private...

  11. Creative Platform Learning (CPL)

    Christensen, Jonna Langeland; Hansen, Søren

    Creative Platform Learning (CPL) er en pædagogisk metode, der skaber foretagsomme og innovative elever, der kan anvende deres kreativitet til at lære nyt. Ifølge den nye skolereform skal Innovation og entreprenørskab tydeliggøres i alle fag. I CPL er det en integreret del af undervisningen...

  12. Games and Platform Decisions

    Hansen, Poul H. Kyvsgård; Mikkola, Juliana Hsuan

    2007-01-01

    is the application of on-line games in order to provide training for decision makers and in order to generate overview over the implications of platform decisions. However, games have to be placed in a context with other methods and we argue that a mixture of games, workshops, and simulations can provide improved...

  13. Shot loading platform analysis

    Norman, B.F.

    1994-01-01

    This document provides the wind/seismic analysis and evaluation for the shot loading platform. Hand calculations were used for the analysis. AISC and UBC load factors were used in this evaluation. The results show that the actual loads are under the allowable loads and all requirements are met

  14. Brake for rollable platform

    Morris, A. L.

    1974-01-01

    Frame-mounted brake is independent of wheels and consists of simple lever-actuated foot. Brake makes good contact with surface even though foot pad is at higher or lower level than wheels, this is particularly important when a rollable platform is used on irregular surface.

  15. CERN Neutrino Platform Hardware

    Nelson, Kevin

    2017-01-01

    My summer research was broadly in CERN's neutrino platform hardware efforts. This project had two main components: detector assembly and data analysis work for ICARUS. Specifically, I worked on assembly for the ProtoDUNE project and monitored the safety of ICARUS as it was transported to Fermilab by analyzing the accelerometer data from its move.

  16. Mobile Platforms and Development Environments

    Helal, Sumi; Li, Wengdong

    2012-01-01

    Mobile platform development has lately become a technological war zone with extremely dynamic and fluid movement, especially in the smart phone and tablet market space. This Synthesis lecture is a guide to the latest developments of the key mobile platforms that are shaping the mobile platform industry. The book covers the three currently dominant native platforms -- iOS, Android and Windows Phone -- along with the device-agnostic HTML5 mobile web platform. The lecture also covers location-based services (LBS) which can be considered as a platform in its own right. The lecture utilizes a sampl

  17. Cyanobacteria as a platform for biofuel production

    Nicole E Nozzi

    2013-09-01

    Full Text Available Cyanobacteria have great potential as a platform for biofuel production because of their fast growth, ability to fix carbon dioxide gas, and their genetic tractability. Furthermore they do not require fermentable sugars or arable land for growth and so competition with cropland would be greatly reduced. In this perspective we discuss the challenges and areas for improvement most pertinent for advancing cyanobacterial fuel production, including: improving genetic parts, carbon fixation, metabolic flux, nutrient requirements on a large scale, and photosynthetic efficiency using natural light.

  18. Metabolic Syndrome

    Metabolic syndrome is a group of conditions that put you at risk for heart disease and diabetes. These conditions ... agree on the definition or cause of metabolic syndrome. The cause might be insulin resistance. Insulin is ...

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

    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

  20. ENERCA: e-learning platform

    Patricia Aguilar-Martínez

    2014-12-01

    Full Text Available ENERCA (the European Network for Rare and Congenital Anemias is a European Commission funded project since 2002. Rare anaemias in Europe comprise haemoglobin disorders, such as thalassemias or sickle cell disease, and other rarer disorders, including inherited diseases of the red cell membrane, of the red cell enzymes, as well as anaemias related to disorders of iron metabolism. Education and training on rare anaemias is one of the main objectives of ENERCA and several educational initiatives have been undertaken during the past and present phases of the project. Among these, the organisation of courses for health professionals, the provision of videos with prevention messages for patients and general practitioners were part of the previous phase, ENERCA 3. In the current phase of the ENERCA project, called e-ENERCA, the workpackage dedicated to Education and Training mainly focuses on training of young physicians, biologists or scientists in the field of diagnosis and management of rare anaemias. The specificity of e-ENERCA is to be based principally on new communication e-tool. Two main educational axes will be developed: the organisation of on-site courses including interactive sessions with voting boxes and the implementation of an online e-learning platform.

  1. Metabolic interactions between cysteamine and epigallocatechin gallate.

    Izzo, Valentina; Pietrocola, Federico; Sica, Valentina; Durand, Sylvère; Lachkar, Sylvie; Enot, David; Bravo-San Pedro, José Manuel; Chery, Alexis; Esposito, Speranza; Raia, Valeria; Maiuri, Luigi; Maiuri, Maria Chiara; Kroemer, Guido

    2017-02-01

    Phase II clinical trials indicate that the combination of cysteamine plus epigallocatechin gallate (EGCG) is effective against cystic fibrosis in patients bearing the most frequent etiological mutation (CFTRΔF508). Here, we investigated the interaction between both agents on cultured respiratory epithelia cells from normal and CFTRΔF508-mutated donors. We observed that the combination of both agents affected metabolic circuits (and in particular the tricarboxylic acid cycle) in a unique way and that cysteamine plus EGCG reduced cytoplasmic protein acetylation more than each of the 2 components alone. In a cell-free system, protein cross-linking activity of EGCG was suppressed by cysteamine. Finally, EGCG was able to enhance the conversion of cysteamine into taurine in metabolic flux experiments. Altogether, these results indicate that multiple pharmacological interactions occur between cysteamine and EGCG, suggesting that they contribute to the unique synergy of both agents in restoring the function of mutated CFTRΔF508.

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

    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.

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

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

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

    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.

  11. Platform Performance and Challenges - using Platforms in Lego Company

    Munk, Lone; Mortensen, Niels Henrik

    2009-01-01

    needs focus on the incentive of using the platform. This problem lacks attention in literature, as well as industry, where assessment criteria do not cover this aspect. Therefore, we recommend including user incentive in platform assessment criteria to these challenges. Concrete solution elements...... ensuring user incentive in platforms is an object for future research...

  12. Available: motorised platform

    The COMPASS collaboration

    2014-01-01

    The COMPASS collaboration would like to offer to a new owner the following useful and fully operational piece of equipment, which is due to be replaced with better adapted equipment.   Please contact Erwin Bielert (erwin.bielert@cern.ch or 160539) for further information.  Motorized platform (FOR FREE):   Fabricated by ACL (Alfredo Cardoso & Cia Ltd) in Portugal. The model number is MeXs 5-­‐30.  Specifications: 5 m wide, 1 m deep, adjustable height (1.5 m if folded). Maximum working floor height: 4 m. conforms to CERN regulations, number LV158. Type LD500, capacity 500 kg and weight 2000 kg.  If no interested party is found before December 2014, the platform will be thrown away.

  13. RemoteLabs Platform

    Nils Crabeel

    2012-03-01

    Full Text Available This paper reports on a first step towards the implementation of a framework for remote experimentation of electric machines – the RemoteLabs platform. This project was focused on the development of two main modules: the user Web-based and the electric machines interfaces. The Web application provides the user with a front-end and interacts with the back-end – the user and experiment persistent data. The electric machines interface is implemented as a distributed client server application where the clients, launched by the Web application, interact with the server modules located in platforms physically connected the electric machines drives. Users can register and authenticate, schedule, specify and run experiments and obtain results in the form of CSV, XML and PDF files. These functionalities were successfully tested with real data, but still without including the electric machines. This inclusion is part of another project scheduled to start soon.

  14. Common tester platform concept.

    Hurst, Michael James

    2008-05-01

    This report summarizes the results of a case study on the doctrine of a common tester platform, a concept of a standardized platform that can be applicable across the broad spectrum of testing requirements throughout the various stages of a weapons program, as well as across the various weapons programs. The common tester concept strives to define an affordable, next-generation design that will meet testing requirements with the flexibility to grow and expand; supporting the initial development stages of a weapons program through to the final production and surveillance stages. This report discusses a concept investing key leveraging technologies and operational concepts combined with prototype tester-development experiences and practical lessons learned gleaned from past weapons programs.

  15. Online stock trading platform

    Ion LUNGU

    2006-01-01

    Full Text Available The Internet is the perfect tool that can assure the market’s transparency for any user who wants to trade on the stock market. The investor can have access to the market news, financial calendar or the press releases of the issuers. A good online trading platform also provides real-time intraday quotes, trading history and technical analysis giving the investor a clearer view of the supply and demand in the market. All this information provides the investor a good image of the market and encourages him to trade. This paper wishes to draft the pieces of an online trading platform and to analyze the impact of developing and implementing one in a brokerage firm.

  16. HPC - Platforms Penta Chart

    Trujillo, Angelina Michelle [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2015-10-08

    Strategy, Planning, Acquiring- very large scale computing platforms come and go and planning for immensely scalable machines often precedes actual procurement by 3 years. Procurement can be another year or more. Integration- After Acquisition, machines must be integrated into the computing environments at LANL. Connection to scalable storage via large scale storage networking, assuring correct and secure operations. Management and Utilization – Ongoing operations, maintenance, and trouble shooting of the hardware and systems software at massive scale is required.

  17. Cloud Robotics Platforms

    Busra Koken

    2015-01-01

    Full Text Available Cloud robotics is a rapidly evolving field that allows robots to offload computation-intensive and storage-intensive jobs into the cloud. Robots are limited in terms of computational capacity, memory and storage. Cloud provides unlimited computation power, memory, storage and especially collaboration opportunity. Cloud-enabled robots are divided into two categories as standalone and networked robots. This article surveys cloud robotic platforms, standalone and networked robotic works such as grasping, simultaneous localization and mapping (SLAM and monitoring.

  18. The Prodiguer Messaging Platform

    Denvil, S.; Greenslade, M. A.; Carenton, N.; Levavasseur, G.; Raciazek, J.

    2015-12-01

    CONVERGENCE is a French multi-partner national project designed to gather HPC and informatics expertise to innovate in the context of running French global climate models with differing grids and at differing resolutions. Efficient and reliable execution of these models and the management and dissemination of model output are some of the complexities that CONVERGENCE aims to resolve.At any one moment in time, researchers affiliated with the Institut Pierre Simon Laplace (IPSL) climate modeling group, are running hundreds of global climate simulations. These simulations execute upon a heterogeneous set of French High Performance Computing (HPC) environments. The IPSL's simulation execution runtime libIGCM (library for IPSL Global Climate Modeling group) has recently been enhanced so as to support hitherto impossible realtime use cases such as simulation monitoring, data publication, metrics collection, simulation control, visualizations … etc. At the core of this enhancement is Prodiguer: an AMQP (Advanced Message Queue Protocol) based event driven asynchronous distributed messaging platform. libIGCM now dispatches copious amounts of information, in the form of messages, to the platform for remote processing by Prodiguer software agents at IPSL servers in Paris. Such processing takes several forms: Persisting message content to database(s); Launching rollback jobs upon simulation failure; Notifying downstream applications; Automation of visualization pipelines; We will describe and/or demonstrate the platform's: Technical implementation; Inherent ease of scalability; Inherent adaptiveness in respect to supervising simulations; Web portal receiving simulation notifications in realtime.

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

    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...

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

    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.

  1. Utilizing platforms in industrialized construction

    Bonev, Martin; Wörösch, Michael; Hvam, Lars

    2015-01-01

    platform strategies, this researchhighlights key aspects of adapting platform-based developed theory to industrialised construction.Building projects use different layers of product, process and logistics platforms to form the right cost– value ratio for the target market application, while modelling...

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

    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...

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

    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...

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

    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.

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

    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.

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

    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.

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

    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

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

    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

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

    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

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

    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

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

    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.

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

    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

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

    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

  14. Microbial metabolomics in open microscale platforms

    Barkal, Layla J.; Theberge, Ashleigh B.; Guo, Chun-Jun; Spraker, Joe; Rappert, Lucas; Berthier, Jean; Brakke, Kenneth A.; Wang, Clay C. C.; Beebe, David J.; Keller, Nancy P.; Berthier, Erwin

    2016-01-01

    The microbial secondary metabolome encompasses great synthetic diversity, empowering microbes to tune their chemical responses to changing microenvironments. Traditional metabolomics methods are ill-equipped to probe a wide variety of environments or environmental dynamics. Here we introduce a class of microscale culture platforms to analyse chemical diversity of fungal and bacterial secondary metabolomes. By leveraging stable biphasic interfaces to integrate microculture with small molecule isolation via liquid–liquid extraction, we enable metabolomics-scale analysis using mass spectrometry. This platform facilitates exploration of culture microenvironments (including rare media typically inaccessible using established methods), unusual organic solvents for metabolite isolation and microbial mutants. Utilizing Aspergillus, a fungal genus known for its rich secondary metabolism, we characterize the effects of culture geometry and growth matrix on secondary metabolism, highlighting the potential use of microscale systems to unlock unknown or cryptic secondary metabolites for natural products discovery. Finally, we demonstrate the potential for this class of microfluidic systems to study interkingdom communication between fungi and bacteria. PMID:26842393

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

    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.

  16. The CERN Neutrino Platform

    Bordoni, Stefania

    2018-01-01

    The long-baseline neutrino programme has been classified as one of the four highest-priority sci- entific objectives in 2013 by the European Strategy for Particle Physics. The Neutrino Platform is the CERN venture to foster and support the next generation of accelerator-based neutrino os- cillation experiments. Part of the present CERN Medium-Term Plan, the Neutrino Platform provide facilities to develop and prototype the next generation of neutrino detectors and contribute to unify the European neu- trino community towards the US and Japanese projects. A significative effort is made on R&D; for LAr TPC technologies: two big LAr TPC prototypes for the DUNE far detector are under con- struction at CERN. Those detectors will be exposed in 2018 to an entirely new and NP-dedicated beam-line from the SPS which will provide electron, muon and hadron beams with energies in the range of sub-GeV to a few GeV. Other projects are also presently under development: one can cite the refurbishing and shipping to the US ...

  17. What is Metabolic Syndrome?

    ... Intramural Research Home / Metabolic Syndrome Metabolic Syndrome Also known as What Is Metabolic syndrome ... metabolic risk factors to be diagnosed with metabolic syndrome. Metabolic Risk Factors A Large Waistline Having a large ...

  18. An integrated biotechnology platform for developing sustainable chemical processes.

    Barton, Nelson R; Burgard, Anthony P; Burk, Mark J; Crater, Jason S; Osterhout, Robin E; Pharkya, Priti; Steer, Brian A; Sun, Jun; Trawick, John D; Van Dien, Stephen J; Yang, Tae Hoon; Yim, Harry

    2015-03-01

    Genomatica has established an integrated computational/experimental metabolic engineering platform to design, create, and optimize novel high performance organisms and bioprocesses. Here we present our platform and its use to develop E. coli strains for production of the industrial chemical 1,4-butanediol (BDO) from sugars. A series of examples are given to demonstrate how a rational approach to strain engineering, including carefully designed diagnostic experiments, provided critical insights about pathway bottlenecks, byproducts, expression balancing, and commercial robustness, leading to a superior BDO production strain and process.

  19. Preparing for a Product Platform

    Fiil-Nielsen, Ole; Munk, Lone; Mortensen, Niels Henrik

    2005-01-01

    on commonalities and similarities in the product family, and variance should be based on customer demands. To relate these terms and to improve the basis on which decisions are made, we need a way of visualizing the hierarchy of the product family as well as the commonality and variance. This visualization method...... of the platform or ensuring that the platform can meet future demands will be very useful in the preparation process of a platform synthesis as well as in the updating or reengineering of an existing product development platform.......Experience in the industry as well as recent related scientific publications show the benefits of product development platforms. Companies use platforms to develop not a single but multiple products (i.e. a product family) simultaneously. When these product development projects are coordinated...

  20. Adoption of Mobile Payment Platforms

    Staykova, Kalina Stefanova; Damsgaard, Jan

    2016-01-01

    Numerous mobile payment solutions, which rely on new disruptive technologies, have been launched on the payment market in recent years. But despite the growing number of mobile payment apps, very few solutions have turned to be successful as the majority of them fail to gain a critical mass...... of users. In this paper, we investigate successful platform adoption strategies by using the Reach and Range Framework for Multi-Sided Platforms as a strategic tool to which mobile payment providers can adhere in order to tackle some of the main challenges they face throughout the evolution...... of their platforms. The analysis indicates that successful mobile payment solutions tend to be launched as one-sided platforms and then gradually be expanded into being two-sided. Our study showcases that the success of mobile payment platforms lies with the ability of the platform to balance the reach (number...

  1. Web Platform Application

    Paulsworth, Ashley [Sunvestment Group, Frederick, MD (United States); Kurtz, Jim [Sunvestment Group, Frederick, MD (United States); Brun de Pontet, Stephanie [Sunvestment Group, Frederick, MD (United States)

    2016-06-15

    Sunvestment Energy Group (previously called Sunvestment Group) was established to create a web application that brings together site hosts, those who will obtain the energy from the solar array, with project developers and funders, including affinity investors. Sunvestment Energy Group (SEG) uses a community-based model that engages with investors who have some affinity with the site host organization. In addition to a financial return, these investors receive non-financial value from their investments and are therefore willing to offer lower cost capital. This enables the site host to enjoy more savings from solar through these less expensive Community Power Purchase Agreements (CPPAs). The purpose of this award was to develop an online platform to bring site hosts and investors together virtually.

  2. Mobile4D platform

    Botha, Adèle

    2010-05-01

    Full Text Available and share their own Internet/Telco service Mashups. digm. This all 2.1.2  SPICE   Service Platform for Innovative Communication Environment was also a European Union’s Sixth Framework Programme (FP6) funded project, which formed a consortium consisting...  OPUCE  SPICE  Twisted   Mobicents  Telco  specific  Minimal Total Life cycle cost  ?  ?  ?  ?  ?  Standards Compliant Solution  ?  ?  ?  ?  ?  Bearer & Device Agnostic  ?  ?  ?  ?  ?  Ease of use and accessibility  ?  ?  ?  ?  ?  Synergies...

  3. Geometric information provider platform

    Meisam Yousefzadeh

    2015-07-01

    Full Text Available Renovation of existing buildings is known as an essential stage in reduction of the energy loss. Considerable part of renovation process depends on geometric reconstruction of building based on semantic parameters. Following many research projects which were focused on parameterizing the energy usage, various energy modelling methods were developed during the last decade. On the other hand, by developing accurate measuring tools such as laser scanners, the interests of having accurate 3D building models are rapidly growing. But the automation of 3D building generation from laser point cloud or detection of specific objects in that is still a challenge.  The goal is designing a platform through which required geometric information can be efficiently produced to support energy simulation software. Developing a reliable procedure which extracts required information from measured data and delivers them to a standard energy modelling system is the main purpose of the project.

  4. Energy Tracking Software Platform

    Ryan Davis; Nathan Bird; Rebecca Birx; Hal Knowles

    2011-04-04

    Acceleration has created an interactive energy tracking and visualization platform that supports decreasing electric, water, and gas usage. Homeowners have access to tools that allow them to gauge their use and track progress toward a smaller energy footprint. Real estate agents have access to consumption data, allowing for sharing a comparison with potential home buyers. Home builders have the opportunity to compare their neighborhood's energy efficiency with competitors. Home energy raters have a tool for gauging the progress of their clients after efficiency changes. And, social groups are able to help encourage members to reduce their energy bills and help their environment. EnergyIT.com is the business umbrella for all energy tracking solutions and is designed to provide information about our energy tracking software and promote sales. CompareAndConserve.com (Gainesville-Green.com) helps homeowners conserve energy through education and competition. ToolsForTenants.com helps renters factor energy usage into their housing decisions.

  5. Sinking offshore platform. Nedsenkbar fralandsplatform

    Einstabland, T.B.; Olsen, O.

    1988-12-19

    The invention deals with a sinking offshore platform of the gravitational type designed for being installed on the sea bed on great depths. The platform consists of at least three inclining pillars placed on a foundation unit. The pillars are at the upper end connected to a tower structure by means of a rigid construction. The tower supports the platform deck. The rigid construction comprises a centre-positioned cylinder connected to the foundation. 11 figs.

  6. Product Platform Screening at LEGO

    Mortensen, Niels Henrik; Steen Jensen, Thomas; Nielsen, Ole Fiil

    2012-01-01

    Product platforms offer great benefits to companies developing new products in highly competitive markets. Literature describes how a single platform can be designed from a technical point of view, but rarely mentions how the process begins. How do companies identify possible platform candidates...... after a few changes had been applied to the initial process layout. This case study shows how companies must focus on a limited selection of simultaneous projects in order to keep focus. Primary stakeholders must be involved from the very beginning, and short presentations of the platform concepts...

  7. Flexible experimental FPGA based platform

    Andersen, Karsten Holm; Nymand, Morten

    2016-01-01

    This paper presents an experimental flexible Field Programmable Gate Array (FPGA) based platform for testing and verifying digital controlled dc-dc converters. The platform supports different types of control strategies, dc-dc converter topologies and switching frequencies. The controller platform...... interface supporting configuration and reading of setup parameters, controller status and the acquisition memory in a simple way. The FPGA based platform, provides an easy way within education or research to use different digital control strategies and different converter topologies controlled by an FPGA...

  8. Introducing Platform Interactions Model for Studying Multi-Sided Platforms

    Staykova, Kalina; Damsgaard, Jan

    2018-01-01

    Multi-Sided Platforms (MSPs) function as socio-technical entities that facilitate direct interactions between various affiliated to them constituencies through developing and managing IT architecture. In this paper, we aim to explain the nature of the platform interactions as key characteristic o...

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

    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.

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

    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.

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

    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.

  12. [Metabolic acidosis].

    Regolisti, Giuseppe; Fani, Filippo; Antoniotti, Riccardo; Castellano, Giuseppe; Cremaschi, Elena; Greco, Paolo; Parenti, Elisabetta; Morabito, Santo; Sabatino, Alice; Fiaccadori, Enrico

    2016-01-01

    Metabolic acidosis is frequently observed in clinical practice, especially among critically ill patients and/or in the course of renal failure. Complex mechanisms are involved, in most cases identifiable by medical history, pathophysiology-based diagnostic reasoning and measure of some key acid-base parameters that are easily available or calculable. On this basis the bedside differential diagnosis of metabolic acidosis should be started from the identification of the two main subtypes of metabolic acidosis: the high anion gap metabolic acidosis and the normal anion gap (or hyperchloremic) metabolic acidosis. Metabolic acidosis, especially in its acute forms with elevated anion gap such as is the case of lactic acidosis, diabetic and acute intoxications, may significantly affect metabolic body homeostasis and patients hemodynamic status, setting the stage for true medical emergencies. The therapeutic approach should be first aimed at early correction of concurrent clinical problems (e.g. fluids and hemodynamic optimization in case of shock, mechanical ventilation in case of concomitant respiratory failure, hemodialysis for acute intoxications etc.), in parallel to the formulation of a diagnosis. In case of severe acidosis, the administration of alkalizing agents should be carefully evaluated, taking into account the risk of side effects, as well as the potential need of renal replacement therapy.

  13. Pre-analytical variables of circulating cell-free nucleosomes containing 5-methylcytosine DNA or histone modification H3K9Me3

    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....

  14. A practice scaffolding interactive platform

    Bundsgaard, Jeppe

    2009-01-01

    A Practice Scaffolding Interactive Platform (PracSIP) is a social learning platform which supports students in collaborative project based learning by simulating a professional practice. A PracSIP puts the core tools of the simulated practice at the students' disposal, it organizes collaboration...

  15. 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.

    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

  16. Paper based electronics platform

    Nassar, Joanna Mohammad

    2017-07-20

    A flexible and non-functionalized low cost paper-based electronic system platform fabricated from common paper, such as paper based sensors, and methods of producing paper based sensors, and methods of sensing using the paper based sensors are provided. A method of producing a paper based sensor can include the steps of: a) providing a conventional paper product to serve as a substrate for the sensor or as an active material for the sensor or both, the paper product not further treated or functionalized; and b) applying a sensing element to the paper substrate, the sensing element selected from the group consisting of a conductive material, the conductive material providing contacts and interconnects, sensitive material film that exhibits sensitivity to pH levels, a compressible and/or porous material disposed between a pair of opposed conductive elements, or a combination of two of more said sensing elements. The method of sensing can further include measuring, using the sensing element, a change in resistance, a change in voltage, a change in current, a change in capacitance, or a combination of any two or more thereof.

  17. Drug Metabolism

    Home; Journals; Resonance – Journal of Science Education; Volume 19; Issue 3. Drug Metabolism: A Fascinating Link Between Chemistry and Biology. Nikhil Taxak Prasad V Bharatam. General Article Volume 19 Issue 3 March 2014 pp 259-282 ...

  18. Drug Metabolism

    IAS Admin

    behind metabolic reactions, importance, and consequences with several ... required for drug action. ... lism, which is catalyzed by enzymes present in the above-men- ... catalyze the transfer of one atom of oxygen to a substrate produc-.

  19. Metabolic Myopathies.

    Tarnopolsky, Mark A

    2016-12-01

    Metabolic myopathies are genetic disorders that impair intermediary metabolism in skeletal muscle. Impairments in glycolysis/glycogenolysis (glycogen-storage disease), fatty acid transport and oxidation (fatty acid oxidation defects), and the mitochondrial respiratory chain (mitochondrial myopathies) represent the majority of known defects. The purpose of this review is to develop a diagnostic and treatment algorithm for the metabolic myopathies. The metabolic myopathies can present in the neonatal and infant period as part of more systemic involvement with hypotonia, hypoglycemia, and encephalopathy; however, most cases present in childhood or in adulthood with exercise intolerance (often with rhabdomyolysis) and weakness. The glycogen-storage diseases present during brief bouts of high-intensity exercise, whereas fatty acid oxidation defects and mitochondrial myopathies present during a long-duration/low-intensity endurance-type activity or during fasting or another metabolically stressful event (eg, surgery, fever). The clinical examination is often normal between acute events, and evaluation involves exercise testing, blood testing (creatine kinase, acylcarnitine profile, lactate, amino acids), urine organic acids (ketones, dicarboxylic acids, 3-methylglutaconic acid), muscle biopsy (histology, ultrastructure, enzyme testing), MRI/spectroscopy, and targeted or untargeted genetic testing. Accurate and early identification of metabolic myopathies can lead to therapeutic interventions with lifestyle and nutritional modification, cofactor treatment, and rapid treatment of rhabdomyolysis.

  20. Animal metabolism

    Walburg, H.E.

    1977-01-01

    Studies on placental transport included the following: clearance of tritiated water as a baseline measurement for transport of materials across perfused placentas; transport of organic and inorganic mercury across the perfused placenta of the guinea pig in late gestation; and transport of cadmium across the perfused placenta of the guinea pig in late gestation. Studies on cadmium absorption and metabolism included the following: intestinal absorption and retention of cadmium in neonatal rats; uptake and distribution of an oral dose of cadmium in postweanling male and female, iron-deficient and normal rats; postnatal viability and growth in rat pups after oral cadmium administration during gestation; and the effect of calcium and phosphorus on the absorption and toxicity of cadmium. Studies on gastrointestinal absorption and mineral metabolism included: uptake and distribution of orally administered plutonium complex compounds in male mice; gastrointestinal absorption of 144 Ce in the newborn mouse, rat, and pig; and gastrointestinal absorption of 95 Nb by rats of different ages. Studies on iodine metabolism included the following: influence of thyroid status and thiocyanate on iodine metabolism in the bovine; effects of simulated fallout radiation on iodine metabolism in dairy cattle; and effects of feeding iodine binding agents on iodine metabolism in the calf

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

    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.

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

    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.

  3. * Comparison of Autologous, Allogeneic, and Cell-Free Scaffold Approaches for Engineered Tendon Repair in a Rabbit Model-A Pilot Study.

    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.

  4. The healing of bony defects by cell-free collagen-based scaffolds compared to stem cell-seeded tissue engineered constructs.

    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.

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

    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.

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

    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

  7. Stratospheric Platforms for Monitoring Purposes

    Konigorski, D.; Gratzel, U.; Obersteiner, M.; Schneidereit, M.

    2010-01-01

    Stratospheric platforms are emerging systems based on challenging technology. Goal is to create a platform, payload, and mission design which is able to complement satellite services on a local scale. Applications are close to traditional satellite business in telecommunication, navigation, science, and earth observation and include for example mobile telecommunications, navigation augmentation, atmospheric research, or border control. Stratospheric platforms could potentially support monitoring activities related to safeguards, e.g. by imagery of surfaces, operational conditions of nuclear facilities, and search for undeclared nuclear activities. Stratospheric platforms are intended to be flown in an altitude band between 16 and 30 km, above 16-20 km to take advantage of usually lower winds facilitating station keeping, below 30 km to limit the challenges to achieve a reasonable payload at acceptable platform sizes. Stratospheric platforms could substitute satellites which are expensive and lack upgrade capabilities for new equipment. Furthermore they have practically an unlimited time over an area of interest. It is intended to keep the platforms operational and maintenance free on a 24/7 basis with an average deployment time of 3 years. Geostationary satellites lack resolution. Potential customers like Armed Forces, National Agencies and commercial customers have indicated interest in the use of stratospheric platforms. Governmental entities are looking for cheaper alternatives to communications and surveillance satellites and stratospheric platforms could offer the following potential advantages: Lower operational cost than satellite or UAV (Unmanned Aerial Vehicles) constellation (fleet required); Faster deployment than satellite constellation; Repositioning capability and ability to loiter as required; Persistent long-term real-time services over a fairly large regional spot; Surge capability: Able to extend capability (either monitoring or communications

  8. ICP (ITER Collaborative Platform)

    Capuano, C.; Carayon, F.; Patel, V. [ITER, 13 - St. Paul-Lez Durance (France)

    2009-07-01

    The ITER organization has the necessity to manage a massive amount of data and processes. Each team requires different process and databases often interconnected with those of others teams. ICP is the current central ITER repository of structured and unstructured data. All data in ICP is served and managed via a web interface that provides global accessibility with a common user friendly interface. This paper will explain the model used by ICP and how it serves the ITER project by providing a robust and agile platform. ICP is developed in ASP.NET using MSSQL Server for data storage. It currently houses 15 data driven applications, 150 different types of record, 500 k objects and 2.5 M references. During European working hours the system averages 150 concurrent users and 20 requests per second. ICP connects to external database applications to provide a single entry point to ITER data and a safe shared storage place to maintain this data long-term. The Core model provides an easy to extend framework to meet the future needs of the Organization. ICP follows a multi-tier architecture, providing logical separation of process. The standard three-tier architecture is expanded, with the data layer separated into data storage, data structure, and data access components. The business or applications logic layer is broken up into a common business functionality layer, a type specific logic layer, and a detached work-flow layer. Finally the presentation tier comprises a presentation adapter layer and an interface layer. Each layer is built up from small blocks which can be combined to create a wide range of more complex functionality. Each new object type developed gains access to a wealth of existing code functionality, while also free to adapt and extend this. The hardware structure is designed to provide complete redundancy, high availability and to handle high load. This document is composed of an abstract followed by the presentation transparencies. (authors)

  9. Platform Expansion Design as Strategic Choice

    Staykova, Kalina S.; Damsgaard, Jan

    2016-01-01

    In this paper, we address how the strategic choice of platform expansion design impacts the subse-quent platform strategy. We identify two distinct approaches to platform expansion – platform bun-dling and platform constellations, which currently co-exist. The purpose of this paper is to outline...

  10. A Typology of Multi-sided Platforms

    Staykova, Kalina Stefanova; Damsgaard, Jan

    2015-01-01

    In this paper we address how the composition of a platform impacts the platform’s business model. By platform’s business model we mean platform features, platform architecture and platform governance. To this end, we construct the Platform Business Model Framework. We apply the framework to three...

  11. The European Photovoltaic Technology Platform

    Nowak, S.; Aulich, H.; Bal, J.L.; Dimmler, B.; Garnier, A.; Jongerden, G.; Luther, J.; Luque, A.; Milner, A.; Nelson, D.; Pataki, I.; Pearsall, N.; Perezagua, E.; Pietruszko, S.; Rehak, J.; Schellekens, E.; Shanker, A.; Silvestrini, G.; Sinke, W.; Willemsen, H.

    2006-05-01

    The European Photovoltaic Technology Platform is one of the European Technology Platforms, a new instrument proposed by the European Commission. European Technology Platforms (ETPs) are a mechanism to bring together all interested stakeholders to develop a long-term vision to address a specific challenge, create a coherent, dynamic strategy to achieve that vision and steer the implementation of an action plan to deliver agreed programmes of activities and optimise the benefits for all parties. The European Photovoltaic Technology Platform has recently been established to define, support and accompany the implementation of a coherent and comprehensive strategic plan for photovoltaics. The platform will mobilise all stakeholders sharing a long-term European vision for PV, helping to ensure that Europe maintains and improves its industrial position. The platform will realise a European Strategic Research Agenda for PV for the next decade(s). Guided by a Steering Committee of 20 high level decision-makers representing all relevant European PV Stakeholders, the European PV Technology Platform comprises 4 Working Groups dealing with the subjects policy and instruments; market deployment; science, technology and applications as well as developing countries and is supported by a secretariat

  12. Vertical Relationships within Platform Marketplaces

    Mark J. Tremblay

    2016-07-01

    Full Text Available In two-sided markets a platform allows consumers and sellers to interact by creating sub-markets within the platform marketplace. For example, Amazon has sub-markets for all of the different product categories available on its site, and smartphones have sub-markets for different types of applications (gaming apps, weather apps, map apps, ridesharing apps, etc.. The network benefits between consumers and sellers depend on the mode of competition within the sub-markets: more competition between sellers lowers product prices, increases the surplus consumers receive from a sub-market, and makes platform membership more desirable for consumers. However, more competition also lowers profits for a seller which makes platform membership less desirable for a seller and reduces seller entry and the number of sub-markets available on the platform marketplace. This dynamic between seller competition within a sub-market and agents’ network benefits leads to platform pricing strategies, participation decisions by consumers and sellers, and welfare results that depend on the mode of competition. Thus, the sub-market structure is important when investigating platform marketplaces.

  13. Platform decisions supported by gaming

    Hansen, Poul H. Kyvsgård; Mikkola, Juliana Hsuan

    2007-01-01

    Platform is an ambiguous multidisciplinary concept. The philosophy behind it is easy to communicate and makes intuitively sense. However, the ease in communication does overshadow the high complexity when the concept is implemented. The practical industrial platform implementation challenge can...... be described as being a configuration problem with a high number of variables. These variables are different in nature; they have contradictory influence on the total performance, and, their importance change over time. Consequently, the specific platform decisions become highly complex and the consequences...

  14. [Metabolic myopathies].

    Papazian, Óscar; Rivas-Chacón, Rafael

    2013-09-06

    To review the metabolic myopathies manifested only by crisis of myalgias, cramps and rigidity of the muscles with decreased voluntary contractions and normal inter crisis neurologic examination in children and adolescents. These metabolic myopathies are autosomic recessive inherited enzymatic deficiencies of the carbohydrates and lipids metabolisms. The end result is a reduction of intra muscle adenosine triphosphate, mainly through mitochondrial oxidative phosphorylation, with decrease of available energy for muscle contraction. The one secondary to carbohydrates intra muscle metabolism disorders are triggered by high intensity brief (fatty acids metabolism disorders are triggered by low intensity prolonged (> 10 min) exercises. The conditions in the first group in order of decreasing frequency are the deficiencies of myophosforilase (GSD V), muscle phosphofructokinase (GSD VII), phosphoglycerate mutase 1 (GSD X) and beta enolase (GSD XIII). The conditions in the second group in order of decreasing frequency are the deficiencies of carnitine palmitoyl transferase II and very long chain acyl CoA dehydrogenase. The differential characteristics of patients in each group and within each group will allow to make the initial presumptive clinical diagnosis in the majority and then to order only the necessary tests to achieve the final diagnosis. Treatment during the crisis includes hydration, glucose and alkalinization of urine if myoglobin in blood and urine are elevated. Prevention includes avoiding exercise which may induce the crisis and fasting. The prognosis is good with the exception of rare cases of acute renal failure due to hipermyoglobinemia because of severe rabdomyolisis.

  15. IRET: requirements for service platforms

    Baresi, Luciano; Ripa, Gianluca; Pasquale, Liliana

    2013-01-01

    peer-reviewed This paper describes IRENE (Indenica Requirements ElicitatioN mEthod), a methodology to elicit and model the requirements of service platforms, and IRET (IREne Tool), the Eclipse-based modeling framework we developed for IRENE

  16. Platform attitude data acquisition system

    Afzulpurkar, S.

    A system for automatic acquisition of underwater platform attitude data has been designed, developed and tested in the laboratory. This is a micro controller based system interfacing dual axis inclinometer, high-resolution digital compass...

  17. Microneedle Platforms for Cell Analysis

    Kavaldzhiev, Mincho

    2017-01-01

    to the development of micro-needle platforms that offer customized fabrication and new capabilities for enhanced cell analyses. The highest degree of geometrical flexibility is achieved with 3D printed micro-needles, which enable optimizing the topographical stress

  18. Elevated Fixed Platform Test Facility

    Federal Laboratory Consortium — The Elevated Fixed Platform (EFP) is a helicopter recovery test facility located at Lakehurst, NJ. It consists of a 60 by 85 foot steel and concrete deck built atop...

  19. Radiographic inspection on offshore platforms

    Soares, Sergio Damasceno; Sperandio, Augusto Gasparoni

    1994-01-01

    One of the great challenges for non-destructive inspection is on offshore platforms, where safety is a critical issue. Inspection by gammagraphy is practically forbidden on the platform deck due to problems to personnel safety and radiological protection. Ir-192 sources are used and the risk of an accident with loss of radioisotope must be considered. It is unfeasible to use gammagraphy, because in case of an accident the rapid evacuation from the platform would be impossible. This problem does not occur when X-ray equipment is used as the radiation source. The limited practicality and portability of the X-ray equipment have prevented its use as a replacement for the gammagraphy. This paper presents the preliminary tests to see the viable use of radiographic tests with constant potential on offshore platforms. (author). 2 refs., 1 fig., 2 tabs, 3 photos

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

    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

  1. Bioma platform advancements during 2017

    FUMAGALLI DAVIDE; NIEMEYER STEFAN

    2017-01-01

    In this report we describe the advancements on the Bioma Framework developed during year 2017. Given that the Bioma platform is quite mature, its core was not recently changed. So that the majority of changes concerns the implementation of the models developed in the platform. Moreover, during 2017 we also set up an alternative version of the framework itself, based on a new developing framework called .NET Core, with the purpose of being able to create a version of Bioma runnable on Linux. ...

  2. The COMET Sleep Research Platform.

    Nichols, Deborah A; DeSalvo, Steven; Miller, Richard A; Jónsson, Darrell; Griffin, Kara S; Hyde, Pamela R; Walsh, James K; Kushida, Clete A

    2014-01-01

    The Comparative Outcomes Management with Electronic Data Technology (COMET) platform is extensible and designed for facilitating multicenter electronic clinical research. Our research goals were the following: (1) to conduct a comparative effectiveness trial (CET) for two obstructive sleep apnea treatments-positive airway pressure versus oral appliance therapy; and (2) to establish a new electronic network infrastructure that would support this study and other clinical research studies. The COMET platform was created to satisfy the needs of CET with a focus on creating a platform that provides comprehensive toolsets, multisite collaboration, and end-to-end data management. The platform also provides medical researchers the ability to visualize and interpret data using business intelligence (BI) tools. COMET is a research platform that is scalable and extensible, and which, in a future version, can accommodate big data sets and enable efficient and effective research across multiple studies and medical specialties. The COMET platform components were designed for an eventual move to a cloud computing infrastructure that enhances sustainability, overall cost effectiveness, and return on investment.

  3. Nucleotide Metabolism

    Martinussen, Jan; Willemoës, M.; Kilstrup, Mogens

    2011-01-01

    Metabolic pathways are connected through their utilization of nucleotides as supplier of energy, allosteric effectors, and their role in activation of intermediates. Therefore, any attempt to exploit a given living organism in a biotechnological process will have an impact on nucleotide metabolis...

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

    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.

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

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

    2013-01-11

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

  6. 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

    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...

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

    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

  8. A rapid and cell-free assay to test the activity of lynch syndrome-associated MSH2 and MSH6 missense variants

    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....

  9. Metabolite damage and repair in metabolic engineering design.

    Sun, Jiayi; Jeffryes, James G; Henry, Christopher S; Bruner, Steven D; Hanson, Andrew D

    2017-11-01

    The necessarily sharp focus of metabolic engineering and metabolic synthetic biology on pathways and their fluxes has tended to divert attention from the damaging enzymatic and chemical side-reactions that pathway metabolites can undergo. Although historically overlooked and underappreciated, such metabolite damage reactions are now known to occur throughout metabolism and to generate (formerly enigmatic) peaks detected in metabolomics datasets. It is also now known that metabolite damage is often countered by dedicated repair enzymes that undo or prevent it. Metabolite damage and repair are highly relevant to engineered pathway design: metabolite damage reactions can reduce flux rates and product yields, and repair enzymes can provide robust, host-independent solutions. Herein, after introducing the core principles of metabolite damage and repair, we use case histories to document how damage and repair processes affect efficient operation of engineered pathways - particularly those that are heterologous, non-natural, or cell-free. We then review how metabolite damage reactions can be predicted, how repair reactions can be prospected, and how metabolite damage and repair can be built into genome-scale metabolic models. Lastly, we propose a versatile 'plug and play' set of well-characterized metabolite repair enzymes to solve metabolite damage problems known or likely to occur in metabolic engineering and synthetic biology projects. Copyright © 2017 International Metabolic Engineering Society. All rights reserved.

  10. Application of real-time PCR of sex-independent insertion-deletion polymorphisms to determine fetal sex using cell-free fetal DNA from maternal plasma.

    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.

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

    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.

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

    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.

  13. Metabolic Surgery

    Pareek, Manan; Schauer, Philip R; Kaplan, Lee M

    2018-01-01

    The alarming rise in the worldwide prevalence of obesity is paralleled by an increasing burden of type 2 diabetes mellitus. Metabolic surgery is the most effective means of obtaining substantial and durable weight loss in individuals with obesity. Randomized trials have recently shown...... the superiority of surgery over medical treatment alone in achieving improved glycemic control, as well as a reduction in cardiovascular risk factors. The mechanisms seem to extend beyond the magnitude of weight loss alone and include improvements in incretin profiles, insulin secretion, and insulin sensitivity....... Moreover, observational data suggest that the reduction in cardiovascular risk factors translates to better patient outcomes. This review describes commonly used metabolic surgical procedures and their current indications and summarizes the evidence related to weight loss and glycemic outcomes. It further...

  14. Metabolic Syndrome

    Sevil Ikinci

    2010-10-01

    Full Text Available Metabolic Syndrome is a combination of risk factors including common etiopathogenesis. These risk factors play different roles in occurence of atherosclerotic diseases, type 2 diabetes, and cancers. Although a compromise can not be achieved on differential diagnosis for MS, the existence of any three criterias enable to diagnose MS. These are abdominal obesity, dislipidemia (hypertrigliceridemia, hypercholesterolemia, and reduced high density lipoprotein hypertension, and elevated fasting blood glucose. According to the results of Metabolic Syndrome Research (METSAR, the overall prevalence of MS in Turkey is 34%; in females 40%, and in males it is 28%. As a result of “Western” diet, and increased frequency of obesity, MS is observed in children and in adolescents both in the world and in Turkey. Resulting in chronic diseases, it is thought that the syndrome can be prevented by healthy lifestyle behaviours. [TAF Prev Med Bull 2010; 9(5.000: 535-540

  15. Stratifying the Develoment of Product Platforms

    Sköld, Martin; Karlsson, Christer

    2013-01-01

    companies develop platforms for different aims, purposes, and product scopes. Following on from this, the requirements for platform development resources, the ways of organizing platform development, and the implications for management styles have not been explored and are presumably varying. To start...... influencing the project length, requirements for platform development resources, principles for organizing, and implications for management styles....

  16. The Educational Platform: Constructing Conceptual Frameworks.

    Peca, Kathy; Isham, Mark

    2001-01-01

    The education faculty at Eastern New Mexico University used educational platforms as a means of developing the unit's conceptual framework. Faculty members developed personal platforms, then synthesized them into one curricular area platform. The resultant unit educational platform became the basis for the unit's conceptual framework, which…

  17. New offshore platform in the Mexican Gulf

    Beisel, T.

    1982-04-01

    After a construction period of only 10 months, the second steel Offshore platform was recently completed in the Mexican Gulf. The pattern for this structure was the Cognac platform. The erection of the new platform, called the 'Cerveza' platform, is described in the article.

  18. The Dynamics of Digital Platform Innovation

    Eaton, Ben

    2016-01-01

    Curated platforms provide an architectural basis for third parties to develop platform complements and for platform owners to control their implementation as a form of open innovation. The refusal to implement complements as innovations can cause tension between platform owners and developers. Th...

  19. Cellular metabolism

    Hildebrand, C.E.; Walters, R.A.

    1977-01-01

    Progress is reported on the following research projects: chromatin structure; the use of circular synthetic polydeoxynucleotides as substrates for the study of DNA repair enzymes; human cellular kinetic response following exposure to DNA-interactive compounds; histone phosphorylation and chromatin structure in cell proliferation; photoaddition products induced in chromatin by uv light; pollutants and genetic information transfer; altered RNA metabolism as a function of cadmium accumulation and intracellular distribution in cultured cells; and thymidylate chromophore destruction by water free radicals

  20. Disentangling Competition Among Platform Driven Strategic Groups

    Kazan, Erol; Tan, Chee-Wee; Lim, Eric

    2015-01-01

    In platform-driven markets, competitive advantage is derived from superior platform design and configurations. For this reason, platform owners strive to create unique and inimitable platform configurals to maintain and extend their competitiveness within network economies. To disentangle firm...... competition within platform-driven markets, we opted for the UK mobile payment market as our empirical setting. By embracing the theoretical lens of strategic groups and digital platforms, this study supplements prior research by deriving a taxonomy of platform-driven strategic groups that is grounded...

  1. Microneedle Platforms for Cell Analysis

    Kavaldzhiev, Mincho

    2017-11-01

    Micro-needle platforms are the core components of many recent drug delivery and gene-editing techniques, which allow for intracellular access, controlled cell membrane stress or mechanical trapping of the nucleus. This dissertation work is devoted to the development of micro-needle platforms that offer customized fabrication and new capabilities for enhanced cell analyses. The highest degree of geometrical flexibility is achieved with 3D printed micro-needles, which enable optimizing the topographical stress environment for cells and cell populations of any size. A fabrication process for 3D-printed micro-needles has been developed as well as a metal coating technique based on standard sputter deposition. This extends the functionalities of the platforms by electrical as well as magnetic features. The micro-needles have been tested on human colon cancer cells (HCT116), showing a high degree of biocompatibility of the platform. Moreover, the capabilities of the 3D-printed micro-needles have been explored for drug delivery via the well-established electroporation technique, by coating the micro-needles with gold. Antibodies and fluorescent dyes have been delivered to HCT116 cells and human embryonic kidney cells with a very high transfection rate up to 90%. In addition, the 3D-printed electroporation platform enables delivery of molecules to suspended cells or adherent cells, with or without electroporation buffer solution, and at ultra-low voltages of 2V. In order to provide a micro-needle platform that exploits existing methods for mass fabrication a custom designed template-based process has been developed. It has been used for the production of gold, iron, nickel and poly-pyrrole micro-needles on silicon and glass substrates. A novel delivery method is introduced that activates the micro-needles by electromagnetic induction, which enables to wirelessly gain intracellular access. The method has been successfully tested on HCT116 cells in culture, where a time

  2. Moodle vs. Social Media Platforms

    Gulieva, Valeria

    Given the competition coming from various social media platforms, it is explored in this paper how students could be encouraged to use Moodle more proactively during their studies. Moodle is a course management system for online learning. It is designed to be a flexible template-based system, which......, known and widely used social platforms. It might be also due to the fact that students do not see the benefits in investing time and efforts in learning the new system. Another reason might be the mandatory nature of Moodle, i.e., it is imposed on students, rather than a free choice – and this might...

  3. Towards a Framework of Digital Platform Competition

    Kazan, Erol; Tan, Chee-Wee; Lim, Eric T. K.

    2016-01-01

    between monopolistic (i.e., Pingit) and federated (i.e., Paym) mobile payment platforms to illustrate its applicability and yield principles on the nature and impact of competition among platform-driven ubiquitous systems. Preliminary findings indicate that monopolistic mobile digital platforms attempt...... to create unique configurals to obtain monopolistic power by tightly coupling platform layers, which are difficult to replicate. Conversely, federated digital platforms compete by dispersing the service layer to harness the collective resources from individual firms. Furthermore, the interaction...

  4. Platform for systems medicine research and diagnostic applications in psychotic disorders-The METSY project

    Frank, Elisabeth; Maier, Dieter; Pajula, Juha

    2018-01-01

    with detailed metabolic characterisation and multi-variate network analysis is essential in order to identify how lipid dysregulation may contribute to psychotic disorders. A decision support system, integrating clinical, neuropsychological and neuroimaging data, was also developed in order to aid clinical......Psychotic disorders are associated with metabolic abnormalities including alterations in glucose and lipid metabolism. A major challenge in the treatment of psychosis is to identify patients with vulnerable metabolic profiles who may be at risk of developing cardiometabolic co...... these endocannabinoid abnormalities relate to metabolic changes in psychosis. Here we review recent research in the field of metabolic co-morbidities in psychotic disorders as well as the methods to study them and potential links to the endocannabinoid system. We also describe the bioinformatics platforms developed...

  5. Zymomonas mobilis: a novel platform for future biorefineries.

    He, Ming Xiong; Wu, Bo; Qin, Han; Ruan, Zhi Yong; Tan, Fu Rong; Wang, Jing Li; Shui, Zong Xia; Dai, Li Chun; Zhu, Qi Li; Pan, Ke; Tang, Xiao Yu; Wang, Wen Guo; Hu, Qi Chun

    2014-01-01

    Biosynthesis of liquid fuels and biomass-based building block chemicals from microorganisms have been regarded as a competitive alternative route to traditional. Zymomonas mobilis possesses a number of desirable characteristics for its special Entner-Doudoroff pathway, which makes it an ideal platform for both metabolic engineering and commercial-scale production of desirable bio-products as the same as Escherichia coli and Saccharomyces cerevisiae based on consideration of future biomass biorefinery. Z. mobilis has been studied extensively on both fundamental and applied level, which will provide a basis for industrial biotechnology in the future. Furthermore, metabolic engineering of Z. mobilis for enhancing bio-ethanol production from biomass resources has been significantly promoted by different methods (i.e. mutagenesis, adaptive laboratory evolution, specific gene knock-out, and metabolic engineering). In addition, the feasibility of representative metabolites, i.e. sorbitol, bionic acid, levan, succinic acid, isobutanol, and isobutanol produced by Z. mobilis and the strategies for strain improvements are also discussed or highlighted in this paper. Moreover, this review will present some guidelines for future developments in the bio-based chemical production using Z. mobilis as a novel industrial platform for future biofineries.

  6. Verkenning locatiegegevens en sociale platforms

    van Loenen, B.; Kilic, Deniz; Van de Velde, Rob

    2017-01-01

    In deze rapportage hebben we ons verdiept in de wereld van de commerciële toepassingen van sensordata, waarin locatiegegevens van smartphones op grote schaal worden vastgelegd. Een beter inzicht in de wereld van de sociale en commerciële platforms zou ervoor moeten zorgen dat deze beter begrepen

  7. 2009 Analysis Platform Review Report

    Ferrell, John [Office of Energy Efficiency and Renewable Energy (EERE), Washington, DC (United States

    2009-12-01

    This document summarizes the recommendations and evaluations provided by an independent external panel of experts at the U.S. Department of Energy Biomass Program’s Analysis platform review meeting, held on February 18, 2009, at the Marriott Residence Inn, National Harbor, Maryland.

  8. 2009 Feedstocks Platform Review Report

    Ferrell, John [Office of Energy Efficiency and Renewable Energy (EERE), Washington, DC (United States)

    2009-12-01

    This document summarizes the recommendations and evaluations provided by an independent external panel of experts at the U.S. Department of Energy Biomass Program‘s Feedstock platform review meeting, held on April 8–10, 2009, at the Grand Hyatt Washington, Washington, D.C.

  9. Platform pricing in matching markets

    Goos, M.; van Cayseele, P.; Willekens, B.

    2011-01-01

    This paper develops a simple model of monopoly platform pricing accounting for two pertinent features of matching markets. 1) The trading process is characterized by search and matching frictions implying limits to positive cross-side network effects and the presence of own-side congestion.

  10. Towards trustworthy health platform cloud

    Deng, M.; Nalin, M.; Petkovic, M.; Baroni, I.; Marco, A.; Jonker, W.; Petkovic, M.

    2012-01-01

    To address today’s major concerns of health service providers regarding security, resilience and data protection when moving on the cloud, we propose an approach to build a trustworthy healthcare platform cloud, based on a trustworthy cloud infrastructure. This paper first highlights the main

  11. 2009 Infrastructure Platform Review Report

    Ferrell, John [Office of Energy Efficiency and Renewable Energy (EERE), Washington, DC (United States)

    2009-12-01

    This document summarizes the recommendations and evaluations provided by an independent external panel of experts at the U.S. Department of Energy Biomass program‘s Infrastructure platform review meeting, held on February 19, 2009, at the Marriott Residence Inn, National Harbor, Maryland.

  12. Metabolite damage and repair in metabolic engineering design

    Sun, Jiayi; Jeffryes, James G.; Henry, Christopher S.; Bruner, Steven D.; Hanson, Andrew D.

    2017-11-01

    The necessarily sharp focus of metabolic engineering and metabolic synthetic biology on pathways and their fluxes has tended to divert attention from the damaging enzymatic and chemical side-reactions that pathway metabolites can undergo. Although historically overlooked and underappreciated, such metabolite damage reactions are now known to occur throughout metabolism and to generate (formerly enigmatic) peaks detected in metabolomics datasets. It is also now known that metabolite damage is often countered by dedicated repair enzymes that undo or prevent it. Metabolite damage and repair are highly relevant to engineered pathway design: metabolite damage reactions can reduce flux rates and product yields, and repair enzymes can provide robust, host-independent solutions. Herein, after introducing the core principles of metabolite damage and repair, we use case histories to document how damage and repair processes affect efficient operation of engineered pathways - particularly those that are heterologous, non-natural, or cell-free. We then review how metabolite damage reactions can be predicted, how repair reactions can be prospected, and how metabolite damage and repair can be built into genome-scale metabolic models. Lastly, we propose a versatile 'plug and play' set of well-characterized metabolite repair enzymes to solve metabolite damage problems known or likely to occur in metabolic engineering and synthetic biology projects.

  13. The anaerobic phototrophic metabolism of 3-chlorobenzoate by Rhodopseudomonas palustris

    Kamal, V S

    1992-10-09

    The degradation of chlorinated aromatic compounds by anaerobic bacteria is now known to be an important mechanism of bioremediation. In an experimental study, a mixed phototrophic culture was found to metabolize 3-chlorobenzoate in the presence of benzoate following adaptation on a benzoate and 3-chlorobenzoate medium for 7 weeks. The dominant bacterial isolate was identified as Rhodopseudomonas palustris. Radioisotopic studies showed [sup 14]C-3-chlorobenzoate was converted by the isolate to [sup 14]CO[sub 2] and cell biomass in the absence of oxygen and in the presence of a cosubstrate red light. Cyclohexane carboxylate was able to replace the cosubstrate, benzoate. The isolate also metabolized 3-chlorobenzoate in the presence of pimelic acid, sodium acetate, and sodium succinate; however, the metabolic rate was reduced. Gas chromatography mass spectrometry and high pressure liquid chromatography indicated the intracellular presence of 3-chlorobenzoate and benzoyl-CoA. Cell-free extracts produced benzoate and benzoyl-CoA. A probable route of 3-chlorobenzoate metabolism via dehalogenation followed by steps similar to the benzoate reductive ring fission pathway is suggested. Comparison of kinetic coefficients showed a higher affinity of the isolate for benzoate. Isolates from representative samples of various freshwater and wastewater ecosystems indicated widespread ecological distribution of R. palustris and the common occurrence of the 3-chlorobenzoate metabolic phenotype. R. palustris was found to grow in mixed anaerobic cultures and retained its 3-chlorobenzoate degradation property. 91 refs., 25 figs., 14 tabs.

  14. Methylation status of the APC and RASSF1A promoter in cell-free circulating DNA and its prognostic role in patients with colorectal cancer.

    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.

  15. 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

    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

  16. 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

    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

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

    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.

  18. Clinical experience of laboratory follow-up with noninvasive prenatal testing using cell-free DNA and positive microdeletion results in 349 cases.

    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.

  19. Effects of heme-PrP complex on cell-free conversion and peroxidase-linked immunodetection of prions in blood-based assays.

    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.

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

    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.

  1. Low-Dose Ionizing Radiation Affects Mesenchymal Stem Cells via Extracellular Oxidized Cell-Free DNA: A Possible Mediator of Bystander Effect and Adaptive Response

    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.

  2. N-Terminal Prodomain of Pfs230 Synthesized Using a Cell-Free System Is Sufficient To Induce Complement-Dependent Malaria Transmission-Blocking Activity▿

    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

  3. N-terminal prodomain of Pfs230 synthesized using a cell-free system is sufficient to induce complement-dependent malaria transmission-blocking activity.

    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.

  4. The usage and current approaches of cell free fetal DNA (cffDNA as a prenatal diagnostic method in fetal aneuploidy screening

    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

  5. Anti-adherence potential of Enterococcus durans cells and its cell-free supernatant on plastic and stainless steel against foodborne pathogens.

    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.

  6. Impact of Cell-Free Fetal DNA Screening on Patients’ Choice of Invasive Procedures after a Positive California Prenatal Screen Result

    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.

  7. 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.

    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.

  8. Quantification of cell-free DNA in blood plasma and DNA damage degree in lymphocytes to evaluate dysregulation of apoptosis in schizophrenia patients.

    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.

  9. 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.

    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.

  10. The diagnostic value of circulating cell free DNA quantification in non-small cell lung cancer: A systematic review with meta-analysis.

    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.

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

    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.

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

    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.

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

    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.

  14. eTumorType, An Algorithm of Discriminating Cancer Types for Circulating Tumor Cells or Cell-free DNAs in Blood

    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.

  15. Fully Integrated Biochip Platforms for Advanced Healthcare

    Giovanni De Micheli

    2012-08-01

    Full Text Available Recent advances in microelectronics and biosensors are enabling developments of innovative biochips for advanced healthcare by providing fully integrated platforms for continuous monitoring of a large set of human disease biomarkers. Continuous monitoring of several human metabolites can be addressed by using fully integrated and minimally invasive devices located in the sub-cutis, typically in the peritoneal region. This extends the techniques of continuous monitoring of glucose currently being pursued with diabetic patients. However, several issues have to be considered in order to succeed in developing fully integrated and minimally invasive implantable devices. These innovative devices require a high-degree of integration, minimal invasive surgery, long-term biocompatibility, security and privacy in data transmission, high reliability, high reproducibility, high specificity, low detection limit and high sensitivity. Recent advances in the field have already proposed possible solutions for several of these issues. The aim of the present paper is to present a broad spectrum of recent results and to propose future directions of development in order to obtain fully implantable systems for the continuous monitoring of the human metabolism in advanced healthcare applications.

  16. Fully integrated biochip platforms for advanced healthcare.

    Carrara, Sandro; Ghoreishizadeh, Sara; Olivo, Jacopo; Taurino, Irene; Baj-Rossi, Camilla; Cavallini, Andrea; de Beeck, Maaike Op; Dehollain, Catherine; Burleson, Wayne; Moussy, Francis Gabriel; Guiseppi-Elie, Anthony; De Micheli, Giovanni

    2012-01-01

    Recent advances in microelectronics and biosensors are enabling developments of innovative biochips for advanced healthcare by providing fully integrated platforms for continuous monitoring of a large set of human disease biomarkers. Continuous monitoring of several human metabolites can be addressed by using fully integrated and minimally invasive devices located in the sub-cutis, typically in the peritoneal region. This extends the techniques of continuous monitoring of glucose currently being pursued with diabetic patients. However, several issues have to be considered in order to succeed in developing fully integrated and minimally invasive implantable devices. These innovative devices require a high-degree of integration, minimal invasive surgery, long-term biocompatibility, security and privacy in data transmission, high reliability, high reproducibility, high specificity, low detection limit and high sensitivity. Recent advances in the field have already proposed possible solutions for several of these issues. The aim of the present paper is to present a broad spectrum of recent results and to propose future directions of development in order to obtain fully implantable systems for the continuous monitoring of the human metabolism in advanced healthcare applications.

  17. Climate News Across Media Platforms

    Eskjær, Mikkel Fugl

    2015-01-01

    In a changing media landscape marked by technological, institutional and cultural convergence, comparative and cross-media content analysis represents a valuable analytical tool in mapping the diverse channels of climate change communication. This paper presents a comparative study of climate...... quantitative and qualitative content analysis the paper documents and explores the extent and character of climate change news across different media platforms. The study aims at contributing to the on-going assessment of how news media are addressing climate change at a time when old and new media...... change news on five different media platforms: newspapers, television, radio, web-news and mobile news. It investigates the themes and actors represented in public climate change communication as well as the diverse possibilities of participating in public debates and information sharing. By combining...

  18. YARP: Yet Another Robot Platform

    Lorenzo Natale

    2008-11-01

    Full Text Available We describe YARP, Yet Another Robot Platform, an open-source project that encapsulates lessons from our experience in building humanoid robots. The goal of YARP is to minimize the effort devoted to infrastructure-level software development by facilitating code reuse, modularity and so maximize research-level development and collaboration. Humanoid robotics is a "bleeding edge" field of research, with constant flux in sensors, actuators, and processors. Code reuse and maintenance is therefore a significant challenge. We describe the main problems we faced and the solutions we adopted. In short, the main features of YARP include support for inter-process communication, image processing as well as a class hierarchy to ease code reuse across different hardware platforms. YARP is currently used and tested on Windows, Linux and QNX6 which are common operating systems used in robotics.

  19. Stabilisation problem in biaxial platform

    Lindner Tymoteusz

    2016-12-01

    Full Text Available The article describes investigation of rolling ball stabilization problem on a biaxial platform. The aim of the control system proposed here is to stabilize ball moving on a plane in equilibrium point. The authors proposed a control algorithm based on cascade PID and they compared it with another control method. The article shows the results of the accuracy of ball stabilization and influence of applied filter on the signal waveform. The application used to detect the ball position measured by digital camera has been written using a cross platform .Net wrapper to the OpenCV image processing library - EmguCV. The authors used the bipolar stepper motor with dedicated electronic controller. The data between the computer and the designed controller are sent with use of the RS232 standard. The control stand is based on ATmega series microcontroller.

  20. SAFETY PLATFORM OF POLISH TRANSPORT

    Katarzyna CHRUZIK

    2016-03-01

    Full Text Available Analyzing the level of Polish transport safety culture can be seen that it is now dependent on the culture of safety management within the organization and the requirements and recommendations of law in this field for different modes of transport (air, rail, road, water. Of the four basic types of transport requirements are widely developed in the aviation, rail, and water – the sea. In order to harmonize the requirements for transport safety so it appears advisable to develop a platform for exchange of safety information for different modes of transport, and the development of good practices multimodal offering the possibility of improving Polish transport safety. Described in the publication of the proposal in addition to the alignment platform experience and knowledge in the field of transport safety in all its kinds, it can also be a tool for perfecting new operators of public transport.

  1. Tax Responses in Platform Industries

    Kind, Hans Jarle; Köthenbürger, Marko; Schjelderup, Guttorm

    that a higher ad valorem tax may undermine a firm's incentive to differentiate its product from that of its competitors. Finally, we demonstrate that the effects of increasing specific taxes may be the opposite of those of increasing value added taxes....... price and thus buy less of the good. The present paper shows that this result need not hold in a two-sided market. On the contrary, a higher ad valorem tax may lower end-user prices and spur sales. Thus, two-sided platform firms may not at all engage in tax shifting via price increases. We further show......Two-sided platform firms serve distinct customer groups that are connected through interdependent demand, and include major businesses such as the media industry, banking, and the software industry. A well known result of tax incidence is that consumers of a more heavily taxed good pay a higher...

  2. Stabilisation problem in biaxial platform

    Lindner, Tymoteusz; Rybarczyk, Dominik; Wyrwał, Daniel

    2016-12-01

    The article describes investigation of rolling ball stabilization problem on a biaxial platform. The aim of the control system proposed here is to stabilize ball moving on a plane in equilibrium point. The authors proposed a control algorithm based on cascade PID and they compared it with another control method. The article shows the results of the accuracy of ball stabilization and influence of applied filter on the signal waveform. The application used to detect the ball position measured by digital camera has been written using a cross platform .Net wrapper to the OpenCV image processing library - EmguCV. The authors used the bipolar stepper motor with dedicated electronic controller. The data between the computer and the designed controller are sent with use of the RS232 standard. The control stand is based on ATmega series microcontroller.

  3. Negotiation platform for personalised advertising

    Sousa, Luís Ventura de; Malheiro, Benedita; Foss, Jerry

    2012-01-01

    This paper describes a multi-agent brokerage platform for near real time advertising personalisation organised in three layers: user interface, agency and marketplace. The personalisation is based on the classification of viewer profiles and advertisements (ads). The goal is to provide viewers with a personalised advertising alignment during programme intervals. The enterprise interface agents upload new ads and negotiation profiles to producer agents and new user and negotiation profiles to ...

  4. Reconfigurable microfluidic platform in ice

    Varejka, M.

    2008-01-01

    Microfluidic devices are popular tools in the biotechnology industry where they provide smaller reagent requirements, high speed of analysis and the possibility for automation. The aim of the project is to make a flexible biocompatible microfluidic platform adapted to different specific applications, mainly analytical and separations which parameters and configuration can be changed multiple times by changing corresponding computer programme. The current project has been sup...

  5. Visual guidance of mobile platforms

    Blissett, Rodney J.

    1993-12-01

    Two systems are described and results presented demonstrating aspects of real-time visual guidance of autonomous mobile platforms. The first approach incorporates prior knowledge in the form of rigid geometrical models linking visual references within the environment. The second approach is based on a continuous synthesis of information extracted from image tokens to generate a coarse-grained world model, from which potential obstacles are inferred. The use of these techniques in workplace applications is discussed.

  6. DATABASE REPLICATION IN HETEROGENOUS PLATFORM

    Hendro Nindito; Evaristus Didik Madyatmadja; Albert Verasius Dian Sano

    2014-01-01

    The application of diverse database technologies in enterprises today is increasingly a common practice. To provide high availability and survavibality of real-time information, a database replication technology that has capability to replicate databases under heterogenous platforms is required. The purpose of this research is to find the technology with such capability. In this research, the data source is stored in MSSQL database server running on Windows. The data will be replicated to MyS...

  7. Interaction of pathogens with host cholesterol metabolism.

    Sviridov, Dmitri; Bukrinsky, Michael

    2014-10-01

    Pathogens of different taxa, from prions to protozoa, target cellular cholesterol metabolism to advance their own development and to impair host immune responses, but also causing metabolic complications, for example, atherosclerosis. This review describes recent findings of how pathogens do it. A common theme in interaction between pathogens and host cholesterol metabolism is pathogens targeting lipid rafts of the host plasma membrane. Many intracellular pathogens use rafts as an entry gate, taking advantage of the endocytic machinery and high abundance of outward-looking molecules that can be used as receptors. At the same time, disruption of the rafts' functional capacity, achieved by the pathogens through a number of various means, impairs the ability of the host to generate immune response, thus helping pathogen to thrive. Pathogens cannot synthesize cholesterol, and salvaging host cholesterol helps pathogens build advanced cholesterol-containing membranes and assembly platforms. Impact on cholesterol metabolism is not limited to the infected cells; proteins and microRNAs secreted by infected cells affect lipid metabolism systemically. Given an essential role that host cholesterol metabolism plays in pathogen development, targeting this interaction may be a viable strategy to fight infections, as well as metabolic complications of the infections.

  8. Small animal radiotherapy research platforms

    Verhaegen, Frank; Granton, Patrick [Department of Radiation Oncology (MAASTRO), GROW-School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht 6201 BN (Netherlands); Tryggestad, Erik, E-mail: frank.verhaegen@maastro.nl [Department of Radiation Oncology and Molecular Radiation Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21231 (United States)

    2011-06-21

    Advances in conformal radiation therapy and advancements in pre-clinical radiotherapy research have recently stimulated the development of precise micro-irradiators for small animals such as mice and rats. These devices are often kilovolt x-ray radiation sources combined with high-resolution CT imaging equipment for image guidance, as the latter allows precise and accurate beam positioning. This is similar to modern human radiotherapy practice. These devices are considered a major step forward compared to the current standard of animal experimentation in cancer radiobiology research. The availability of this novel equipment enables a wide variety of pre-clinical experiments on the synergy of radiation with other therapies, complex radiation schemes, sub-target boost studies, hypofractionated radiotherapy, contrast-enhanced radiotherapy and studies of relative biological effectiveness, to name just a few examples. In this review we discuss the required irradiation and imaging capabilities of small animal radiation research platforms. We describe the need for improved small animal radiotherapy research and highlight pioneering efforts, some of which led recently to commercially available prototypes. From this, it will be clear that much further development is still needed, on both the irradiation side and imaging side. We discuss at length the need for improved treatment planning tools for small animal platforms, and the current lack of a standard therein. Finally, we mention some recent experimental work using the early animal radiation research platforms, and the potential they offer for advancing radiobiology research. (topical review)

  9. Small animal radiotherapy research platforms

    Verhaegen, Frank; Granton, Patrick; Tryggestad, Erik

    2011-06-01

    Advances in conformal radiation therapy and advancements in pre-clinical radiotherapy research have recently stimulated the development of precise micro-irradiators for small animals such as mice and rats. These devices are often kilovolt x-ray radiation sources combined with high-resolution CT imaging equipment for image guidance, as the latter allows precise and accurate beam positioning. This is similar to modern human radiotherapy practice. These devices are considered a major step forward compared to the current standard of animal experimentation in cancer radiobiology research. The availability of this novel equipment enables a wide variety of pre-clinical experiments on the synergy of radiation with other therapies, complex radiation schemes, sub-target boost studies, hypofractionated radiotherapy, contrast-enhanced radiotherapy and studies of relative biological effectiveness, to name just a few examples. In this review we discuss the required irradiation and imaging capabilities of small animal radiation research platforms. We describe the need for improved small animal radiotherapy research and highlight pioneering efforts, some of which led recently to commercially available prototypes. From this, it will be clear that much further development is still needed, on both the irradiation side and imaging side. We discuss at length the need for improved treatment planning tools for small animal platforms, and the current lack of a standard therein. Finally, we mention some recent experimental work using the early animal radiation research platforms, and the potential they offer for advancing radiobiology research.

  10. Small animal radiotherapy research platforms

    Verhaegen, Frank; Granton, Patrick; Tryggestad, Erik

    2011-01-01

    Advances in conformal radiation therapy and advancements in pre-clinical radiotherapy research have recently stimulated the development of precise micro-irradiators for small animals such as mice and rats. These devices are often kilovolt x-ray radiation sources combined with high-resolution CT imaging equipment for image guidance, as the latter allows precise and accurate beam positioning. This is similar to modern human radiotherapy practice. These devices are considered a major step forward compared to the current standard of animal experimentation in cancer radiobiology research. The availability of this novel equipment enables a wide variety of pre-clinical experiments on the synergy of radiation with other therapies, complex radiation schemes, sub-target boost studies, hypofractionated radiotherapy, contrast-enhanced radiotherapy and studies of relative biological effectiveness, to name just a few examples. In this review we discuss the required irradiation and imaging capabilities of small animal radiation research platforms. We describe the need for improved small animal radiotherapy research and highlight pioneering efforts, some of which led recently to commercially available prototypes. From this, it will be clear that much further development is still needed, on both the irradiation side and imaging side. We discuss at length the need for improved treatment planning tools for small animal platforms, and the current lack of a standard therein. Finally, we mention some recent experimental work using the early animal radiation research platforms, and the potential they offer for advancing radiobiology research. (topical review)

  11. Benchmarking computer platforms for lattice QCD applications

    Hasenbusch, M.; Jansen, K.; Pleiter, D.; Wegner, P.; Wettig, T.

    2003-09-01

    We define a benchmark suite for lattice QCD and report on benchmark results from several computer platforms. The platforms considered are apeNEXT, CRAY T3E, Hitachi SR8000, IBM p690, PC-Clusters, and QCDOC. (orig.)

  12. Benchmarking computer platforms for lattice QCD applications

    Hasenbusch, M.; Jansen, K.; Pleiter, D.; Stueben, H.; Wegner, P.; Wettig, T.; Wittig, H.

    2004-01-01

    We define a benchmark suite for lattice QCD and report on benchmark results from several computer platforms. The platforms considered are apeNEXT, CRAY T3E; Hitachi SR8000, IBM p690, PC-Clusters, and QCDOC

  13. The Innovative Capabilities Of Digital Payment Platforms

    Kazan, Erol

    2015-01-01

    This study presents a model for studying the innovative capabilities of digital payment platforms in regards to open innovation integration and commercialization. We perceive digital platforms as layered modular IT artifacts, where platform governance and the configuration of platform layers impact...... the support for open innovation. The proposed model has been employed in a comparative case study between two digital payment platforms: Apple Pay and Google Wallet. The findings suggest that digital payment platforms make use of boundary resources to be highly integrative or integratable, which supports...... the intended conjoint commercialization efforts. Furthermore, the architectural design of digital platforms impacts the access to commercialization, resulting to an exclusion or inclusion strategy in accessing value opportunities. Our findings contribute to the open innovation and digital platform literature...

  14. Hierarchical DSE for multi-ASIP platforms

    Micconi, Laura; Corvino, Rosilde; Gangadharan, Deepak

    2013-01-01

    This work proposes a hierarchical Design Space Exploration (DSE) for the design of multi-processor platforms targeted to specific applications with strict timing and area constraints. In particular, it considers platforms integrating multiple Application Specific Instruction Set Processors (ASIPs...

  15. The Logic of Digital Platform Disruption

    Kazan, Erol; Tan, Chee-Wee; Lim, Eric T. K.

    Digital platforms are disruptive IT artifacts, because they facilitate the quick release of innovative platform derivatives from third parties (e.g., apps). This study endeavours to unravel the disruptive potential, caused by distinct designs and configurations of digital platforms on market...... environments. We postulate that the disruptive potential of digital platforms is determined by the degree of alignment among the business, technology and platform profiles. Furthermore, we argue that the design and configuration of the aforementioned three elements dictates the extent to which open innovation...... is permitted. To shed light on the disruptive potential of digital platforms, we opted for payment platforms as our unit of analysis. Through interviews with experts and payment providers, we seek to gain an in-depth appreciation of how contemporary digital payment platforms are designed and configured...

  16. Software development on the SAP HANA platform

    Walker, Mark

    2013-01-01

    Software Development on the SAP HANA Platform is a general tutorial guide to SAP HANA.This book is written for beginners to the SAP HANA platform. No knowledge of SAP HANA is necessary to start using this book.

  17. Towards a Disruptive Digital Platform Model

    Kazan, Erol

    that digital platforms leverage on three strategic design elements (i.e., business, architecture, and technology design) to create supportive conditions for facilitating disruption. To shed light on disruptive digital platforms, I opted for payment platforms as my empirical context and unit of analysis......Digital platforms are layered modular information technology architectures that support disruption. Digital platforms are particularly disruptive, as they facilitate the quick release of digital innovations that may replace established innovations. Yet, despite their support for disruption, we have...... not fully understood how such digital platforms can be strategically designed and configured to facilitate disruption. To that end, this thesis endeavors to unravel disruptive digital platforms from the supply perspective that are grounded on strategic digital platform design elements. I suggest...

  18. Platforms for Persistent Communications, Surveillance and Reconnaissance

    Campbell, William; Vehlow, Chuck; Wartell, Mike; Adler, Allen; Swan, Pete; Wynn, Bob; Beriwaln, Madhu; Collier, Darrell; Gallagher, Herb; Glaser, Gary; Scalera, Steve; Puthoff, Jolene

    2008-01-01

    ...). The Army Science Board investigated capabilities of platforms deployed in space near space and lower altitudes and assessed tradeoffs among benefits weaknesses costs and logistics burdens associated with platform types...

  19. Software Development Process Improvement in Datacom Platform

    Trabelsi, Walid

    2008-01-01

    Masteroppgave i Informasjons- og Kommunikasjonsteknologi 2008, Universitetet i Agder, Grimstad Ericsson Mobile Platform (EMP) is responsible of the development of a software platform and also to some extend responsible for related hardware parts. EMP is developing the data communication parts of the platform which is used by EMP customers. The platform development is done in large development programs and each program span over a quite a long time period. However, as we see eve...

  20. Assembly procedure for Shot Loading Platform

    Routh, R.D.

    1995-01-01

    This supporting document describes the assembly procedure for the Shot Loading Platform. The Shot Loading Platform is used by multiple equipment removal projects to load shielding shot in the annular spaces of the equipment storage containers. The platform height is adjustable to accommodate different sizes of storage containers and transport assemblies

  1. Reverse engineering of the robot base platform

    Anwar A Rahman; Azizul Rahman A Aziz; Mohd Arif Hamzah; Muhd Nor Atan; Fadil Ismail; Rosli Darmawan

    2009-01-01

    The robot base platform used to place the robotic arm version 2 was imported through a local company. The robot base platform is used as a reference for reverse egineering development for a smaller size robot. The paper will discuss the reverse engineering design process and parameters involved in the development of the robot base platform. (Author)

  2. Carbohydrate Metabolism Disorders

    ... metabolic disorder, something goes wrong with this process. Carbohydrate metabolism disorders are a group of metabolic disorders. Normally your enzymes break carbohydrates down into glucose (a type of sugar). If ...

  3. Comprehensive metabolic panel

    Metabolic panel - comprehensive; Chem-20; SMA20; Sequential multi-channel analysis with computer-20; SMAC20; Metabolic panel 20 ... Chernecky CC, Berger BJ. Comprehensive metabolic panel (CMP) - blood. In: ... Tests and Diagnostic Procedures . 6th ed. St Louis, MO: ...

  4. 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.

    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.

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

    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.

  6. An Algorithm Measuring Donor Cell-Free DNA in Plasma of Cellular and Solid Organ Transplant Recipients That Does Not Require Donor or Recipient Genotyping

    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

  7. Cell-Free DNA-Based Non-invasive Prenatal Screening for Common Aneuploidies in a Canadian Province: A Cost-Effectiveness Analysis.

    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.

  8. Pyrrhocoricin, a proline-rich antimicrobial peptide derived from insect, inhibits the translation process in the cell-free Escherichia coli protein synthesis system.

    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.

  9. Highly sensitive detection of ESR1 mutations in cell-free DNA from patients with metastatic breast cancer using molecular barcode sequencing.

    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.

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

    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

  12. 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.

    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

  13. 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.

    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

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

    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.

  15. The price of performance: a cost and performance analysis of the implementation of cell-free fetal DNA testing for Down syndrome in Ontario, Canada.

    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.

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

    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.

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

    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.

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

    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

  19. Prognostic role of APC and RASSF1A promoter methylation status in cell free circulating DNA of operable gastric cancer patients.

    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.

  20. Follicle-stimulating hormone (FSH) unmasks specific high affinity FSH-binding sites in cell-free membrane preparations of porcine granulosa cells

    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.

  1. eTumorType, An Algorithm of Discriminating Cancer Types for Circulating Tumor Cells or Cell-free DNAs in Blood.

    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.

  2. Product Platform Development in Industrial Networks

    Karlsson, Christer; Skold, Martin

    2011-01-01

    The article examines the strategic issues involved in the deployment of product platform development in an industrial network. The move entails identifying the types and characteristics of generically different product platform strategies and clarifying strategic motives and differences. Number o...... of platforms and product brands serve as the key dimensions when distinguishing the different strategies. Each strategy has its own challenges and raises various issues to deal with.......The article examines the strategic issues involved in the deployment of product platform development in an industrial network. The move entails identifying the types and characteristics of generically different product platform strategies and clarifying strategic motives and differences. Number...

  3. HPLC-MS-Based Metabonomics Reveals Disordered Lipid Metabolism in Patients with Metabolic Syndrome

    Xinjie Zhao

    2011-12-01

    Full Text Available Ultra-high performance liquid chromatography/ quadrupole time of flight mass spectrometry-based metabonomics platform was employed to profile the plasma metabolites of patients with metabolic syndrome and the healthy controls. Data analysis revealed lots of differential metabolites between the two groups, and most of them were identified as lipids. Several fatty acids and lysophosphatidylcholines were of higher plasma levels in the patient group, indicating the occurrence of insulin resistance and inflammation. The identified ether phospholipids were decreased in the patient group, reflecting the oxidative stress and some metabolic disorders. These identified metabolites can also be used to aid diagnosis of patients with metabolic syndrome. These results showed that metabonomics was a promising and powerful method to study metabolic syndrome.

  4. The ESA Geohazard Exploitation Platform

    Bally, Philippe; Laur, Henri; Mathieu, Pierre-Philippe; Pinto, Salvatore

    2015-04-01

    Earthquakes represent one of the world's most significant hazards in terms both of loss of life and damages. In the first decade of the 21st century, earthquakes accounted for 60 percent of fatalities from natural disasters, according to the United Nations International Strategy for Disaster Reduction (UNISDR). To support mitigation activities designed to assess and reduce risks and improve response in emergency situations, satellite EO can be used to provide a broad range of geo-information services. This includes for instance crustal block boundary mapping to better characterize active faults, strain rate mapping to assess how rapidly faults are deforming, soil vulnerability mapping to help estimate how the soil is behaving in reaction to seismic phenomena, geo-information to assess the extent and intensity of the earthquake impact on man-made structures and formulate assumptions on the evolution of the seismic sequence, i.e. where local aftershocks or future main shocks (on nearby faults) are most likely to occur. In May 2012, the European Space Agency and the GEO Secretariat convened the International Forum on Satellite EO for Geohazards now known as the Santorini Conference. The event was the continuation of a series of international workshops such as those organized by the Geohazards Theme of the Integrated Global Observing Strategy Partnership. In Santorini the seismic community has set out a vision of the EO contribution to an operational global seismic risk program, which lead to the Geohazard Supersites and Natural Laboratories (GSNL) initiative. The initial contribution of ESA to suuport the GSNL was the first Supersites Exploitation Platform (SSEP) system in the framework of Grid Processing On Demand (GPOD), now followed by the Geohazard Exploitation Platform (GEP). In this presentation, we will describe the contribution of the GEP for exploiting satellite EO for geohazard risk assessment. It is supporting the GEO Supersites and has been further

  5. Vaccine platform recombinant measles virus.

    Mühlebach, Michael D

    2017-10-01

    The classic development of vaccines is lengthy, tedious, and may not necessarily be successful as demonstrated by the case of HIV. This is especially a problem for emerging pathogens that are newly introduced into the human population and carry the inherent risk of pandemic spread in a naïve population. For such situations, a considerable number of different platform technologies are under development. These are also under development for pathogens, where directly derived vaccines are regarded as too complicated or even dangerous due to the induction of inefficient or unwanted immune responses causing considerable side-effects as for dengue virus. Among platform technologies are plasmid-based DNA vaccines, RNA replicons, single-round infectious vector particles, or replicating vaccine-based vectors encoding (a) critical antigen(s) of the target pathogens. Among the latter, recombinant measles viruses derived from vaccine strains have been tested. Measles vaccines are among the most effective and safest life-attenuated vaccines known. Therefore, the development of Schwarz-, Moraten-, or AIK-C-strain derived recombinant vaccines against a wide range of mostly viral, but also bacterial pathogens was quite straightforward. These vaccines generally induce powerful humoral and cellular immune responses in appropriate animal models, i.e., transgenic mice or non-human primates. Also in the recent first clinical phase I trial, the results have been quite encouraging. The trial indicated the expected safety and efficacy also in human patients, interestingly independent from the level of prevalent anti-measles immunity before the trial. Thereby, recombinant measles vaccines expressing additional antigens are a promising platform for future vaccines.

  6. Towards a Framework of Digital Platform Disruption

    Kazan, Erol; Tan, Chee-Wee; Lim, Eric T. K.

    2014-01-01

    Digital platforms are disruptive information technology (IT) artifacts that erode conventional business logic associated with traditional market structures. This paper presents a framework for examining the disruptive potential of digital platforms whereby we postulate that the strategic interplay...... digital platforms purposely decouple platform layers, to foster open innovation and accelerate market disruption. This paper therefore represents a first concrete step aimed at unravelling the disruptive potential of digital platforms....... of governance regimes and platform layers is deterministic of whether disruptive derivatives are permitted to flourish. This framework has been employed in a comparative case study between centralized (i.e., PayPal) and decentralized (i.e., Coinkite) digital payment platforms to illustrate its applicability...

  7. Turbine engine airfoil and platform assembly

    Campbell, Christian X [Oviedo, FL; James, Allister W [Chuluota, FL; Morrison, Jay A [Oviedo, FL

    2012-07-31

    A turbine airfoil (22A) is formed by a first process using a first material. A platform (30A) is formed by a second process using a second material that may be different from the first material. The platform (30A) is assembled around a shank (23A) of the airfoil. One or more pins (36A) extend from the platform into holes (28) in the shank (23A). The platform may be formed in two portions (32A, 34A) and placed around the shank, enclosing it. The two platform portions may be bonded to each other. Alternately, the platform (30B) may be cast around the shank (23B) using a metal alloy with better castability than that of the blade and shank, which may be specialized for thermal tolerance. The pins (36A-36D) or holes for them do not extend to an outer surface (31) of the platform, avoiding stress concentrations.

  8. Platforms for Innovation and Internationalization

    Rasmussen, Erik Stavnsager; Petersen, Nicolaj Hannesbo

    2017-01-01

    The high-tech global startup has many challenges related to both innovation and internationalization. From a Danish cluster of Welfare Tech firms, eight innovative and international firms were selected and interviewed. Such firms typically have to be agile and operate in virtual networks in almost...... all parts of their value chains. This article contributes to the understanding of how innovation and internationalization to a great extent are interlinked. The firms have developed a core product or service offering, which the firms often describe as “a platform”. Around the platform, they develop...

  9. PRISMES. The INES microgrid platform

    Barruel, F.; Vervaart, M.; Merten, J. [INES, 73 - Le Bourget-du-lac (FR). Lab. for Solar Systems (L2S)

    2010-07-01

    The massive penetration of fluctuating renewable energies will challenge the low voltage grids. Innovative solutions to address this issue need to be developed and tested. They may consist of intelligent energy management systems and the use of storage systems. For this purpose the PRISMES platform has been designed and implemented at the INES headquarter in Chambery. It is a universal test and development tool, such as stand alone hybrid systems and grid connected PV systems combined with a storage system and loads. This paper describes the PRISMES facility which will start operation in autumn 2008. (orig.)

  10. Microalgal Metabolic Network Model Refinement through High-Throughput Functional Metabolic Profiling

    Chaiboonchoe, Amphun; Dohai, Bushra Saeed; Cai, Hong; Nelson, David R.; Jijakli, Kenan; Salehi-Ashtiani, Kourosh

    2014-01-01

    Metabolic modeling provides the means to define metabolic processes at a systems level; however, genome-scale metabolic models often remain incomplete in their description of metabolic networks and may include reactions that are experimentally unverified. This shortcoming is exacerbated in reconstructed models of newly isolated algal species, as there may be little to no biochemical evidence available for the metabolism of such isolates. The phenotype microarray (PM) technology (Biolog, Hayward, CA, USA) provides an efficient, high-throughput method to functionally define cellular metabolic activities in response to a large array of entry metabolites. The platform can experimentally verify many of the unverified reactions in a network model as well as identify missing or new reactions in the reconstructed metabolic model. The PM technology has been used for metabolic phenotyping of non-photosynthetic bacteria and fungi, but it has not been reported for the phenotyping of microalgae. Here, we introduce the use of PM assays in a systematic way to the study of microalgae, applying it specifically to the green microalgal model species Chlamydomonas reinhardtii. The results obtained in this study validate a number of existing annotated metabolic reactions and identify a number of novel and unexpected metabolites. The obtained information was used to expand and refine the existing COBRA-based C. reinhardtii metabolic network model iRC1080. Over 254 reactions were added to the network, and the effects of these additions on flux distribution within the network are described. The novel reactions include the support of metabolism by a number of d-amino acids, l-dipeptides, and l-tripeptides as nitrogen sources, as well as support of cellular respiration by cysteamine-S-phosphate as a phosphorus source. The protocol developed here can be used as a foundation to functionally profile other microalgae such as known microalgae mutants and novel isolates.

  11. Microalgal Metabolic Network Model Refinement through High-Throughput Functional Metabolic Profiling

    Chaiboonchoe, Amphun; Dohai, Bushra Saeed; Cai, Hong; Nelson, David R. [Division of Science and Math, New York University Abu Dhabi, Abu Dhabi (United Arab Emirates); Center for Genomics and Systems Biology (CGSB), New York University Abu Dhabi Institute, Abu Dhabi (United Arab Emirates); Jijakli, Kenan [Division of Science and Math, New York University Abu Dhabi, Abu Dhabi (United Arab Emirates); Center for Genomics and Systems Biology (CGSB), New York University Abu Dhabi Institute, Abu Dhabi (United Arab Emirates); Engineering Division, Biofinery, Manhattan, KS (United States); Salehi-Ashtiani, Kourosh, E-mail: ksa3@nyu.edu [Division of Science and Math, New York University Abu Dhabi, Abu Dhabi (United Arab Emirates); Center for Genomics and Systems Biology (CGSB), New York University Abu Dhabi Institute, Abu Dhabi (United Arab Emirates)

    2014-12-10

    Metabolic modeling provides the means to define metabolic processes at a systems level; however, genome-scale metabolic models often remain incomplete in their description of metabolic networks and may include reactions that are experimentally unverified. This shortcoming is exacerbated in reconstructed models of newly isolated algal species, as there may be little to no biochemical evidence available for the metabolism of such isolates. The phenotype microarray (PM) technology (Biolog, Hayward, CA, USA) provides an efficient, high-throughput method to functionally define cellular metabolic activities in response to a large array of entry metabolites. The platform can experimentally verify many of the unverified reactions in a network model as well as identify missing or new reactions in the reconstructed metabolic model. The PM technology has been used for metabolic phenotyping of non-photosynthetic bacteria and fungi, but it has not been reported for the phenotyping of microalgae. Here, we introduce the use of PM assays in a systematic way to the study of microalgae, applying it specifically to the green microalgal model species Chlamydomonas reinhardtii. The results obtained in this study validate a number of existing annotated metabolic reactions and identify a number of novel and unexpected metabolites. The obtained information was used to expand and refine the existing COBRA-based C. reinhardtii metabolic network model iRC1080. Over 254 reactions were added to the network, and the effects of these additions on flux distribution within the network are described. The novel reactions include the support of metabolism by a number of d-amino acids, l-dipeptides, and l-tripeptides as nitrogen sources, as well as support of cellular respiration by cysteamine-S-phosphate as a phosphorus source. The protocol developed here can be used as a foundation to functionally profile other microalgae such as known microalgae mutants and novel isolates.

  12. ENERCA: e-learning platform

    Patricia Aguilar-Martínez

    2014-01-01

    ENERCA (the European Network for Rare and Congenital Anemias) is a European Commission funded project since 2002. Rare anaemias in Europe comprise haemoglobin disorders, such as thalassemias or sickle cell disease, and other rarer disorders, including inherited diseases of the red cell membrane, of the red cell enzymes, as well as anaemias related to disorders of iron metabolism. Education and training on rare anaemias is one of the main objectives of ENERCA and several educational initiative...

  13. Corynebacterium glutamicum for Sustainable Bioproduction: From Metabolic Physiology to Systems Metabolic Engineering.

    Becker, Judith; Gießelmann, Gideon; Hoffmann, Sarah Lisa; Wittmann, Christoph

    Since its discovery 60 years ago, Corynebacterium glutamicum has evolved into a workhorse for industrial biotechnology. Traditionally well known for its remarkable capacity to produce amino acids, this Gram-positive soil bacterium, has become a flexible, efficient production platform for various bulk and fine chemicals, materials, and biofuels. The central turnstile of all these achievements is our excellent understanding of its metabolism and physiology. This knowledge base, together with innovative systems metabolic engineering concepts, which integrate systems and synthetic biology into strain engineering, has upgraded C. glutamicum into one of the most successful industrial microorganisms in the world.

  14. Autonomous Landing on Moving Platforms

    Mendoza Chavez, Gilberto

    2016-08-01

    This thesis investigates autonomous landing of a micro air vehicle (MAV) on a nonstationary ground platform. Unmanned aerial vehicles (UAVs) and micro air vehicles (MAVs) are becoming every day more ubiquitous. Nonetheless, many applications still require specialized human pilots or supervisors. Current research is focusing on augmenting the scope of tasks that these vehicles are able to accomplish autonomously. Precise autonomous landing on moving platforms is essential for self-deployment and recovery of MAVs, but it remains a challenging task for both autonomous and piloted vehicles. Model Predictive Control (MPC) is a widely used and effective scheme to control constrained systems. One of its variants, output-feedback tube-based MPC, ensures robust stability for systems with bounded disturbances under system state reconstruction. This thesis proposes a MAV control strategy based on this variant of MPC to perform rapid and precise autonomous landing on moving targets whose nominal (uncommitted) trajectory and velocity are slowly varying. The proposed approach is demonstrated on an experimental setup.

  15. 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.

    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.

  16. Mass spectrometry and metabolism of leukotriene B4

    Harper, T.W.

    1985-01-01

    Leukotriene B 4 (LTB 4 ) is a potent and powerful chemotactic factor for polymorphonuclear leukocytes and has been hypothesized to play a role as a mediator of the inflammatory response. A cell free system capable of generating leukotrienes from exogenous arachidonic acid was developed using both rat basophilic leukemia cells and murine mastocytoma cells. The cell free leukotriene generating system was used to prepare leukotrienes for mass spectral studies using electron impact (EI), chemical ionization (Cl), negative ion chemical ionization (NCl) and both positive and negative ion fast atom bombardment mass spectrometry (FAB-MS). Although all of the techniques examined yielded useful information, NCl was found to be the most useful for sensitive quantitation of leukotrienes, whereas Cl and El are more useful for unambiguous structural characterization of hydroxy fatty acid leukotrienes. Investigation of the in vivo metabolism of LTB 4 in the mouse and guinea pig indicated that less than 10% of the injected radiolabel from 3 H-LTB 4 was excreted via the urine and feces. Approximately 30% of the urinary radioactivity was volatile, probably due to loss of the tritium label as water. In the monkey, approximately 10% of the injected radiolabel from 14 C-LTB 4 was excreted in the urine within 5 1/2 hours. In this study, approximately 10% of the urinary radiolabel was volatile, with a large number of unidentified non-volatile products also observed. The isolated perfused rat lung was found to rapidly metabolize leukotriene C 4 to leukotrienes D 4 and E 4 and to leukotriene C 4 sulfoxide

  17. 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.

    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.

  18. 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.

    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.

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

    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.

  20. 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.

    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.

  1. Implications Enzymatic Degradation of the Endothelial Glycocalyx on the Microvascular Hemodynamics and the Arteriolar Red Cell Free Layer of the Rat Cremaster Muscle

    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

  2. The value of fluorimetry (Qubit) and spectrophotometry (NanoDrop) in the quantification of cell-free DNA (cfDNA) in malignant melanoma and prostate cancer patients.

    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.

  3. Implications Enzymatic Degradation of the Endothelial Glycocalyx on the Microvascular Hemodynamics and the Arteriolar Red Cell Free Layer of the Rat Cremaster Muscle.

    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

  4. An open source platform for multi-scale spatially distributed simulations of microbial ecosystems

    Segre, Daniel [Boston Univ., MA (United States)

    2014-08-14

    The goal of this project was to develop a tool for facilitating simulation, validation and discovery of multiscale dynamical processes in microbial ecosystems. This led to the development of an open-source software platform for Computation Of Microbial Ecosystems in Time and Space (COMETS). COMETS performs spatially distributed time-dependent flux balance based simulations of microbial metabolism. Our plan involved building the software platform itself, calibrating and testing it through comparison with experimental data, and integrating simulations and experiments to address important open questions on the evolution and dynamics of cross-feeding interactions between microbial species.

  5. Digital platforms as enablers for digital transformation

    Hossain, Mokter; Lassen, Astrid Heidemann

    transformation is crucial. This study aims at exploring how organizations are driven towards transformation in various ways to embrace digital platforms for ideas, technologies, and knowledge. It shows the opportunities and challenges digital platforms bring in organizations. It also highlights underlying......Digital platforms offer new ways for organizations to collaborate with the external environment for ideas, technologies, and knowledge. They provide new possibilities and competence but they also bring new challenges for organizations. Understanding the role of these platforms in digital...... mechanisms and potential outcomes of various digital platforms. The contribution of the submission is valuable for scholars to understand and further explore this area. It provides insight for practitioners to capture value through digital platforms and accelerate the pace of organizations’ digital...

  6. Platform development: implications for portfolio management

    Hsuan, Juliana; Hansen, Poul H. Kyvsgård

    2007-01-01

    " The challenge of implementing industrial platforms in practice can be described as a configuration problem caused by a considerable number of variables, which often have contradictory influences on the total performance of the firm. Consequently, the specific platform decisions become extremely...... complex, possibly increasing the strategic risks for the firm. This paper reports preliminary findings on platform management process at LEGO, a Danish toy company. Specifically, we report the process of applying games combined with simulations and workshops in the platform development. We also propose...... a framework, based on the portfolio management thinking to evaluate the degree of modularity embedded in a given platform and to which extent it is aligned with other platforms."...

  7. Platform economy in Denmark – precarious employment?

    Rasmussen, Stine; Madsen, Per Kongshøj

    limited. Nevertheless the labour offered through the platforms has a precarious character for instance in terms of lower wages and poorer rights and protection compared to the labour at the traditional, offline labour market. One important issue here is also the confusion as to whether the worker......This paper takes a labour market perspective on the emerging concept of the 'sharing economy' or 'platform economy', which we use as a more appropriate term for the phenomenon. Platform economy is in the article understood as those business models that have emerged since the millennium, where...... digital platforms serve as the link between persons wanting to make use of certain activities, services etc. and those owning them and we only have an interest in the work-related platforms. That means platforms, where paid work is offered and demanded. International examples of this new phenomenon...

  8. A Virtual Commissioning Learning Platform

    Mortensen, Steffen; Madsen, Ole

    2018-01-01

    The introduction of reconfigurable manufacturing systems (RMS), Industry 4.0 and the associated technologies requires the establishment of new competencies. Towards that goal, Aalborg University (AAU) has developed an Industry 4.0 learning factory, the AAU Smart Production Lab. The AAU Smart...... Production Lab integrates a number of Industry 4.0 technologies for learning and research purposes. One of the many techniques is virtual commissioning. Virtual commissioning uses a virtual plant model and real controllers (PLCs) enabling a full emulation of the manufacturing system for verification. Virtual...... commissioning can lower the commissioning time up to 63%, allowing faster time to market. However, virtual commission is still missing industrial impact one of the reasons being lack of competencies and integration experiences. The paper presents the setup of the virtual commissioning learning platform...

  9. Generational Transitions in Platform Markets

    Kretschmer, Tobias; Claussen, Jörg

    2016-01-01

    The introduction of a new product generation forces incumbents in platform markets to rebuild their installed base of complementary products. Using three different data sets on the U.S. market for video game consoles, we show that incumbents can, to some extent, substitute for rebuilding their new...... installed base by making their new products backward compatible, which lets them draw on the installed base of software for the parent generation. However, while this positive direct effect of backward compatibility dominates in our setting, we also observe a (weaker) negative indirect effect working...... through the reduced supply of new software. We find that both effects are moderated by the age of the new technological generation and by the technological leap between generations: backward compatibility becomes less important with increasing console age and larger technological improvement between...

  10. SERS sensors for DVD platform

    Brøgger, Anna Line

    This Ph.D. thesis explores the engineering of a portable sensor system for detection of rare and small molecules. The Ph.D. project is part of the research project 'Multi-Sensor DVD platform' (MUSE), aiming to integrate different sensors on a rotating disc. The sensors are chosen to complement each...... other, creating more reliable and stable results for the end user. The rotating disc comprises microfluidic channels, which can be utilized for handling and manipulating liquid samples such as blood or water. The focus of this Ph.D. thesis, is on the integration of one specific sensor on a rotating disc....... The sensor is based upon surface enhanced Raman spectroscopy (SERS), which detects molecular vibrations. The aim of this thesis is to cover the different aspects of the sensor system. SERS substrates, consisting of nanopillars with gold or silver caps on top, have been fabricated by standard micro and nano...

  11. Language Geography from Microblogging Platforms

    Mocanu, Delia; Baronchelli, Andrea; Perra, Nicola; Gonçalves, Bruno; Vespignani, Alessandro

    2013-03-01

    Microblogging platforms have now become major open source indicators for complex social interactions. With the advent of smartphones, the everincreasing mobile Internet traffic gives us the unprecedented opportunity to complement studies of complex social phenomena with real-time location information. In this work, we show that the data nowadays accessible allows for detailed studies at different scales, ranging from country-level aggregate analysis to the analysis of linguistic communities withing specific neighborhoods. The high resolution and coverage of this data permits us to investigate such issues as the linguistic homogeneity of different countries, touristic seasonal patterns within countries, and the geographical distribution of different languages in bilingual regions. This work highlights the potentialities of geolocalized studies of open data sources that can provide an extremely detailed picture of the language geography.

  12. Quantum photonics hybrid integration platform

    Murray, E.; Floether, F. F. [Cambridge Research Laboratory, Toshiba Research Europe Limited, 208 Science Park, Milton Road, Cambridge CB4 0GZ (United Kingdom); Cavendish Laboratory, University of Cambridge, J.J. Thomson Avenue, Cambridge CB3 0HE (United Kingdom); Ellis, D. J. P.; Meany, T.; Bennett, A. J., E-mail: anthony.bennet@crl.toshiba.co.uk; Shields, A. J. [Cambridge Research Laboratory, Toshiba Research Europe Limited, 208 Science Park, Milton Road, Cambridge CB4 0GZ (United Kingdom); Lee, J. P. [Cambridge Research Laboratory, Toshiba Research Europe Limited, 208 Science Park, Milton Road, Cambridge CB4 0GZ (United Kingdom); Engineering Department, University of Cambridge, 9 J. J. Thomson Avenue, Cambridge CB3 0FA (United Kingdom); Griffiths, J. P.; Jones, G. A. C.; Farrer, I.; Ritchie, D. A. [Cavendish Laboratory, University of Cambridge, J.J. Thomson Avenue, Cambridge CB3 0HE (United Kingdom)

    2015-10-26

    Fundamental to integrated photonic quantum computing is an on-chip method for routing and modulating quantum light emission. We demonstrate a hybrid integration platform consisting of arbitrarily designed waveguide circuits and single-photon sources. InAs quantum dots (QD) embedded in GaAs are bonded to a SiON waveguide chip such that the QD emission is coupled to the waveguide mode. The waveguides are SiON core embedded in a SiO{sub 2} cladding. A tuneable Mach Zehnder interferometer (MZI) modulates the emission between two output ports and can act as a path-encoded qubit preparation device. The single-photon nature of the emission was verified using the on-chip MZI as a beamsplitter in a Hanbury Brown and Twiss measurement.

  13. Understanding Platform-Based Digital Currencies

    Ben Fung; Hanna Halaburda

    2014-01-01

    Given technological advances and the widespread use of the Internet, various digital currencies have emerged. In most cases, Internet platforms such as Facebook and Amazon restrict the functionality of their digital currencies to enhance the business model and maximize their profits. While platform-based digital currencies could increase the efficiency of retail payments, they could also raise some important policy issues if they were to become widely used outside of the platform. Thus, it is...

  14. Applied architecture patterns on the Microsoft platform

    Dovgal, Andre; Noriskin, Gregor

    2014-01-01

    Presented in a scenario-driven tutorial way, we lead you through fictitious example problems and present you with the best solutions. This book is intended for architects, developers, and managers who need to improve their knowledge of the Microsoft application platform. This book will appeal to anyone, especially consultants, who want to get up to speed on selecting the most appropriate platform for a particular problem. A good understanding of the general Windows platform and development technologies would be helpful.

  15. Gas turbine bucket with impingement cooled platform

    Jones, Raphael Durand

    2002-01-01

    In a turbine bucket having an airfoil portion and a root portion, with a substantially planar platform at an interface between the airfoil portion and root portion, a platform cooling arrangement including at least one bore in the root portion and at least one impingement cooling tube seated in the bore, the tube extending beyond the bore with an outlet in close proximity to a targeted area on an underside of the platform.

  16. Launching platforms for user-generated content

    Batista, Guilherme Luís Caroço

    2015-01-01

    Field lab: Entrepreneurial and innovative ventures This paper intends to discuss and absorb the Best Practices employed by successful User- Generated Content (UGC)1 platforms and constitute a guide on how to launch a platform without having a cyclical lack of content and users. Research shows that companies have resorted to integration with mature UGC platforms, and providing content by themselves, in an initial state. I conclude that integration possibilities should be explore...

  17. Disentangling Competition Among Platform Driven Strategic Groups

    Kazan, Erol; Tan, Chee-Wee; Lim, Eric T. K.

    2018-01-01

    for the mobile payment market in the United Kingdom as our empirical setting. By conceptualizing digital platforms as layered modular architectures and embracing the theoretical lens of strategic groups, this study supplements prior research by deriving a taxonomy of platform profiles that is grounded...... delivery architecture. The preceding attributes of value creation architecture and value delivery architecture aided us in identifying six profiles associated with mobile payment platforms, which in turn led us to advance three competitive strategies that could be pursued by digital platforms in network...

  18. DESIGN OF OFFSHORE CONCRETE GRAVITY PLATFORMS

    gas industry. Manufacturing and construction methods are discussed. Current ... Keywords: concrete gravity platform, offshore, foundation design, manufacturing, ... forms are used to support production drilling ... Manufacture and Construction.

  19. Helicopter flight simulation motion platform requirements

    Schroeder, Jeffery Allyn

    Flight simulators attempt to reproduce in-flight pilot-vehicle behavior on the ground. This reproduction is challenging for helicopter simulators, as the pilot is often inextricably dependent on external cues for pilot-vehicle stabilization. One important simulator cue is platform motion; however, its required fidelity is unknown. To determine the required motion fidelity, several unique experiments were performed. A large displacement motion platform was used that allowed pilots to fly tasks with matched motion and visual cues. Then, the platform motion was modified to give cues varying from full motion to no motion. Several key results were found. First, lateral and vertical translational platform cues had significant effects on fidelity. Their presence improved performance and reduced pilot workload. Second, yaw and roll rotational platform cues were not as important as the translational platform cues. In particular, the yaw rotational motion platform cue did not appear at all useful in improving performance or reducing workload. Third, when the lateral translational platform cue was combined with visual yaw rotational cues, pilots believed the platform was rotating when it was not. Thus, simulator systems can be made more efficient by proper combination of platform and visual cues. Fourth, motion fidelity specifications were revised that now provide simulator users with a better prediction of motion fidelity based upon the frequency responses of their motion control laws. Fifth, vertical platform motion affected pilot estimates of steady-state altitude during altitude repositionings. This refutes the view that pilots estimate altitude and altitude rate in simulation solely from visual cues. Finally, the combined results led to a general method for configuring helicopter motion systems and for developing simulator tasks that more likely represent actual flight. The overall results can serve as a guide to future simulator designers and to today's operators.

  20. Platform for systems medicine research and diagnostic applications in psychotic disorders-The METSY project.

    Frank, Elisabeth; Maier, Dieter; Pajula, Juha; Suvitaival, Tommi; Borgan, Faith; Butz-Ostendorf, Markus; Fischer, Alexander; Hietala, Jarmo; Howes, Oliver; Hyötyläinen, Tuulia; Janssen, Joost; Laurikainen, Heikki; Moreno, Carmen; Suvisaari, Jaana; Van Gils, Mark; Orešič, Matej

    2018-04-01

    Psychotic disorders are associated with metabolic abnormalities including alterations in glucose and lipid metabolism. A major challenge in the treatment of psychosis is to identify patients with vulnerable metabolic profiles who may be at risk of developing cardiometabolic co-morbidities. It is established that both central and peripheral metabolic organs use lipids to control energy balance and regulate peripheral insulin sensitivity. The endocannabinoid system, implicated in the regulation of glucose and lipid metabolism, has been shown to be dysregulated in psychosis. It is currently unclear how these endocannabinoid abnormalities relate to metabolic changes in psychosis. Here we review recent research in the field of metabolic co-morbidities in psychotic disorders as well as the methods to study them and potential links to the endocannabinoid system. We also describe the bioinformatics platforms developed in the EU project METSY for the investigations of the biological etiology in patients at risk of psychosis and in first episode psychosis patients. The METSY project was established with the aim to identify and evaluate multi-modal peripheral and neuroimaging markers that may be able to predict the onset and prognosis of psychiatric and metabolic symptoms in patients at risk of developing psychosis and first episode psychosis patients. Given the intrinsic complexity and widespread role of lipid metabolism, a systems biology approach which combines molecular, structural and functional neuroimaging methods with detailed metabolic characterisation and multi-variate network analysis is essential in order to identify how lipid dysregulation may contribute to psychotic disorders. A decision support system, integrating clinical, neuropsychological and neuroimaging data, was also developed in order to aid clinical decision making in psychosis. Knowledge of common and specific mechanisms may aid the etiopathogenic understanding of psychotic and metabolic disorders