Process for assembly and transformation into Saccharomyces cerevisiae of a synthetic yeast artificial chromosome containing a multigene cassette to express enzymes that enhance xylose utilization designed for an automated pla

USDA-ARS?s Scientific Manuscript database

A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system ...

Modification and identification of a vector for making a large phage antibody library.

PubMed

Zhang, Guo-min; Chen, Yü-ping; Guan, Yuan-zhi; Wang, Yan; An, Yun-qing

2007-11-20

The large phage antibody library is used to obtain high-affinity human antibody, and the Loxp/cre site-specific recombination system is a potential method for constructing a large phage antibody library. In the present study, a phage antibody library vector pDF was reconstructed to construct diabody more quickly and conveniently without injury to homologous recombination and the expression function of the vector and thus to integrate construction of the large phage antibody library with the preparation of diabodies. scFv was obtained by overlap polymerase chain reaction (PCR) amplification with the newly designed VL and VH extension primers. loxp511 was flanked by VL and VH and the endonuclease ACC III encoding sequences were introduced on both sides of loxp511. scFv was cloned into the vector pDF to obtain the vector pDscFv. The vector expression function was identified and the feasibility of diabody preparation was evaluated. A large phage antibody library was constructed in pDscFv. Several antigens were used to screen the antibody library and the quality of the antibody library was evaluated. The phage antibody library expression vector pDscFv was successfully constructed and confirmed to express functional scFv. The large phage antibody library constructed using this vector was of high diversity. Screening of the library on 6 antigens confirmed the generation of specific antibodies to these antigens. Two antibodies were subjected to enzymatic digestion and were prepared into diabody with functional expression. The reconstructed vector pDscFv retains its recombination capability and expression function and can be used to construct large phage antibody libraries. It can be used as a convenient and quick method for preparing diabodies after simple enzymatic digestion, which facilitates clinical trials and application of antibody therapy.

Construction, expression and in vitro biological behaviors of Ig scFv fragment in patients with chronic B cell leukemia.

PubMed

Zhu, Lijuan; Liao, Wenjun; Zhu, Huifen; Lei, Ping; Wang, Zhihua; Shao, Jingfang; Zhang, Yue; Shen, Guanxin

2006-01-01

The expression vector of SmIg scFv fragment was constructed in patient with B cell chronic lymphocyte leukemia (B-CLL) and expressed in E. coli to obtain scFv fragment, and the effect of the protein on the proliferation of stimulated peripheral blood mononuclear cells (PBMC) was investigated in vitro. Two pairs of primers were designed, and variable region genes of light chain and heavy chain were amplified by PCR respectively from the pGEM-T vectors previously constructed in our laboratory which containing light chain gene or Fd fragment of heavy chain gene. The PCR product was digested, purified and inserted into pHEN2 vector to construct the soluble expression vector pHEN2-scFv. After the induction by IPTG, the scFv protein was identified by SDS-PAGE electrophoresis and purified by Ni-NTA-Chromatography. MTT was used to determine the effect of purified protein on the proliferation of stimulated PBMC in vitro. Plasmid PCR and restriction enzyme digestion of pHEN2-scFv revealed the pHEN2-scFv vector was constructed successfully. Id-scFv protein was expressed in positive clone after induced by IPTG. SDS-PAGE analysis showed that the relative molecular weight of fusion protein was about 30 kD (1 kD= 0.9921 ku), which was consistent with the theoretically predicted value. Proliferation of PBMC could be induced by purified Id-scFv. It was suggested that the expression vector of SmIg scFv fragment was constructed successfully, and scFv protein was expressed and secreted from E. coli, which could induce proliferation of PBMC. This may lay an experimental foundation for further research of Id-HSP complex vaccine for B-CLL.

Construction of pDUO: A bicistronic shuttle vector series for dual expression of recombinant proteins

USDA-ARS?s Scientific Manuscript database

Our ability to genetically manipulate microbial systems is often hampered by the availability of genetic tools. Thus, there is a need for the continued expansion of our molecular tool box. In support of this expansion, this study reports the design, construction, and validation of a new shuttle vect...

76 FR 2096 - Record of Decision for the Environmental Impact Statement for the Proposed Abengoa Biorefinery...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-01-12

... the design, construction, and startup of a commercial-scale integrated biorefinery to be located near... responsibilities under EPAct 2005, DOE issued a funding opportunity announcement in February 2006 for the design... proposed transmission line be designed to protect migratory birds and raptors. A few commenters expressed...

An Overview of Starfish: A Table-Centric Tool for Interactive Synthesis

NASA Technical Reports Server (NTRS)

Tsow, Alex

2008-01-01

Engineering is an interactive process that requires intelligent interaction at many levels. My thesis [1] advances an engineering discipline for high-level synthesis and architectural decomposition that integrates perspicuous representation, designer interaction, and mathematical rigor. Starfish, the software prototype for the design method, implements a table-centric transformation system for reorganizing control-dominated system expressions into high-level architectures. Based on the digital design derivation (DDD) system a designer-guided synthesis technique that applies correctness preserving transformations to synchronous data flow specifications expressed as co- recursive stream equations Starfish enhances user interaction and extends the reachable design space by incorporating four innovations: behavior tables, serialization tables, data refinement, and operator retiming. Behavior tables express systems of co-recursive stream equations as a table of guarded signal updates. Developers and users of the DDD system used manually constructed behavior tables to help them decide which transformations to apply and how to specify them. These design exercises produced several formally constructed hardware implementations: the FM9001 microprocessor, an SECD machine for evaluating LISP, and the SchemEngine, garbage collected machine for interpreting a byte-code representation of compiled Scheme programs. Bose and Tuna, two of DDD s developers, have subsequently commercialized the design derivation methodology at Derivation Systems, Inc. (DSI). DSI has formally derived and validated PCI bus interfaces and a Java byte-code processor; they further executed a contract to prototype SPIDER-NASA's ultra-reliable communications bus. To date, most derivations from DDD and DRS have targeted hardware due to its synchronous design paradigm. However, Starfish expressions are independent of the synchronization mechanism; there is no commitment to hardware or globally broadcast clocks. Though software back-ends for design derivation are limited to the DDD stream-interpreter, targeting synchronous or real-time software is not substantively different from targeting hardware.

Integrated pathway-based transcription regulation network mining and visualization based on gene expression profiles.

PubMed

Kibinge, Nelson; Ono, Naoaki; Horie, Masafumi; Sato, Tetsuo; Sugiura, Tadao; Altaf-Ul-Amin, Md; Saito, Akira; Kanaya, Shigehiko

2016-06-01

Conventionally, workflows examining transcription regulation networks from gene expression data involve distinct analytical steps. There is a need for pipelines that unify data mining and inference deduction into a singular framework to enhance interpretation and hypotheses generation. We propose a workflow that merges network construction with gene expression data mining focusing on regulation processes in the context of transcription factor driven gene regulation. The pipeline implements pathway-based modularization of expression profiles into functional units to improve biological interpretation. The integrated workflow was implemented as a web application software (TransReguloNet) with functions that enable pathway visualization and comparison of transcription factor activity between sample conditions defined in the experimental design. The pipeline merges differential expression, network construction, pathway-based abstraction, clustering and visualization. The framework was applied in analysis of actual expression datasets related to lung, breast and prostrate cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

High-Yield Resveratrol Production in Engineered Escherichia coli ▿ †

PubMed Central

Lim, Chin Giaw; Fowler, Zachary L.; Hueller, Thomas; Schaffer, Steffen; Koffas, Mattheos A. G.

2011-01-01

Plant polyphenols have been the subject of several recent scientific investigations since many of the molecules in this class have been found to be highly active in the human body, with a plethora of health-promoting activities against a variety of diseases, including heart disease, diabetes, and cancer, and with even the potential to slow aging. Further development of these potent natural therapeutics hinges on the formation of robust industrial production platforms designed using specifically selected as well as engineered protein sources along with the construction of optimal expression platforms. In this work, we first report the investigation of various stilbene synthases from an array of plant species considering structure-activity relationships, their expression efficiency in microorganisms, and their ability to synthesize resveratrol. Second, we looked into the construct environment of recombinantly expressed stilbene synthases, including different promoters, construct designs, and host strains, to create an Escherichia coli strain capable of producing superior resveratrol titers sufficient for commercial usage. Further improvement of metabolic capabilities of the recombinant strain aimed at improving the intracellular malonyl-coenzyme A pool, a resveratrol precursor, resulted in a final improved titer of 2.3 g/liter resveratrol. PMID:21441338

The road plan model: Information model for planning road building activities

NASA Technical Reports Server (NTRS)

Azinhal, Rafaela K.; Moura-Pires, Fernando

1994-01-01

The general building contractor is presented with an information model as an approach for deriving a high-level work plan of construction activities applied to road building. Road construction activities are represented in a Road Plan Model (RPM), which is modeled in the ISO standard STEP/EXPRESS and adopts various concepts from the GARM notation. The integration with the preceding road design stage and the succeeding phase of resource scheduling is discussed within the framework of a Road Construction Model. Construction knowledge is applied to the road design and the terrain model of the surrounding road infrastructure for the instantiation of the RPM. Issues regarding the implementation of a road planner application supporting the RPM are discussed.

Design and construction of 2A peptide-linked multicistronic vectors.

PubMed

Szymczak-Workman, Andrea L; Vignali, Kate M; Vignali, Dario A A

2012-02-01

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. This article describes the design and construction of 2A peptide-linked multicistronic vectors, which can be used to express multiple proteins from a single open reading frame (ORF). The small 2A peptide sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence. Expression of more than two genes using conventional approaches has several limitations, most notably imbalanced protein expression and large size. The use of 2A peptide sequences alleviates these concerns. They are small (18-22 amino acids) and have divergent amino-terminal sequences, which minimizes the chance for homologous recombination and allows for multiple, different 2A peptide sequences to be used within a single vector. Importantly, separation of genes placed between 2A peptide sequences is nearly 100%, which allows for stoichiometric and concordant expression of the genes, regardless of the order of placement within the vector.

Functionalized PEI Nanoparticles For Delivery Of IGF-1R-Targeted siRNA's to UPAR-Expressing Tumors In Vitro And In Vivo

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

Giblin, Michael F

2012-12-14

This proposal addressed the use of imaging technologies to develop therapeutic nanoparticle constructs which could reduce expression of molecules within the cancer cell important in tumor progression. The proposal described new labeling techniques that would result in therapeutic constructs which could be tracked both within targeted cells individually as well as within the individuals being treated. Representing a new generation of dual-labeled in vivo imaging agent, the constructs envisioned here would allow microPET imaging of targeted receptor expression as well as fluorescent imaging of silencing complexes targeting IGF-1R mRNA's. As such, this proposal was highly relevant to the Office ofmore » Biological and Environmental Research (BER) goals of facilitating improvements in radiotracer design in order to solve critical problems in biology and nuclear medicine.« less

Design and Implementation of a Student-Generated Virtual Museum in a Language Curriculum to Enhance Collaborative Multimodal Meaning-Making

ERIC Educational Resources Information Center

Ho, Caroline M. L.; Nelson, Mark Evan; Mueller-Wittig, Wolfgang

2011-01-01

This paper reports on a study, "MUSE", which involved Secondary (Grade 7) students in designing and constructing a virtual museum. It presents a description and evaluation of the design and implementation of the technologically-mediated intervention within a language curriculum that emphasizes multimodal meaning-making and expression.…

Design verification of SIFT

NASA Technical Reports Server (NTRS)

Moser, Louise; Melliar-Smith, Michael; Schwartz, Richard

1987-01-01

A SIFT reliable aircraft control computer system, designed to meet the ultrahigh reliability required for safety critical flight control applications by use of processor replications and voting, was constructed for SRI, and delivered to NASA Langley for evaluation in the AIRLAB. To increase confidence in the reliability projections for SIFT, produced by a Markov reliability model, SRI constructed a formal specification, defining the meaning of reliability in the context of flight control. A further series of specifications defined, in increasing detail, the design of SIFT down to pre- and post-conditions on Pascal code procedures. Mechanically checked mathematical proofs were constructed to demonstrate that the more detailed design specifications for SIFT do indeed imply the formal reliability requirement. An additional specification defined some of the assumptions made about SIFT by the Markov model, and further proofs were constructed to show that these assumptions, as expressed by that specification, did indeed follow from the more detailed design specifications for SIFT. This report provides an outline of the methodology used for this hierarchical specification and proof, and describes the various specifications and proofs performed.

Validation of a Theory of Planned Behavior-Based Questionnaire to Examine Factors Associated With Milk Expression.

PubMed

Bai, Yeon K; Dinour, Lauren M

2017-11-01

A proper assessment of multidimensional needs for breastfeeding mothers in various settings is crucial to facilitate and support breastfeeding and its exclusivity. The theory of planned behavior (TPB) has been used frequently to measure factors associated with breastfeeding. Full utility of the TPB requires accurate measurement of theory constructs. Research aim: This study aimed to develop and confirm the psychometric properties of an instrument, Milk Expression on Campus, based on the TPB and to establish the reliability and validity of the instrument. In spring 2015, 218 breastfeeding (current or in the recent past) employees and students at one university campus in northern New Jersey completed the online questionnaire containing demography and theory-based items. Internal consistency (α) and split-half reliability ( r) tests and factor analyses established and confirmed the reliability and construct validity of this instrument. Milk Expression on Campus showed strong and significant reliabilities as a full scale (α = .78, r = .74, p < .001) and theory construct subscales. Validity was confirmed as psychometric properties corresponded to the factors extracted from the scale. Four factors extracted from the direct construct subscales accounted for 79.49% of the total variability. Four distinct factors from the indirect construct subscales accounted for 73.68% of the total variability. Milk Expression on Campus can serve as a model TPB-based instrument to examine factors associated with women's milk expression behavior. The utility of this instrument extends to designing effective promotion programs to foster breastfeeding and milk expression behaviors in diverse settings.

Designing synthetic RNA for delivery by nanoparticles

NASA Astrophysics Data System (ADS)

Jedrzejczyk, Dominika; Gendaszewska-Darmach, Edyta; Pawlowska, Roza; Chworos, Arkadiusz

2017-03-01

The rapid development of synthetic biology and nanobiotechnology has led to the construction of various synthetic RNA nanoparticles of different functionalities and potential applications. As they occur naturally, nucleic acids are an attractive construction material for biocompatible nanoscaffold and nanomachine design. In this review, we provide an overview of the types of RNA and nucleic acid’s nanoparticle design, with the focus on relevant nanostructures utilized for gene-expression regulation in cellular models. Structural analysis and modeling is addressed along with the tools available for RNA structural prediction. The functionalization of RNA-based nanoparticles leading to prospective applications of such constructs in potential therapies is shown. The route from the nanoparticle design and modeling through synthesis and functionalization to cellular application is also described. For a better understanding of the fate of targeted RNA after delivery, an overview of RNA processing inside the cell is also provided.

Engineering a Functional Small RNA Negative Autoregulation Network with Model-Guided Design.

PubMed

Hu, Chelsea Y; Takahashi, Melissa K; Zhang, Yan; Lucks, Julius B

2018-05-22

RNA regulators are powerful components of the synthetic biology toolbox. Here, we expand the repertoire of synthetic gene networks built from these regulators by constructing a transcriptional negative autoregulation (NAR) network out of small RNAs (sRNAs). NAR network motifs are core motifs of natural genetic networks, and are known for reducing network response time and steady state signal. Here we use cell-free transcription-translation (TX-TL) reactions and a computational model to design and prototype sRNA NAR constructs. Using parameter sensitivity analysis, we design a simple set of experiments that allow us to accurately predict NAR function in TX-TL. We transfer successful network designs into Escherichia coli and show that our sRNA transcriptional network reduces both network response time and steady-state gene expression. This work broadens our ability to construct increasingly sophisticated RNA genetic networks with predictable function.

Fibrin gels exhibit improved biological, structural, and mechanical properties compared with collagen gels in cell-based tendon tissue-engineered constructs.

PubMed

Breidenbach, Andrew P; Dyment, Nathaniel A; Lu, Yinhui; Rao, Marepalli; Shearn, Jason T; Rowe, David W; Kadler, Karl E; Butler, David L

2015-02-01

The prevalence of tendon and ligament injuries and inadequacies of current treatments is driving the need for alternative strategies such as tissue engineering. Fibrin and collagen biopolymers have been popular materials for creating tissue-engineered constructs (TECs), as they exhibit advantages of biocompatibility and flexibility in construct design. Unfortunately, a few studies have directly compared these materials for tendon and ligament applications. Therefore, this study aims at determining how collagen versus fibrin hydrogels affect the biological, structural, and mechanical properties of TECs during formation in vitro. Our findings show that tendon and ligament progenitor cells seeded in fibrin constructs exhibit improved tenogenic gene expression patterns compared with their collagen-based counterparts for approximately 14 days in culture. Fibrin-based constructs also exhibit improved cell-derived collagen alignment, increased linear modulus (2.2-fold greater) compared with collagen-based constructs. Cyclic tensile loading, which promotes the maturation of tendon constructs in a previous work, exhibits a material-dependent effect in this study. Fibrin constructs show trending reductions in mechanical, biological, and structural properties, whereas collagen constructs only show improved tenogenic expression in the presence of mechanical stimulation. These findings highlight that components of the mechanical stimulus (e.g., strain amplitude or time of initiation) need to be tailored to the material and cell type. Given the improvements in tenogenic expression, extracellular matrix organization, and material properties during static culture, in vitro findings presented here suggest that fibrin-based constructs may be a more suitable alternative to collagen-based constructs for tissue-engineered tendon/ligament repair.

Fibrin Gels Exhibit Improved Biological, Structural, and Mechanical Properties Compared with Collagen Gels in Cell-Based Tendon Tissue-Engineered Constructs

PubMed Central

Dyment, Nathaniel A.; Lu, Yinhui; Rao, Marepalli; Shearn, Jason T.; Rowe, David W.; Kadler, Karl E.; Butler, David L.

2015-01-01

The prevalence of tendon and ligament injuries and inadequacies of current treatments is driving the need for alternative strategies such as tissue engineering. Fibrin and collagen biopolymers have been popular materials for creating tissue-engineered constructs (TECs), as they exhibit advantages of biocompatibility and flexibility in construct design. Unfortunately, a few studies have directly compared these materials for tendon and ligament applications. Therefore, this study aims at determining how collagen versus fibrin hydrogels affect the biological, structural, and mechanical properties of TECs during formation in vitro. Our findings show that tendon and ligament progenitor cells seeded in fibrin constructs exhibit improved tenogenic gene expression patterns compared with their collagen-based counterparts for approximately 14 days in culture. Fibrin-based constructs also exhibit improved cell-derived collagen alignment, increased linear modulus (2.2-fold greater) compared with collagen-based constructs. Cyclic tensile loading, which promotes the maturation of tendon constructs in a previous work, exhibits a material-dependent effect in this study. Fibrin constructs show trending reductions in mechanical, biological, and structural properties, whereas collagen constructs only show improved tenogenic expression in the presence of mechanical stimulation. These findings highlight that components of the mechanical stimulus (e.g., strain amplitude or time of initiation) need to be tailored to the material and cell type. Given the improvements in tenogenic expression, extracellular matrix organization, and material properties during static culture, in vitro findings presented here suggest that fibrin-based constructs may be a more suitable alternative to collagen-based constructs for tissue-engineered tendon/ligament repair. PMID:25266738

Polyester: simulating RNA-seq datasets with differential transcript expression.

PubMed

Frazee, Alyssa C; Jaffe, Andrew E; Langmead, Ben; Leek, Jeffrey T

2015-09-01

Statistical methods development for differential expression analysis of RNA sequencing (RNA-seq) requires software tools to assess accuracy and error rate control. Since true differential expression status is often unknown in experimental datasets, artificially constructed datasets must be utilized, either by generating costly spike-in experiments or by simulating RNA-seq data. Polyester is an R package designed to simulate RNA-seq data, beginning with an experimental design and ending with collections of RNA-seq reads. Its main advantage is the ability to simulate reads indicating isoform-level differential expression across biological replicates for a variety of experimental designs. Data generated by Polyester is a reasonable approximation to real RNA-seq data and standard differential expression workflows can recover differential expression set in the simulation by the user. Polyester is freely available from Bioconductor (http://bioconductor.org/). jtleek@gmail.com Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

A modular toolset for recombination transgenesis and neurogenetic analysis of Drosophila.

PubMed

Wang, Ji-Wu; Beck, Erin S; McCabe, Brian D

2012-01-01

Transgenic Drosophila have contributed extensively to our understanding of nervous system development, physiology and behavior in addition to being valuable models of human neurological disease. Here, we have generated a novel series of modular transgenic vectors designed to optimize and accelerate the production and analysis of transgenes in Drosophila. We constructed a novel vector backbone, pBID, that allows both phiC31 targeted transgene integration and incorporates insulator sequences to ensure specific and uniform transgene expression. Upon this framework, we have built a series of constructs that are either backwards compatible with existing restriction enzyme based vectors or utilize Gateway recombination technology for high-throughput cloning. These vectors allow for endogenous promoter or Gal4 targeted expression of transgenic proteins with or without fluorescent protein or epitope tags. In addition, we have generated constructs that facilitate transgenic splice isoform specific RNA inhibition of gene expression. We demonstrate the utility of these constructs to analyze proteins involved in nervous system development, physiology and neurodegenerative disease. We expect that these reagents will facilitate the proficiency and sophistication of Drosophila genetic analysis in both the nervous system and other tissues.

A More Powerful Test in Three-Level Cluster Randomized Designs

ERIC Educational Resources Information Center

Konstantopoulos, Spyros

2011-01-01

Field experiments that involve nested structures frequently assign treatment conditions to entire groups (such as schools). A key aspect of the design of such experiments includes knowledge of the clustering effects that are often expressed via intraclass correlation. This study provides methods for constructing a more powerful test for the…

Development of a domain-specific genetic language to design Chlamydomonas reinhardtii expression vectors.

PubMed

Wilson, Mandy L; Okumoto, Sakiko; Adam, Laura; Peccoud, Jean

2014-01-15

Expression vectors used in different biotechnology applications are designed with domain-specific rules. For instance, promoters, origins of replication or homologous recombination sites are host-specific. Similarly, chromosomal integration or viral delivery of an expression cassette imposes specific structural constraints. As de novo gene synthesis and synthetic biology methods permeate many biotechnology specialties, the design of application-specific expression vectors becomes the new norm. In this context, it is desirable to formalize vector design strategies applicable in different domains. Using the design of constructs to express genes in the chloroplast of Chlamydomonas reinhardtii as an example, we show that a vector design strategy can be formalized as a domain-specific language. We have developed a graphical editor of context-free grammars usable by biologists without prior exposure to language theory. This environment makes it possible for biologists to iteratively improve their design strategies throughout the course of a project. It is also possible to ensure that vectors designed with early iterations of the language are consistent with the latest iteration of the language. The context-free grammar editor is part of the GenoCAD application. A public instance of GenoCAD is available at http://www.genocad.org. GenoCAD source code is available from SourceForge and licensed under the Apache v2.0 open source license.

End-to-end automated microfluidic platform for synthetic biology: from design to functional analysis

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

Linshiz, Gregory; Jensen, Erik; Stawski, Nina

Synthetic biology aims to engineer biological systems for desired behaviors. The construction of these systems can be complex, often requiring genetic reprogramming, extensive de novo DNA synthesis, and functional screening. Here, we present a programmable, multipurpose microfluidic platform and associated software and apply the platform to major steps of the synthetic biology research cycle: design, construction, testing, and analysis. We show the platform’s capabilities for multiple automated DNA assembly methods, including a new method for Isothermal Hierarchical DNA Construction, and for Escherichia coli and Saccharomyces cerevisiae transformation. The platform enables the automated control of cellular growth, gene expression induction, andmore » proteogenic and metabolic output analysis. Finally, taken together, we demonstrate the microfluidic platform’s potential to provide end-to-end solutions for synthetic biology research, from design to functional analysis.« less

End-to-end automated microfluidic platform for synthetic biology: from design to functional analysis

DOE PAGES

Linshiz, Gregory; Jensen, Erik; Stawski, Nina; ...

2016-02-02

Synthetic biology aims to engineer biological systems for desired behaviors. The construction of these systems can be complex, often requiring genetic reprogramming, extensive de novo DNA synthesis, and functional screening. Here, we present a programmable, multipurpose microfluidic platform and associated software and apply the platform to major steps of the synthetic biology research cycle: design, construction, testing, and analysis. We show the platform’s capabilities for multiple automated DNA assembly methods, including a new method for Isothermal Hierarchical DNA Construction, and for Escherichia coli and Saccharomyces cerevisiae transformation. The platform enables the automated control of cellular growth, gene expression induction, andmore » proteogenic and metabolic output analysis. Finally, taken together, we demonstrate the microfluidic platform’s potential to provide end-to-end solutions for synthetic biology research, from design to functional analysis.« less

Double promoter expression systems for recombinant protein production by industrial microorganisms.

PubMed

Öztürk, Sibel; Ergün, Burcu Gündüz; Çalık, Pınar

2017-10-01

Using double promoter expression systems is a promising approach to increase heterologous protein production. In this review, current double promoter expression systems for the production of recombinant proteins (r-proteins) by industrially important bacteria, Bacillus subtilis and Escherichia coli; and yeasts, Saccharomyces cerevisiae and Pichia pastoris, are discussed by assessing their potentials and drawbacks. Double promoter expression systems need to be designed to maintain a higher specific product formation rate within the production domain. While bacterial double promoter systems have been constructed as chimeric tandem promoters, yeast dual promoter systems have been developed as separate expression cassettes. To increase production and productivity, the optimal transcriptional activity should be justified either by simultaneously satisfying the requirements of both promoters, or by consecutively stimulating the changeover from one to another in a biphasic process or via successive-iterations. Thus, considering the dynamics of a fermentation process, double promoters can be classified according to their operational mechanisms, as: i) consecutively operating double promoter systems, and ii) simultaneously operating double promoter systems. Among these metabolic design strategies, extending the expression period with two promoters activated under different conditions, or enhancing the transcriptional activity with two promoters activated under similar conditions within the production domain, can be applied independently from the host. Novel studies with new insights, which aim a rational systematic design and construction of dual promoter expression vectors with tailored transcriptional activity, will empower r-protein production with enhanced production and productivity. Finally, the current state-of-the-art review emphasizes the advantages of double promoter systems along with the necessity for discovering new promoters for the development of more effective and adaptive processes to meet the increasing demand of r-protein industry.

Ribosomal Binding Site Switching: An Effective Strategy for High-Throughput Cloning Constructions

PubMed Central

Li, Yunlong; Zhang, Yong; Lu, Pei; Rayner, Simon; Chen, Shiyun

2012-01-01

Direct cloning of PCR fragments by TA cloning or blunt end ligation are two simple methods which would greatly benefit high-throughput (HTP) cloning constructions if the efficiency can be improved. In this study, we have developed a ribosomal binding site (RBS) switching strategy for direct cloning of PCR fragments. RBS is an A/G rich region upstream of the translational start codon and is essential for gene expression. Change from A/G to T/C in the RBS blocks its activity and thereby abolishes gene expression. Based on this property, we introduced an inactive RBS upstream of a selectable marker gene, and designed a fragment insertion site within this inactive RBS. Forward and reverse insertions of specifically tailed fragments will respectively form an active and inactive RBS, thus all background from vector self-ligation and fragment reverse insertions will be eliminated due to the non-expression of the marker gene. The effectiveness of our strategy for TA cloning and blunt end ligation are confirmed. Application of this strategy to gene over-expression, a bacterial two-hybrid system, a bacterial one-hybrid system, and promoter bank construction are also verified. The advantages of this simple procedure, together with its low cost and high efficiency, makes our strategy extremely useful in HTP cloning constructions. PMID:23185557

Design, Construction and Cloning of Truncated ORF2 and tPAsp-PADRE-Truncated ORF2 Gene Cassette From Hepatitis E Virus in the pVAX1 Expression Vector

PubMed Central

Farshadpour, Fatemeh; Makvandi, Manoochehr; Taherkhani, Reza

2015-01-01

Background: Hepatitis E Virus (HEV) is the causative agent of enterically transmitted acute hepatitis and has high mortality rate of up to 30% among pregnant women. Therefore, development of a novel vaccine is a desirable goal. Objectives: The aim of this study was to construct tPAsp-PADRE-truncated open reading frame 2 (ORF2) and truncated ORF2 DNA plasmid, which can assist future studies with the preparation of an effective vaccine against Hepatitis E Virus. Materials and Methods: A synthetic codon-optimized gene cassette encoding tPAsp-PADRE-truncated ORF2 protein was designed, constructed and analyzed by some bioinformatics software. Furthermore, a codon-optimized truncated ORF2 gene was amplified by the polymerase chain reaction (PCR), with a specific primer from the previous construct. The constructs were sub-cloned in the pVAX1 expression vector and finally expressed in eukaryotic cells. Results: Sequence analysis and bioinformatics studies of the codon-optimized gene cassette revealed that codon adaptation index (CAI), GC content, and frequency of optimal codon usage (Fop) value were improved, and performance of the secretory signal was confirmed. Cloning and sub-cloning of the tPAsp-PADRE-truncated ORF2 gene cassette and truncated ORF2 gene were confirmed by colony PCR, restriction enzymes digestion and DNA sequencing of the recombinant plasmids pVAX-tPAsp-PADRE-truncated ORF2 (aa 112-660) and pVAX-truncated ORF2 (aa 112-660). The expression of truncated ORF2 protein in eukaryotic cells was approved by an Immunofluorescence assay (IFA) and the reverse transcriptase polymerase chain reaction (RT-PCR) method. Conclusions: The results of this study demonstrated that the tPAsp-PADRE-truncated ORF2 gene cassette and the truncated ORF2 gene in recombinant plasmids are successfully expressed in eukaryotic cells. The immunogenicity of the two recombinant plasmids with different formulations will be evaluated as a novel DNA vaccine in future investigations. PMID:26865938

Design criteria for synthetic riboswitches acting on transcription

PubMed Central

Wachsmuth, Manja; Domin, Gesine; Lorenz, Ronny; Serfling, Robert; Findeiß, Sven; Stadler, Peter F; Mörl, Mario

2015-01-01

Riboswitches are RNA-based regulators of gene expression composed of a ligand-sensing aptamer domain followed by an overlapping expression platform. The regulation occurs at either the level of transcription (by formation of terminator or antiterminator structures) or translation (by presentation or sequestering of the ribosomal binding site). Due to a modular composition, these elements can be manipulated by combining different aptamers and expression platforms and therefore represent useful tools to regulate gene expression in synthetic biology. Using computationally designed theophylline-dependent riboswitches we show that 2 parameters, terminator hairpin stability and folding traps, have a major impact on the functionality of the designed constructs. These have to be considered very carefully during design phase. Furthermore, a combination of several copies of individual riboswitches leads to a much improved activation ratio between induced and uninduced gene activity and to a linear dose-dependent increase in reporter gene expression. Such serial arrangements of synthetic riboswitches closely resemble their natural counterparts and may form the basis for simple quantitative read out systems for the detection of specific target molecules in the cell. PMID:25826571

Guidelines for the successful generation of protein–ligand complex crystals

PubMed Central

Müller, Ilka

2017-01-01

With continuous technical improvements at synchrotron facilities, data-collection rates have increased dramatically. This makes it possible to collect diffraction data for hundreds of protein–ligand complexes within a day, provided that a suitable crystal system is at hand. However, developing a suitable crystal system can prove challenging, exceeding the timescale of data collection by several orders of magnitude. Firstly, a useful crystallization construct of the protein of interest needs to be chosen and its expression and purification optimized, before screening for suitable crystallization and soaking conditions can start. This article reviews recent publications analysing large data sets of crystallization trials, with the aim of identifying factors that do or do not make a good crystallization construct, and gives guidance in the design of an expression construct. It provides an overview of common protein-expression systems, addresses how ligand binding can be both help and hindrance for protein purification, and describes ligand co-crystallization and soaking, with an emphasis on troubleshooting. PMID:28177304

Role of caspase-9 in the effector caspases and genome expressions, and growth of bovine skeletal myoblasts.

PubMed

Van Ba, Hoa; Hwang, Inho

2014-02-01

Caspase-9 has been reported as the key regulator of apoptosis, however, its role in skeletal myoblast development and molecular involvements during cell growth still remains unknown. The current study aimed to present the key role of caspase-9 in the expressions of apoptotic caspases and genome, and cell viability during myoblast growth using RNA interference mediated silencing. Three small interference RNA sequences (siRNAs) targeting caspase-9 gene was designed and ligated into pSilencer plasmid vector to construct shRNA expression constructs. Cells were transfected with the constructs for 48 h. Results indicated that all three siRNAs could silence the caspase-9 mRNA expression significantly. Particularly, the mRNA expression level of caspase-9 in the cells transfected by shRNA1, shRNA2 and shRNA3 constructs were reduced by 37.85%, 68.20% and 58.14%, respectively. Suppression of caspase-9 led to the significant increases in the mRNA and protein expressions of effector caspase-3, whereas the reduction in mRNA and protein expressions of caspase-7. The microarray results showed that the suppression of caspase-9 resulted in significant upregulations of cell proliferation-, adhesion-, growth-, development- and division-regulating genes, whereas the reduction in the expressions of cell death program- and stress response-regulating genes. Furthermore, cell viability was significantly increased following the transfection. These data suggest that caspase-9 could play an important role in the control of cell growth, and knockdown of caspase-9 may have genuine potential in the treatment of skeletal muscle atrophy. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

Make-Her-Spaces as Hybrid Places: Designing and Resisting Self Constructions in Urban Classrooms

ERIC Educational Resources Information Center

Norris, Aaminah

2014-01-01

This qualitative study analyzes how an urban schoolteacher guided her 19 tenth grade Latina and African American young women in developing positive self-concepts as expressed through the implementation of design thinking processes. This work examines how young women who had limited access to digital media negotiated their identities as they…

Efficient search, mapping, and optimization of multi-protein genetic systems in diverse bacteria

PubMed Central

Farasat, Iman; Kushwaha, Manish; Collens, Jason; Easterbrook, Michael; Guido, Matthew; Salis, Howard M

2014-01-01

Developing predictive models of multi-protein genetic systems to understand and optimize their behavior remains a combinatorial challenge, particularly when measurement throughput is limited. We developed a computational approach to build predictive models and identify optimal sequences and expression levels, while circumventing combinatorial explosion. Maximally informative genetic system variants were first designed by the RBS Library Calculator, an algorithm to design sequences for efficiently searching a multi-protein expression space across a > 10,000-fold range with tailored search parameters and well-predicted translation rates. We validated the algorithm's predictions by characterizing 646 genetic system variants, encoded in plasmids and genomes, expressed in six gram-positive and gram-negative bacterial hosts. We then combined the search algorithm with system-level kinetic modeling, requiring the construction and characterization of 73 variants to build a sequence-expression-activity map (SEAMAP) for a biosynthesis pathway. Using model predictions, we designed and characterized 47 additional pathway variants to navigate its activity space, find optimal expression regions with desired activity response curves, and relieve rate-limiting steps in metabolism. Creating sequence-expression-activity maps accelerates the optimization of many protein systems and allows previous measurements to quantitatively inform future designs. PMID:24952589

An Automated Pipeline for Engineering Many-Enzyme Pathways: Computational Sequence Design, Pathway Expression-Flux Mapping, and Scalable Pathway Optimization.

PubMed

Halper, Sean M; Cetnar, Daniel P; Salis, Howard M

2018-01-01

Engineering many-enzyme metabolic pathways suffers from the design curse of dimensionality. There are an astronomical number of synonymous DNA sequence choices, though relatively few will express an evolutionary robust, maximally productive pathway without metabolic bottlenecks. To solve this challenge, we have developed an integrated, automated computational-experimental pipeline that identifies a pathway's optimal DNA sequence without high-throughput screening or many cycles of design-build-test. The first step applies our Operon Calculator algorithm to design a host-specific evolutionary robust bacterial operon sequence with maximally tunable enzyme expression levels. The second step applies our RBS Library Calculator algorithm to systematically vary enzyme expression levels with the smallest-sized library. After characterizing a small number of constructed pathway variants, measurements are supplied to our Pathway Map Calculator algorithm, which then parameterizes a kinetic metabolic model that ultimately predicts the pathway's optimal enzyme expression levels and DNA sequences. Altogether, our algorithms provide the ability to efficiently map the pathway's sequence-expression-activity space and predict DNA sequences with desired metabolic fluxes. Here, we provide a step-by-step guide to applying the Pathway Optimization Pipeline on a desired multi-enzyme pathway in a bacterial host.

[Construction and selection of effective mouse Smad6 recombinant lenti-virus interference vectors].

PubMed

Yu, Jing; Qi, Mengchun; Deng, Jiupeng; Liu, Gang; Chen, Huaiqing

2010-10-01

This experiment was designed to construct mouse Smad6 recombinant RNA interference vectors and determine their interference effects on bone marrow mesenchymal stem cells (BMSCs). Three recombinant Smad6 RNA interference vectors were constructed by molecular clone techniques with a lenti-virus vector expressing green fluorescent protein (GFP), and the correctness of recombinant vectors was verified by DNA sequencing. Mouse BMSCs were used for transfection experiments and BMP-2 was in use for osteogenic induction of MSCs. The transfection efficiency of recombinant vectors was examined by Laser confocal scanning microscope and the interference effect of recombinant vectors on Smad6 gene expression was determined by real-time RT-PCR and Western blot, respectively. Three Smad6 recombinant RNA interference vectors were successfully constructed and their correctness was proved by DNA sequencing. After transfection, GFPs were effectively expressed in MSCs and all of three recombinant vectors gained high transfection efficiency (> 95%). Both real-time PCR and Western blot examination indicated that among three recombinant vectors, No. 2 Svector had the best interference effect and the interference effect was nearly 91% at protein level. In conclusion, Mouse recombinant Smad6 RNA interference (RNAi) vector was successfully constructed and it provided an effective tool for further studies on BMP signal pathways.

Biomimetic 3D tissue printing for soft tissue regeneration.

PubMed

Pati, Falguni; Ha, Dong-Heon; Jang, Jinah; Han, Hyun Ho; Rhie, Jong-Won; Cho, Dong-Woo

2015-09-01

Engineered adipose tissue constructs that are capable of reconstructing soft tissue with adequate volume would be worthwhile in plastic and reconstructive surgery. Tissue printing offers the possibility of fabricating anatomically relevant tissue constructs by delivering suitable matrix materials and living cells. Here, we devise a biomimetic approach for printing adipose tissue constructs employing decellularized adipose tissue (DAT) matrix bioink encapsulating human adipose tissue-derived mesenchymal stem cells (hASCs). We designed and printed precisely-defined and flexible dome-shaped structures with engineered porosity using DAT bioink that facilitated high cell viability over 2 weeks and induced expression of standard adipogenic genes without any supplemented adipogenic factors. The printed DAT constructs expressed adipogenic genes more intensely than did non-printed DAT gel. To evaluate the efficacy of our printed tissue constructs for adipose tissue regeneration, we implanted them subcutaneously in mice. The constructs did not induce chronic inflammation or cytotoxicity postimplantation, but supported positive tissue infiltration, constructive tissue remodeling, and adipose tissue formation. This study demonstrates that direct printing of spatially on-demand customized tissue analogs is a promising approach to soft tissue regeneration. Copyright © 2015 Elsevier Ltd. All rights reserved.

Regulation of endogenous human gene expression by ligand-inducible TALE transcription factors.

PubMed

Mercer, Andrew C; Gaj, Thomas; Sirk, Shannon J; Lamb, Brian M; Barbas, Carlos F

2014-10-17

The construction of increasingly sophisticated synthetic biological circuits is dependent on the development of extensible tools capable of providing specific control of gene expression in eukaryotic cells. Here, we describe a new class of synthetic transcription factors that activate gene expression in response to extracellular chemical stimuli. These inducible activators consist of customizable transcription activator-like effector (TALE) proteins combined with steroid hormone receptor ligand-binding domains. We demonstrate that these ligand-responsive TALE transcription factors allow for tunable and conditional control of gene activation and can be used to regulate the expression of endogenous genes in human cells. Since TALEs can be designed to recognize any contiguous DNA sequence, the conditional gene regulatory system described herein will enable the design of advanced synthetic gene networks.

A Modular Toolset for Recombination Transgenesis and Neurogenetic Analysis of Drosophila

PubMed Central

Wang, Ji-Wu; Beck, Erin S.; McCabe, Brian D.

2012-01-01

Transgenic Drosophila have contributed extensively to our understanding of nervous system development, physiology and behavior in addition to being valuable models of human neurological disease. Here, we have generated a novel series of modular transgenic vectors designed to optimize and accelerate the production and analysis of transgenes in Drosophila. We constructed a novel vector backbone, pBID, that allows both phiC31 targeted transgene integration and incorporates insulator sequences to ensure specific and uniform transgene expression. Upon this framework, we have built a series of constructs that are either backwards compatible with existing restriction enzyme based vectors or utilize Gateway recombination technology for high-throughput cloning. These vectors allow for endogenous promoter or Gal4 targeted expression of transgenic proteins with or without fluorescent protein or epitope tags. In addition, we have generated constructs that facilitate transgenic splice isoform specific RNA inhibition of gene expression. We demonstrate the utility of these constructs to analyze proteins involved in nervous system development, physiology and neurodegenerative disease. We expect that these reagents will facilitate the proficiency and sophistication of Drosophila genetic analysis in both the nervous system and other tissues. PMID:22848718

How to make a good animation: A grounded cognition model of how visual representation design affects the construction of abstract physics knowledge

NASA Astrophysics Data System (ADS)

Chen, Zhongzhou; Gladding, Gary

2014-06-01

Visual representations play a critical role in teaching physics. However, since we do not have a satisfactory understanding of how visual perception impacts the construction of abstract knowledge, most visual representations used in instructions are either created based on existing conventions or designed according to the instructor's intuition, which leads to a significant variance in their effectiveness. In this paper we propose a cognitive mechanism based on grounded cognition, suggesting that visual perception affects understanding by activating "perceptual symbols": the basic cognitive unit used by the brain to construct a concept. A good visual representation activates perceptual symbols that are essential for the construction of the represented concept, whereas a bad representation does the opposite. As a proof of concept, we conducted a clinical experiment in which participants received three different versions of a multimedia tutorial teaching the integral expression of electric potential. The three versions were only different by the details of the visual representation design, only one of which contained perceptual features that activate perceptual symbols essential for constructing the idea of "accumulation." On a following post-test, participants receiving this version of tutorial significantly outperformed those who received the other two versions of tutorials designed to mimic conventional visual representations used in classrooms.

[Expression of Dengue virus type 2 nonstructural protein 3 and isolation of host proteins interacting with it].

PubMed

Weng, Daihui; Lei, Yingfeng; Dong, Yangchao; Han, Peijun; Ye, Chuantao; Yang, Jing; Wang, Yuan; Yin, Wen

2015-12-01

To construct the plasmid expressing the fusion protein of Dengue virus type 2 (DENV2) nonstructural protein 3 (NS3) with affinity tag, and isolate the cellular proteins interacting with NS3 protein using tandem affinity purification (TAP) assay. Primers for amplifying NS3 gene were designed according to the sequence of DENV2 genome and chemically synthesized. The NS3 fragments, after amplified by PCR with DENV2 cDNA as template, were digested and cloned into the mammalian eukaryotic expression vector pCI-SF with the tandem affinity tag (FLAG-StrepII). The recombinant pCI-NS3-SF was transiently transformed by Lipofectamine(TM) 2000 into HEK293T cells, and the expression of the fusion protein was confirmed by Western blotting. Cellular proteins that interacted with NS3 were isolated and purified by TAP assay. The eukaryotic expression vector expressing NS3 protein was successfully constructed. The host proteins interacting with NS3 protein were isolated by TAP system. TAP is an efficient method to isolate the cellular proteins interacting with DENV2 NS3.

A triplex ribozyme expression system based on a single hairpin ribozyme.

PubMed

Aquino-Jarquin, Guillermo; Benítez-Hess, María Luisa; DiPaolo, Joseph A; Alvarez-Salas, Luis M

2008-09-01

Triplex ribozyme (RZ) configurations allow for the individual activity of trans-acting RZs in multiple expression cassettes (multiplex), thereby increasing target cleavage relative to conventionally expressed RZs. Although hairpin RZs have been advantageously compared to hammerhead RZs, their longer size and structural features complicated triplex design. We present a triplex expression system based on a single hairpin RZ with transcleavage capability and simple engineering. The system was tested in vitro using cis- and trans-cleavage kinetic assays against a known target RNA from HPV-16 E6/E7 mRNA. Single and multiplex triplex RZ constructs were more efficient in cleaving the target than tandem-cloned hairpin RZs, suggesting that the release of individual RZs enhanced trans-cleavage kinetics. Multiplex systems constructed with two different hairpin RZs resulted in better trans-cleavage compared to standard double-RZ constructs. In addition, the triplex RZ performed cis- and trans-cleavage in cervical cancer cells. The use of triplex configurations with multiplex RZs permit differential targeting of the same or different RNA, thus improving potential use against unstable targets. This prototype will provide the basis for the development of future RZ-based therapies and technologies.

The quality estimation of exterior wall’s and window filling’s construction design

NASA Astrophysics Data System (ADS)

Saltykov, Ivan; Bovsunovskaya, Maria

2017-10-01

The article reveals the term of “artificial envelope” in dwelling building. Authors offer a complex multifactorial approach to the design quality estimation of external fencing structures, which is based on various parameters impact. These referred parameters are: functional, exploitation, cost, and also, the environmental index is among them. The quality design index Qк is inputting for the complex characteristic of observed above parameters. The mathematical relation of this index from these parameters is the target function for the quality design estimation. For instance, the article shows the search of optimal variant for wall and window designs in small, middle and large square dwelling premises of economic class buildings. The graphs of target function single parameters are expressed for the three types of residual chamber’s dimensions. As a result of the showing example, there is a choice of window opening’s dimensions, which make the wall’s and window’s constructions properly correspondent to the producible complex requirements. The authors reveal the comparison of recommended window filling’s square in accordance with the building standards, and the square, due to the finding of the optimal variant of the design quality index. The multifactorial approach for optimal design searching, which is mentioned in this article, can be used in consideration of various construction elements of dwelling buildings in accounting of suitable climate, social and economic construction area features.

Designing a Soluble Near Full-Length HIV-1 GP41 Trimer

DTIC Science & Technology

2012-11-26

envelope; gp41 trimer; bacteriophage T4 display; prehairpin fusion intermediate. Background: The envelope glycoprotein gp41 is a key component of...protein into trimers and defined oligomers. These gp41 trimers were displayed on bacteriophage T4 capsid nanoparticles by attaching to the small...Construction of the Expression Vectors —All the gp41 constructs were generated by splicing-by- overlap extension PCR using wild-type HXB2 gp41 DNA

A system decomposition approach to the design of functional observers

NASA Astrophysics Data System (ADS)

Fernando, Tyrone; Trinh, Hieu

2014-09-01

This paper reports a system decomposition that allows the construction of a minimum-order functional observer using a state observer design approach. The system decomposition translates the functional observer design problem to that of a state observer for a smaller decomposed subsystem. Functional observability indices are introduced, and a closed-form expression for the minimum order required for a functional observer is derived in terms of those functional observability indices.

Design and construction of functional AAV vectors.

PubMed

Gray, John T; Zolotukhin, Serge

2011-01-01

Using the basic principles of molecular biology and laboratory techniques presented in this chapter, researchers should be able to create a wide variety of AAV vectors for both clinical and basic research applications. Basic vector design concepts are covered for both protein coding gene expression and small non-coding RNA gene expression cassettes. AAV plasmid vector backbones (available via AddGene) are described, along with critical sequence details for a variety of modular expression components that can be inserted as needed for specific applications. Protocols are provided for assembling the various DNA components into AAV vector plasmids in Escherichia coli, as well as for transferring these vector sequences into baculovirus genomes for large-scale production of AAV in the insect cell production system.

A highly tunable system for the simultaneous expression of multiple enzymes in Saccharomyces cerevisiae.

PubMed

Ito, Yoichiro; Yamanishi, Mamoru; Ikeuchi, Akinori; Matsuyama, Takashi

2015-01-16

Control of the expression levels of multiple enzymes in transgenic yeasts is essential for the effective production of complex molecules through fermentation. Here, we propose a tunable strategy for the control of expression levels based on the design of terminator regions and other gene-expression control elements in Saccharomyces cerevisiae. Our genome-integrated system, which is capable of producing high expression levels over a wide dynamic range, will broadly enable metabolic engineering and synthetic biology. We demonstrated that the activities of multiple cellulases and the production of ethanol were doubled in a transgenic yeast constructed with our system compared with those achieved with a standard expression system.

Analysis, annotation, and profiling of the oat seed transcriptome

USDA-ARS?s Scientific Manuscript database

Novel high-throughput next generation sequencing (NGS) technologies are providing opportunities to explore genomes and transcriptomes in a cost-effective manner. To construct a gene expression atlas of developing oat (Avena sativa) seeds, two software packages specifically designed for RNA-seq (Trin...

Secretion of a recombinant protein without a signal peptide by the exocrine glands of transgenic rabbits

PubMed Central

Iski, Gergely; Lipták, Nándor; Gócza, Elen; Kues, Wilfried A.; Bősze, Zsuzsanna

2017-01-01

Transgenic rabbits carrying mammary gland specific gene constructs are extensively used for excreting recombinant proteins into the milk. Here, we report refined phenotyping of previously generated Venus transposon-carrying transgenic rabbits with particular emphasis on the secretion of the reporter protein by exocrine glands, such as mammary, salivary, tear and seminal glands. The Sleeping Beauty (SB) transposon transgenic construct contains the Venus fluorophore cDNA, but without a signal peptide for the secretory pathway, driven by the ubiquitous CAGGS (CAG) promoter. Despite the absence of a signal peptide, the fluorophore protein was readily detected in milk, tear, saliva and seminal fluids. The expression pattern was verified by Western blot analysis. Mammary gland epithelial cells of SB-CAG-Venus transgenic lactating does also showed Venus-specific expression by tissue histology and fluorescence microscopy. In summary, the SB-CAG-Venus transgenic rabbits secrete the recombinant protein by different glands. This finding has relevance not only for the understanding of the biological function of exocrine glands, but also for the design of constructs for expression of recombinant proteins in dairy animals. PMID:29077768

[Construction of EZH2 Knockout Animal Model by CRISPR/Cas9 Technology].

PubMed

Meng, Fanrong; Zhao, Dan; Zhou, Qinghua; Liu, Zhe

2018-05-20

It has been proven that CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9) system was the modern gene-editing technology through the constitutive expression of nucleases Cas9 in the mammalian, which binds to the specific site in the genome mediated by single-guide RNA (sgRNA) at desired genomic loci. The aim of this study is that the animal model of EZH2 gene knockout was constructed using CRISPR/Cas9 technology. In this study, we designed two single-guide RNAs targeting the Exon3 and Exon4 of EZH2 gene. Then, their gene-targeting efficiency were detected by SURVEYOR assay. The lentivirus was perfused into the lungs of mice by using a bronchial tube and detected by immunohistochemistry and qRT-PCR. The experimental results of NIH-3T3 cells verify that the designed sgEZH2 can efficiently effect the cleavage of target DNA by Cas9 in vitro. The immunohistochemistry and qRT-PCR results showed that the EZH2 expression in experimental group was significantly decreased in the mouse lung tissue. The study successfully designed two sgRNA which can play a knock-out EZH2 function. An EZH2 knockout animal model was successfully constructed by CRISPR/Cas9 system, and it will be an effective animal model for studying the functions and mechanisms of EZH2.

An ancient rule for constructing dodecagonal quasiperiodic patterns

NASA Astrophysics Data System (ADS)

Ajlouni, Rima

2017-02-01

The discovery of complex dodecagonal patterns in historical Islamic architecture is generating a renewed interest into understanding the mathematical principles of traditional Islamic geometry. By employing a compass and a straightedge, ancient craftsmen utilized consistent design principles that allowed for diverse geometric expressions to be realized throughout the ancient world. Derived from these principles, a global multi-level structural model is proposed that provides a general guiding principle for constructing a wide variety of infinite dodecagon-based quasiperiodic patterns.

Effects of transmission-blocking vaccines simultaneously targeting pre- and post-fertilization antigens in the rodent malaria parasite Plasmodium yoelii.

PubMed

Zheng, Li; Pang, Wei; Qi, Zanmei; Luo, Enjie; Cui, Liwang; Cao, Yaming

2016-08-08

Transmission-blocking vaccine (TBV) is a promising strategy for interrupting the malaria transmission cycle. Current TBV candidates include both pre- and post-fertilization antigens expressed during sexual development of the malaria parasites. We tested whether a TBV design combining two sexual-stage antigens has better transmission-blocking activity. Using the rodent malaria model Plasmodium yoelii, we pursued a DNA vaccination strategy with genes encoding the gametocyte antigen Pys48/45 and the major ookinete surface protein Pys25. Immunization of mice with DNA constructs expression either Pys48/45 or Pys25 elicited strong antibody responses, which specifically recognized a ~45 and ~25 kDa protein from gametocyte and ookinete lysates, respectively. Immune sera from mice immunized with DNA constructs expressing Pys48/45 and Pys25 individually and in combination displayed evident transmission-blocking activity in in vitro ookinete culture and direct mosquito feeding experiments. With both assays, the Pys25 sera had higher transmission-blocking activity than the Pys48/45 sera. Intriguingly, compared with the immunization with the individual DNA vaccines, immunization with both DNA constructs produced lower antibody responses against individual antigens. The resultant immune sera from the composite vaccination had significantly lower transmission-blocking activity than those from Pys25 DNA immunization group, albeit the activity was substantially higher than that from the Pys48 DNA vaccination group. This result suggested that vaccination with the two DNA constructs did not achieve a synergistic effect, but rather caused interference in inducing antigen-specific antibody responses. This result has important implications for future design of composite vaccines targeting different sexual antigens.

Transgenic carrot expressing fusion protein comprising M. tuberculosis antigens induces immune response in mice.

PubMed

Permyakova, Natalia V; Zagorskaya, Alla A; Belavin, Pavel A; Uvarova, Elena A; Nosareva, Olesya V; Nesterov, Andrey E; Novikovskaya, Anna A; Zav'yalov, Evgeniy L; Moshkin, Mikhail P; Deineko, Elena V

2015-01-01

Tuberculosis remains one of the major infectious diseases, which continues to pose a major global health problem. Transgenic plants may serve as bioreactors to produce heterologous proteins including antibodies, antigens, and hormones. In the present study, a genetic construct has been designed that comprises the Mycobacterium tuberculosis genes cfp10, esat6 and dIFN gene, which encode deltaferon, a recombinant analog of the human γ-interferon designed for expression in plant tissues. This construct was transferred to the carrot (Daucus carota L.) genome by Agrobacterium-mediated transformation. This study demonstrates that the fusion protein CFP10-ESAT6-dIFN is synthesized in the transgenic carrot storage roots. The protein is able to induce both humoral and cell-mediated immune responses in laboratory animals (mice) when administered either orally or by injection. It should be emphasized that M. tuberculosis antigens contained in the fusion protein have no cytotoxic effect on peripheral blood mononuclear cells.

The research on construction and application of machining process knowledge base

NASA Astrophysics Data System (ADS)

Zhao, Tan; Qiao, Lihong; Qie, Yifan; Guo, Kai

2018-03-01

In order to realize the application of knowledge in machining process design, from the perspective of knowledge in the application of computer aided process planning(CAPP), a hierarchical structure of knowledge classification is established according to the characteristics of mechanical engineering field. The expression of machining process knowledge is structured by means of production rules and the object-oriented methods. Three kinds of knowledge base models are constructed according to the representation of machining process knowledge. In this paper, the definition and classification of machining process knowledge, knowledge model, and the application flow of the process design based on the knowledge base are given, and the main steps of the design decision of the machine tool are carried out as an application by using the knowledge base.

Design, Assembly, and Characterization of TALE-Based Transcriptional Activators and Repressors.

PubMed

Thakore, Pratiksha I; Gersbach, Charles A

2016-01-01

Transcription activator-like effectors (TALEs) are modular DNA-binding proteins that can be fused to a variety of effector domains to regulate the epigenome. Nucleotide recognition by TALE monomers follows a simple cipher, making this a powerful and versatile method to activate or repress gene expression. Described here are methods to design, assemble, and test TALE transcription factors (TALE-TFs) for control of endogenous gene expression. In this protocol, TALE arrays are constructed by Golden Gate cloning and tested for activity by transfection and quantitative RT-PCR. These methods for engineering TALE-TFs are useful for studies in reverse genetics and genomics, synthetic biology, and gene therapy.

A reverse engineering approach to optimize experiments for the construction of biological regulatory networks.

PubMed

Zhang, Xiaomeng; Shao, Bin; Wu, Yangle; Qi, Ouyang

2013-01-01

One of the major objectives in systems biology is to understand the relation between the topological structures and the dynamics of biological regulatory networks. In this context, various mathematical tools have been developed to deduct structures of regulatory networks from microarray expression data. In general, from a single data set, one cannot deduct the whole network structure; additional expression data are usually needed. Thus how to design a microarray expression experiment in order to get the most information is a practical problem in systems biology. Here we propose three methods, namely, maximum distance method, trajectory entropy method, and sampling method, to derive the optimal initial conditions for experiments. The performance of these methods is tested and evaluated in three well-known regulatory networks (budding yeast cell cycle, fission yeast cell cycle, and E. coli. SOS network). Based on the evaluation, we propose an efficient strategy for the design of microarray expression experiments.

Modular Ligation Extension of Guide RNA Operons (LEGO) for Multiplexed dCas9 Regulation of Metabolic Pathways in Saccharomyces cerevisiae.

PubMed

Deaner, Matthew; Holzman, Allison; Alper, Hal S

2018-04-16

Metabolic engineering typically utilizes a suboptimal step-wise gene target optimization approach to parse a highly connected and regulated cellular metabolism. While the endonuclease-null CRISPR/Cas system has enabled gene expression perturbations without genetic modification, it has been mostly limited to small sets of gene targets in eukaryotes due to inefficient methods to assemble and express large sgRNA operons. In this work, we develop a TEF1p-tRNA expression system and demonstrate that the use of tRNAs as splicing elements flanking sgRNAs provides higher efficiency than both Pol III and ribozyme-based expression across a variety of single sgRNA and multiplexed contexts. Next, we devise and validate a scheme to allow modular construction of tRNA-sgRNA (TST) operons using an iterative Type IIs digestion/ligation extension approach, termed CRISPR-Ligation Extension of sgRNA Operons (LEGO). This approach enables facile construction of large TST operons. We demonstrate this utility by constructing a metabolic rewiring prototype for 2,3-butanediol production in 2 distinct yeast strain backgrounds. These results demonstrate that our approach can act as a surrogate for traditional genetic modification on a much shorter design-cycle timescale. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A compact, in vivo screen of all 6-mers reveals drivers of tissue-specific expression and guides synthetic regulatory element design.

PubMed

Smith, Robin P; Riesenfeld, Samantha J; Holloway, Alisha K; Li, Qiang; Murphy, Karl K; Feliciano, Natalie M; Orecchia, Lorenzo; Oksenberg, Nir; Pollard, Katherine S; Ahituv, Nadav

2013-07-18

Large-scale annotation efforts have improved our ability to coarsely predict regulatory elements throughout vertebrate genomes. However, it is unclear how complex spatiotemporal patterns of gene expression driven by these elements emerge from the activity of short, transcription factor binding sequences. We describe a comprehensive promoter extension assay in which the regulatory potential of all 6 base-pair (bp) sequences was tested in the context of a minimal promoter. To enable this large-scale screen, we developed algorithms that use a reverse-complement aware decomposition of the de Bruijn graph to design a library of DNA oligomers incorporating every 6-bp sequence exactly once. Our library multiplexes all 4,096 unique 6-mers into 184 double-stranded 15-bp oligomers, which is sufficiently compact for in vivo testing. We injected each multiplexed construct into zebrafish embryos and scored GFP expression in 15 tissues at two developmental time points. Twenty-seven constructs produced consistent expression patterns, with the majority doing so in only one tissue. Functional sequences are enriched near biologically relevant genes, match motifs for developmental transcription factors, and are required for enhancer activity. By concatenating tissue-specific functional sequences, we generated completely synthetic enhancers for the notochord, epidermis, spinal cord, forebrain and otic lateral line, and show that short regulatory sequences do not always function modularly. This work introduces a unique in vivo catalog of short, functional regulatory sequences and demonstrates several important principles of regulatory element organization. Furthermore, we provide resources for designing compact, reverse-complement aware k-mer libraries.

Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits.

PubMed

Freed, Emily F; Winkler, James D; Weiss, Sophie J; Garst, Andrew D; Mutalik, Vivek K; Arkin, Adam P; Knight, Rob; Gill, Ryan T

2015-11-20

The reliable engineering of biological systems requires quantitative mapping of predictable and context-independent expression over a broad range of protein expression levels. However, current techniques for modifying expression levels are cumbersome and are not amenable to high-throughput approaches. Here we present major improvements to current techniques through the design and construction of E. coli genome-wide libraries using synthetic DNA cassettes that can tune expression over a ∼10(4) range. The cassettes also contain molecular barcodes that are optimized for next-generation sequencing, enabling rapid and quantitative tracking of alleles that have the highest fitness advantage. We show these libraries can be used to determine which genes and expression levels confer greater fitness to E. coli under different growth conditions.

Advances in genetic circuit design: novel biochemistries, deep part mining, and precision gene expression.

PubMed

Nielsen, Alec A K; Segall-Shapiro, Thomas H; Voigt, Christopher A

2013-12-01

Cells use regulatory networks to perform computational operations to respond to their environment. Reliably manipulating such networks would be valuable for many applications in biotechnology; for example, in having genes turn on only under a defined set of conditions or implementing dynamic or temporal control of expression. Still, building such synthetic regulatory circuits remains one of the most difficult challenges in genetic engineering and as a result they have not found widespread application. Here, we review recent advances that address the key challenges in the forward design of genetic circuits. First, we look at new design concepts, including the construction of layered digital and analog circuits, and new approaches to control circuit response functions. Second, we review recent work to apply part mining and computational design to expand the number of regulators that can be used together within one cell. Finally, we describe new approaches to obtain precise gene expression and to reduce context dependence that will accelerate circuit design by more reliably balancing regulators while reducing toxicity. Copyright © 2013. Published by Elsevier Ltd.

An Important Question for Student Journalists: Must Freedom, Rules/Law Inevitably Conflict?

ERIC Educational Resources Information Center

Martinson, David L.

2001-01-01

Considers the importance of developing a well constructed publications policy and notes that a carefully designed policy will provide some mechanism for addressing questions regarding the inevitable conflicts that will arise when students express a desire to publish certain controversial material. (SC)

Initial Intervention Outcomes of the Dependable Strengths Project.

ERIC Educational Resources Information Center

McMurrer, James E., Jr.

Dependable Strengths Articulation Process (DSAP) is a self-development process designed to facilitate positive self-constructions and improved personal functioning. This study examined the effectiveness of DSAP interventions. Participants (N=30) were college graduates who had expressed a desire to improve their educational and career planning and…

A Demonstration of Delay and Constructive Modeling Effects in Distributed Interactive Simulation.

DTIC Science & Technology

1998-02-01

with the Armstrong Laboratory Design Technology Branch, Veda Incorporated, and Science Applications International Corporation (SAIC). SAIC was working...The authors express special thanks to Mr. Dave O’Quinn of Veda Incorporated who provided quality simulation engineering support, and to Mr. David...platform employed in the study was the Engineering Design Simulator (EDSM) shown in Figure 3. Developed by Veda Inc., the EDSM is a single-seat

Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH) fusion to gfp (green fluorescent protein)

PubMed Central

Jugder, Bat-Erdene; Welch, Jeffrey; Braidy, Nady

2016-01-01

Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2). Amongst a number of promising candidates for application in the oxidation of H2 is a soluble [Ni–Fe] uptake hydrogenase (SH) produced by Cupriavidus necator H16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP) reporter system to characterise PSH promoter activity using several gene cloning approaches. A PSH promoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSH promoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinant C. necator H16 cells. Here we report the first successful fluorescent reporter system to study PSH promoter activity in C. necator H16. The fusion construct allowed for the design of a simple screening assay to evaluate PSH activity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression. PMID:27547572

Why the bigger live longer and travel farther: animals, vehicles, rivers and the winds.

PubMed

Bejan, Adrian

2012-01-01

Here we show that constructal-law physics unifies the design of animate and inanimate movement by requiring that larger bodies move farther, and their movement on the landscape last longer. The life span of mammals must scale as the body mass (M) raised to the power 1/4, and the distance traveled during the lifetime must increase with body size. The same size effect on life span and distance traveled holds for the other flows that move mass on earth: atmospheric and oceanic jets and plumes, river basins, animals and human operated vehicles. The physics is the same for all flow systems on the landscape: the scaling rules of "design" are expressions of the natural tendency of all flow systems to generate designs that facilitate flow access. This natural tendency is the constructal law of design and evolution in nature. Larger bodies are more efficient movers of mass on the landscape.

Identification and purification of a soluble region of BubR1: a critical component of the mitotic checkpoint complex.

PubMed

Yoon, Jongchul; Kang, Yup; Kim, Kyunggon; Park, Jungeun; Kim, Youngsoo

2005-11-01

The mitotic checkpoint complex (MCC) ensures the fidelity of chromosomal segregation, by delaying the onset of anaphase until all sister chromatids have been properly attached to the mitotic spindle. In essence, this MCC-induced delay is achieved via the inhibition of the anaphase-promoting complex (APC). Among the components of the MCC, BubR1 plays two major roles in the functions of the mitotic checkpoint. First, BubR1 is able to inhibit APC activity, either by itself or as a component of the MCC, by sequestering a APC coactivator, known as Cdc20. Second, BubR1 activates mitotic checkpoint signaling cascades by binding to the centromere-associated protein E, a microtubule motor protein. Obtaining highly soluble BubR1 is a prerequisite for the study of its structure. BubR1 is a multi-domain protein, which includes a KEN box motif, a mad3-like region, a Bub3 binding domain, and a kinase domain. We obtained a soluble BubR1 construct using a three-step expression strategy. First, we obtained two constructs from BLAST sequence homology searches, both of which were expressed abundantly in the inclusion bodies. We then adjusted the lengths of the two constructs by secondary structure prediction, thereby generating partially soluble constructs. Third, we optimized the solubility of the two constructs by either chopping or adding a few residues at the C-terminus. Finally, we obtained a highly soluble BubR1 construct via the Escherichia coli expression system, which allowed for a yield of 10.8 mg/L culture. This report may provide insight into the design of highly soluble constructs of insoluble multi-domain proteins.

Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly

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

Torella, JP; Boehm, CR; Lienert, F

2013-12-28

In vitro recombination methods have enabled one-step construction of large DNA sequences from multiple parts. Although synthetic biological circuits can in principle be assembled in the same fashion, they typically contain repeated sequence elements such as standard promoters and terminators that interfere with homologous recombination. Here we use a computational approach to design synthetic, biologically inactive unique nucleotide sequences (UNSes) that facilitate accurate ordered assembly. Importantly, our designed UNSes make it possible to assemble parts with repeated terminator and insulator sequences, and thereby create insulated functional genetic circuits in bacteria and mammalian cells. Using UNS-guided assembly to construct repeating promoter-gene-terminatormore » parts, we systematically varied gene expression to optimize production of a deoxychromoviridans biosynthetic pathway in Escherichia coli. We then used this system to construct complex eukaryotic AND-logic gates for genomic integration into embryonic stem cells. Construction was performed by using a standardized series of UNS-bearing BioBrick-compatible vectors, which enable modular assembly and facilitate reuse of individual parts. UNS-guided isothermal assembly is broadly applicable to the construction and optimization of genetic circuits and particularly those requiring tight insulation, such as complex biosynthetic pathways, sensors, counters and logic gates.« less

An Individualized Problem-Solving Approach for Teaching Choral Phrase Shaping: An Experimental Study

ERIC Educational Resources Information Center

Broomhead, Paul

2009-01-01

This experiment tested a treatment designed to improve choral students' expressiveness regarding keyword emphasis and phrase shaping. The treatment was founded upon the constructivist belief that students actively construct conceptual knowledge through problem solving. Participants were 46 university students randomly selected from a nonauditioned…

Expression in the Visual Arts.

ERIC Educational Resources Information Center

Millward, Peter; Parton, Anthony

2001-01-01

Focuses on how the construction of understanding can be supported through the visual arts in the context of the British National Curriculum for Art and Design. Giving students experience working with art materials is not, in itself, sufficient; the experience must be shaped in order for students to develop artistic understanding and appreciation.…

Democratic Education: An (Im)Possibility that yet Remains to Come

ERIC Educational Resources Information Center

Friedrich, Daniel; Jaastad, Bryn; Popkewitz, Thomas S.

2010-01-01

Efforts to develop democratic schools have moved along particular rules and standards of "reasoning" even when expressed through different ideological and paradigmatic lines. From attempts to make a democratic education to critical pedagogy, different approaches overlap in their historical construction of the reason of schooling: designing society…

An Assertiveness Inventory for Adults

ERIC Educational Resources Information Center

Gay, Melvin L.; And Others

1975-01-01

The Adult Self-Expression Scale is a 48-item, self-report measure of assertiveness designed for use with adults in general. Scale was found to have high test-retest reliability and moderate-to-high construct validity, as established by correlations with Adjective Check List scales and by a discriminant analysis procedure. (Author)

Adnectin-Based Design of Chimeric Antigen Receptor for T Cell Engineering.

PubMed

Han, Xiaolu; Cinay, Gunce E; Zhao, Yifan; Guo, Yunfei; Zhang, Xiaoyang; Wang, Pin

2017-11-01

Although chimeric antigen receptor (CAR)-engineered T cell therapy has achieved encouraging clinical trial results for treating hematological cancers, further optimization can likely expand this therapeutic success to more patients and other cancer types. Most CAR constructs used in clinical trials incorporate single chain variable fragment (scFv) as the extracellular antigen recognition domain. The immunogenicity of nonhuman scFv could cause host rejection against CAR T cells and compromise their persistence and efficacy. The limited availability of scFvs and slow discovery of new monoclonal antibodies also limit the development of novel CAR constructs. Adnectin, a class of affinity molecules derived from the tenth type III domain of human fibronectin, can be an alternative to scFv as an antigen-binding moiety in the design of CAR molecules. We constructed adnectin-based CARs targeting epithelial growth factor receptor (EGFR) and found that compared to scFv-based CAR, T cells engineered with adnectin-based CARs exhibited equivalent cell-killing activity against target H292 lung cancer cells in vitro and had comparable antitumor efficacy in xenograft tumor-bearing mice in vivo. In addition, with optimal affinity tuning, adnectin-based CAR showed higher selectivity on target cells with high EGFR expression than on those with low expression. This new design of adnectin CARs can potentially facilitate the development of T cell immunotherapy for cancer and other diseases. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Engineering Extracellular Expression Systems in Escherichia coli Based on Transcriptome Analysis and Cell Growth State.

PubMed

Gao, Wen; Yin, Jun; Bao, Lichen; Wang, Qun; Hou, Shan; Yue, Yali; Yao, Wenbing; Gao, Xiangdong

2018-05-18

Escherichia coli extracellular expression systems have a number of advantages over other systems, such as lower pyrogen levels and a simple purification process. Various approaches, such as the generation of leaky mutants via chromosomal engineering, have been explored for this expression system. However, extracellular protein yields in leaky mutants are relatively low compared to that in intracellular expression systems and therefore need to be improved. In this work, we describe the construction, characterization, and mechanism of enhanced extracellular expression in Escherichia coli. On the basis of the localizations, functions, and transcription levels of cell envelope proteins, we systematically elucidated the effects of multiple gene deletions on cell growth and extracellular expression using modified CRISPR/Cas9-based genome editing and a FlAsH labeling assay. High extracellular yields of heterologous proteins of different sizes were obtained by screening multiple gene mutations. The enhancement of extracellular secretion was associated with the derepression of translation and translocation. This work utilized universal methods in the design of extracellular expression systems for genes not directly associated with protein synthesis that were used to generate strains with higher protein expression capability. We anticipate that extracellular expression systems may help to shed light on the poorly understood aspects of these secretion processes as well as to further assist in the construction of engineered prokaryotic cells for efficient extracellular production of heterologous proteins.

Characterization of complex systems using the design of experiments approach: transient protein expression in tobacco as a case study.

PubMed

Buyel, Johannes Felix; Fischer, Rainer

2014-01-31

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

Achieving large dynamic range control of gene expression with a compact RNA transcription–translation regulator

PubMed Central

2017-01-01

Abstract RNA transcriptional regulators are emerging as versatile components for genetic network construction. However, these regulators suffer from incomplete repression in their OFF state, making their dynamic range less than that of their protein counterparts. This incomplete repression causes expression leak, which impedes the construction of larger synthetic regulatory networks as leak propagation can interfere with desired network function. To address this, we demonstrate how naturally derived antisense RNA-mediated transcriptional regulators can be configured to regulate both transcription and translation in a single compact RNA mechanism that functions in Escherichia coli. Using in vivo gene expression assays, we show that a combination of transcriptional termination and ribosome binding site sequestration increases repression from 85% to 98%, or activation from 10-fold to over 900-fold, in response to cognate antisense RNAs. We also show that orthogonal repressive versions of this mechanism can be created through engineering minimal antisense RNAs. Finally, to demonstrate the utility of this mechanism, we use it to reduce network leak in an RNA-only cascade. We anticipate these regulators will find broad use as synthetic biology moves beyond parts engineering to the design and construction of more sophisticated regulatory networks. PMID:28387839

Computational design of RNA parts, devices, and transcripts with kinetic folding algorithms implemented on multiprocessor clusters.

PubMed

Thimmaiah, Tim; Voje, William E; Carothers, James M

2015-01-01

With progress toward inexpensive, large-scale DNA assembly, the demand for simulation tools that allow the rapid construction of synthetic biological devices with predictable behaviors continues to increase. By combining engineered transcript components, such as ribosome binding sites, transcriptional terminators, ligand-binding aptamers, catalytic ribozymes, and aptamer-controlled ribozymes (aptazymes), gene expression in bacteria can be fine-tuned, with many corollaries and applications in yeast and mammalian cells. The successful design of genetic constructs that implement these kinds of RNA-based control mechanisms requires modeling and analyzing kinetically determined co-transcriptional folding pathways. Transcript design methods using stochastic kinetic folding simulations to search spacer sequence libraries for motifs enabling the assembly of RNA component parts into static ribozyme- and dynamic aptazyme-regulated expression devices with quantitatively predictable functions (rREDs and aREDs, respectively) have been described (Carothers et al., Science 334:1716-1719, 2011). Here, we provide a detailed practical procedure for computational transcript design by illustrating a high throughput, multiprocessor approach for evaluating spacer sequences and generating functional rREDs. This chapter is written as a tutorial, complete with pseudo-code and step-by-step instructions for setting up a computational cluster with an Amazon, Inc. web server and performing the large numbers of kinefold-based stochastic kinetic co-transcriptional folding simulations needed to design functional rREDs and aREDs. The method described here should be broadly applicable for designing and analyzing a variety of synthetic RNA parts, devices and transcripts.

Real-Time MENTAT programming language and architecture

NASA Technical Reports Server (NTRS)

Grimshaw, Andrew S.; Silberman, Ami; Liu, Jane W. S.

1989-01-01

Real-time MENTAT, a programming environment designed to simplify the task of programming real-time applications in distributed and parallel environments, is described. It is based on the same data-driven computation model and object-oriented programming paradigm as MENTAT. It provides an easy-to-use mechanism to exploit parallelism, language constructs for the expression and enforcement of timing constraints, and run-time support for scheduling and exciting real-time programs. The real-time MENTAT programming language is an extended C++. The extensions are added to facilitate automatic detection of data flow and generation of data flow graphs, to express the timing constraints of individual granules of computation, and to provide scheduling directives for the runtime system. A high-level view of the real-time MENTAT system architecture and programming language constructs is provided.

EMMA: An Extensible Mammalian Modular Assembly Toolkit for the Rapid Design and Production of Diverse Expression Vectors.

PubMed

Martella, Andrea; Matjusaitis, Mantas; Auxillos, Jamie; Pollard, Steven M; Cai, Yizhi

2017-07-21

Mammalian plasmid expression vectors are critical reagents underpinning many facets of research across biology, biomedical research, and the biotechnology industry. Traditional cloning methods often require laborious manual design and assembly of plasmids using tailored sequential cloning steps. This process can be protracted, complicated, expensive, and error-prone. New tools and strategies that facilitate the efficient design and production of bespoke vectors would help relieve a current bottleneck for researchers. To address this, we have developed an extensible mammalian modular assembly kit (EMMA). This enables rapid and efficient modular assembly of mammalian expression vectors in a one-tube, one-step golden-gate cloning reaction, using a standardized library of compatible genetic parts. The high modularity, flexibility, and extensibility of EMMA provide a simple method for the production of functionally diverse mammalian expression vectors. We demonstrate the value of this toolkit by constructing and validating a range of representative vectors, such as transient and stable expression vectors (transposon based vectors), targeting vectors, inducible systems, polycistronic expression cassettes, fusion proteins, and fluorescent reporters. The method also supports simple assembly combinatorial libraries and hierarchical assembly for production of larger multigenetic cargos. In summary, EMMA is compatible with automated production, and novel genetic parts can be easily incorporated, providing new opportunities for mammalian synthetic biology.

Programming gene expression with combinatorial promoters

PubMed Central

Cox, Robert Sidney; Surette, Michael G; Elowitz, Michael B

2007-01-01

Promoters control the expression of genes in response to one or more transcription factors (TFs). The architecture of a promoter is the arrangement and type of binding sites within it. To understand natural genetic circuits and to design promoters for synthetic biology, it is essential to understand the relationship between promoter function and architecture. We constructed a combinatorial library of random promoter architectures. We characterized 288 promoters in Escherichia coli, each containing up to three inputs from four different TFs. The library design allowed for multiple −10 and −35 boxes, and we observed varied promoter strength over five decades. To further analyze the functional repertoire, we defined a representation of promoter function in terms of regulatory range, logic type, and symmetry. Using these results, we identified heuristic rules for programming gene expression with combinatorial promoters. PMID:18004278

Construction of regulatory networks using expression time-series data of a genotyped population.

PubMed

Yeung, Ka Yee; Dombek, Kenneth M; Lo, Kenneth; Mittler, John E; Zhu, Jun; Schadt, Eric E; Bumgarner, Roger E; Raftery, Adrian E

2011-11-29

The inference of regulatory and biochemical networks from large-scale genomics data is a basic problem in molecular biology. The goal is to generate testable hypotheses of gene-to-gene influences and subsequently to design bench experiments to confirm these network predictions. Coexpression of genes in large-scale gene-expression data implies coregulation and potential gene-gene interactions, but provide little information about the direction of influences. Here, we use both time-series data and genetics data to infer directionality of edges in regulatory networks: time-series data contain information about the chronological order of regulatory events and genetics data allow us to map DNA variations to variations at the RNA level. We generate microarray data measuring time-dependent gene-expression levels in 95 genotyped yeast segregants subjected to a drug perturbation. We develop a Bayesian model averaging regression algorithm that incorporates external information from diverse data types to infer regulatory networks from the time-series and genetics data. Our algorithm is capable of generating feedback loops. We show that our inferred network recovers existing and novel regulatory relationships. Following network construction, we generate independent microarray data on selected deletion mutants to prospectively test network predictions. We demonstrate the potential of our network to discover de novo transcription-factor binding sites. Applying our construction method to previously published data demonstrates that our method is competitive with leading network construction algorithms in the literature.

Design, Assembly, and Characterization of TALE-Based Transcriptional Activators and Repressors

PubMed Central

Thakore, Pratiksha I.; Gersbach, Charles A.

2016-01-01

Transcription activator-like effectors (TALEs) are modular DNA-binding proteins that can be fused to a variety of effector domains to regulate the epigenome. Nucleotide recognition by TALE monomers follows a simple cipher, making this a powerful and versatile method to activate or repress gene expression. Described here are methods to design, assemble, and test TALE transcription factors (TALE-TFs) for control of endogenous gene expression. In this protocol, TALE arrays are constructed by Golden Gate cloning and tested for activity by transfection and quantitative RT-PCR. These methods for engineering TALE-TFs are useful for studies in reverse genetics and genomics, synthetic biology, and gene therapy. PMID:26443215

MultiSite Gateway-Compatible Cell Type-Specific Gene-Inducible System for Plants1[OPEN

PubMed Central

Siligato, Riccardo; Wang, Xin; Yadav, Shri Ram; Lehesranta, Satu; Ma, Guojie; Ursache, Robertas; Sevilem, Iris; Zhang, Jing; Gorte, Maartje; Prasad, Kalika; Heidstra, Renze

2016-01-01

A powerful method to study gene function is expression or overexpression in an inducible, cell type-specific system followed by observation of consequent phenotypic changes and visualization of linked reporters in the target tissue. Multiple inducible gene overexpression systems have been developed for plants, but very few of these combine plant selection markers, control of expression domains, access to multiple promoters and protein fusion reporters, chemical induction, and high-throughput cloning capabilities. Here, we introduce a MultiSite Gateway-compatible inducible system for Arabidopsis (Arabidopsis thaliana) plants that provides the capability to generate such constructs in a single cloning step. The system is based on the tightly controlled, estrogen-inducible XVE system. We demonstrate that the transformants generated with this system exhibit the expected cell type-specific expression, similar to what is observed with constitutively expressed native promoters. With this new system, cloning of inducible constructs is no longer limited to a few special cases but can be used as a standard approach when gene function is studied. In addition, we present a set of entry clones consisting of histochemical and fluorescent reporter variants designed for gene and promoter expression studies. PMID:26644504

BioCreative V track 4: a shared task for the extraction of causal network information using the Biological Expression Language.

PubMed

Rinaldi, Fabio; Ellendorff, Tilia Renate; Madan, Sumit; Clematide, Simon; van der Lek, Adrian; Mevissen, Theo; Fluck, Juliane

2016-01-01

Automatic extraction of biological network information is one of the most desired and most complex tasks in biological and medical text mining. Track 4 at BioCreative V attempts to approach this complexity using fragments of large-scale manually curated biological networks, represented in Biological Expression Language (BEL), as training and test data. BEL is an advanced knowledge representation format which has been designed to be both human readable and machine processable. The specific goal of track 4 was to evaluate text mining systems capable of automatically constructing BEL statements from given evidence text, and of retrieving evidence text for given BEL statements. Given the complexity of the task, we designed an evaluation methodology which gives credit to partially correct statements. We identified various levels of information expressed by BEL statements, such as entities, functions, relations, and introduced an evaluation framework which rewards systems capable of delivering useful BEL fragments at each of these levels. The aim of this evaluation method is to help identify the characteristics of the systems which, if combined, would be most useful for achieving the overall goal of automatically constructing causal biological networks from text. © The Author(s) 2016. Published by Oxford University Press.

Experimental design for three-color and four-color gene expression microarrays.

PubMed

Woo, Yong; Krueger, Winfried; Kaur, Anupinder; Churchill, Gary

2005-06-01

Three-color microarrays, compared with two-color microarrays, can increase design efficiency and power to detect differential expression without additional samples and arrays. Furthermore, three-color microarray technology is currently available at a reasonable cost. Despite the potential advantages, clear guidelines for designing and analyzing three-color experiments do not exist. We propose a three- and a four-color cyclic design (loop) and a complementary graphical representation to help design experiments that are balanced, efficient and robust to hybridization failures. In theory, three-color loop designs are more efficient than two-color loop designs. Experiments using both two- and three-color platforms were performed in parallel and their outputs were analyzed using linear mixed model analysis in R/MAANOVA. These results demonstrate that three-color experiments using the same number of samples (and fewer arrays) will perform as efficiently as two-color experiments. The improved efficiency of the design is somewhat offset by a reduced dynamic range and increased variability in the three-color experimental system. This result suggests that, with minor technological improvements, three-color microarrays using loop designs could detect differential expression more efficiently than two-color loop designs. http://www.jax.org/staff/churchill/labsite/software Multicolor cyclic design construction methods and examples along with additional results of the experiment are provided at http://www.jax.org/staff/churchill/labsite/pubs/yong.

Hypoxia-inducible bidirectional shRNA expression vector delivery using PEI/chitosan-TBA copolymers for colorectal Cancer gene therapy.

PubMed

Javan, Bita; Atyabi, Fatemeh; Shahbazi, Majid

2018-06-01

This investigation was conducted to construct a hypoxia/colorectal dual-specific bidirectional short hairpin RNA (shRNA) expression vector and to transfect it into the colon cancer cell line HT-29 with PEI/chitosan-TBA nanoparticles for the simultaneous knock down of β-catenin and Bcl-2 under hypoxia. To construct a pRNA-bipHRE-CEA vector, the carcinoma embryonic antigen (CEA) promoter designed in two directions and the vascular endothelial growth factor (VEGF) enhancer were inserted between two promoters for hypoxic cancer specific gene expression. To confirm the therapeutic effect of the dual-specific vector, β-catenin and Bcl-2 shRNAs were inserted downstream of each promoter. The physicochemical properties, the cytotoxicity, and the transfection efficiency of these PEI/chitosan-TBA nanoparticles were investigated. In addition, the antitumor effects of the designed vector on the expression of β-catenin and Bcl-2, cell cycle distribution, and apoptosis were investigated in vitro. The silencing effect of the hypoxia-response shRNA expression vector was relatively low (18%-25%) under normoxia, whereas it was significantly increased to approximately 50%-60% in the HT-29 cell line. Moreover, the cancer cells showed significant G0/G1 arrest and increased apoptosis due to gene silencing under hypoxia. Furthermore, MTS assay, fluorescence microscopy images, and flow cytometry analyses confirmed that the PEI/chitosan-TBA blend system provided effective transfection with low cytotoxicity. This novel hypoxia-responsive shRNA expression vector may be useful for RNA interference (RNAi)-based cancer gene therapy in hypoxic colorectal tumors. Moreover, the PEI/chitosan-TBA copolymer might be a promising gene carrier for use in gene transfer in vivo. Copyright © 2018. Published by Elsevier Inc.

COL1A1 transgene expression in stably transfected osteoblastic cells. Relative contributions of first intron, 3'-flanking sequences, and sequences derived from the body of the human COL1A1 minigene

NASA Technical Reports Server (NTRS)

Breault, D. T.; Lichtler, A. C.; Rowe, D. W.

1997-01-01

Collagen reporter gene constructs have be used to identify cell-specific sequences needed for transcriptional activation. The elements required for endogenous levels of COL1A1 expression, however, have not been elucidated. The human COL1A1 minigene is expressed at high levels and likely harbors sequence elements required for endogenous levels of activity. Using stably transfected osteoblastic Py1a cells, we studied a series of constructs (pOBColCAT) designed to characterize further the elements required for high level of expression. pOBColCAT, which contains the COL1A1 first intron, was expressed at 50-100-fold higher levels than ColCAT 3.6, which lacks the first intron. This difference is best explained by improved mRNA processing rather than a transcriptional effect. Furthermore, variation in activity observed with the intron deletion constructs is best explained by altered mRNA splicing. Two major regions of the human COL1A1 minigene, the 3'-flanking sequences and the minigene body, were introduced into pOBColCAT to assess both transcriptional enhancing activity and the effect on mRNA stability. Analysis of the minigene body, which includes the first five exons and introns fused with the terminal six introns and exons, revealed an orientation-independent 5-fold increase in CAT activity. In contrast the 3'-flanking sequences gave rise to a modest 61% increase in CAT activity. Neither region increased the mRNA half-life of the parent construct, suggesting that CAT-specific mRNA instability elements may serve as dominant negative regulators of stability. This study suggests that other sites within the body of the COL1A1 minigene are important for high expression, e.g. during periods of rapid extracellular matrix production.

12 CFR 390.41 - Construction of time limits.

Code of Federal Regulations, 2012 CFR

2012-01-01

... time prescribed by this subpart, the date of the act or event that commences the designated period of... follows: (1) If service is made by first class, registered, or certified mail, add three calendar days to the prescribed period; (2) If service is made by express mail or overnight delivery service, add one...

12 CFR 390.41 - Construction of time limits.

Code of Federal Regulations, 2014 CFR

2014-01-01

... time prescribed by this subpart, the date of the act or event that commences the designated period of... follows: (1) If service is made by first class, registered, or certified mail, add three calendar days to the prescribed period; (2) If service is made by express mail or overnight delivery service, add one...

12 CFR 1081.114 - Construction of time limits.

Code of Federal Regulations, 2012 CFR

2012-01-01

... statute, the date of the act or event that commences the designated period of time is not included. The... follows: (1) If service is made by first class, registered, or certified mail, add three calendar days to the prescribed period; (2) If service is made by express mail or overnight delivery service, add one...

12 CFR 109.12 - Construction of time limits.

Code of Federal Regulations, 2012 CFR

2012-01-01

... or event that commences the designated period of time is not included. The last day so computed is... certified mail, add three calendar days to the prescribed period; (2) If service is made by express mail or overnight delivery service, add one calendar day to the prescribed period; or (3) If service is made by...

Design and Implementation of Technology Enabled Affective Learning Using Fusion of Bio-Physical and Facial Expression

ERIC Educational Resources Information Center

Ray, Arindam; Chakrabarti, Amlan

2016-01-01

Technology Enabled Learning is a cognitive, constructive, systematic, collaborative learning procedure, which transforms teaching-learning pedagogy where role of emotion is very often neglected. Emotion plays significant role in the cognitive process of human being, so the transformation is incomplete without capturing the learner's emotional…

Geometry of Thin Nematic Elastomer Sheets

NASA Astrophysics Data System (ADS)

Aharoni, Hillel; Sharon, Eran; Kupferman, Raz

A thin sheet of nematic elastomer attains 3D configurations depending on the nematic director field upon heating. In this talk we describe the intrinsic geometry of such a sheet, and derive an expression for the metric induced by general smooth nematic director fields. Furthermore, we investigate the reverse problem of constructing a director field that induces a specified 2D geometry. We provide an explicit analytical recipe for constructing any surface of revolution using this method. We demonstrate how the design of an arbitrary 2D geometry is accessible using approximate numerical methods.

Vector modifications to eliminate transposase expression following piggyBac-mediated transgenesis

PubMed Central

Chakraborty, Syandan; Ji, HaYeun; Chen, Jack; Gersbach, Charles A.; Leong, Kam W.

2014-01-01

Transgene insertion plays an important role in gene therapy and in biological studies. Transposon-based systems that integrate transgenes by transposase-catalyzed “cut-and-paste” mechanism have emerged as an attractive system for transgenesis. Hyperactive piggyBac transposon is particularly promising due to its ability to integrate large transgenes with high efficiency. However, prolonged expression of transposase can become a potential source of genotoxic effects due to uncontrolled transposition of the integrated transgene from one chromosomal locus to another. In this study we propose a vector design to decrease post-transposition expression of transposase and to eliminate the cells that have residual transposase expression. We design a single plasmid construct that combines the transposase and the transpositioning transgene element to share a single polyA sequence for termination. Consequently, the separation of the transposase element from the polyA sequence after transposition leads to its deactivation. We also co-express Herpes Simplex Virus thymidine kinase (HSV-tk) with the transposase. Therefore, cells having residual transposase expression can be eliminated by the administration of ganciclovir. We demonstrate the utility of this combination transposon system by integrating and expressing a model therapeutic gene, human coagulation Factor IX, in HEK293T cells. PMID:25492703

Design and cloning strategies for constructing shRNA expression vectors

PubMed Central

McIntyre, Glen J; Fanning, Gregory C

2006-01-01

Background Short hairpin RNA (shRNA) encoded within an expression vector has proven an effective means of harnessing the RNA interference (RNAi) pathway in mammalian cells. A survey of the literature revealed that shRNA vector construction can be hindered by high mutation rates and the ensuing sequencing is often problematic. Current options for constructing shRNA vectors include the use of annealed complementary oligonucleotides (74 % of surveyed studies), a PCR approach using hairpin containing primers (22 %) and primer extension of hairpin templates (4 %). Results We considered primer extension the most attractive method in terms of cost. However, in initial experiments we encountered a mutation frequency of 50 % compared to a reported 20 – 40 % for other strategies. By modifying the technique to be an isothermal reaction using the DNA polymerase Phi29, we reduced the error rate to 10 %, making primer extension the most efficient and cost-effective approach tested. We also found that inclusion of a restriction site in the loop could be exploited for confirming construct integrity by automated sequencing, while maintaining intended gene suppression. Conclusion In this study we detail simple improvements for constructing and sequencing shRNA that overcome current limitations. We also compare the advantages of our solutions against proposed alternatives. Our technical modifications will be of tangible benefit to researchers looking for a more efficient and reliable shRNA construction process. PMID:16396676

Database construction for PromoterCAD: synthetic promoter design for mammals and plants.

PubMed

Nishikata, Koro; Cox, Robert Sidney; Shimoyama, Sayoko; Yoshida, Yuko; Matsui, Minami; Makita, Yuko; Toyoda, Tetsuro

2014-03-21

Synthetic promoters can control a gene's timing, location, and expression level. The PromoterCAD web server ( http://promotercad.org ) allows the design of synthetic promoters to control plant gene expression, by novel arrangement of cis-regulatory elements. Recently, we have expanded PromoterCAD's scope with additional plant and animal data: (1) PLACE (Plant Cis-acting Regulatory DNA Elements), including various sized sequence motifs; (2) PEDB (Mammalian Promoter/Enhancer Database), including gene expression data for mammalian tissues. The plant PromoterCAD data now contains 22 000 Arabidopsis thaliana genes, 2 200 000 microarray measurements in 20 growth conditions and 79 tissue organs and developmental stages, while the new mammalian PromoterCAD data contains 679 Mus musculus genes and 65 000 microarray measurements in 96 tissue organs and cell types ( http://promotercad.org/mammal/ ). This work presents step-by-step instructions for adding both regulatory motif and gene expression data to PromoterCAD, to illustrate how users can expand PromoterCAD functionality for their own applications and organisms.

Effects of cell type and configuration on anabolic and catabolic activity in 3D co-culture of mesenchymal stem cells and nucleus pulposus cells.

PubMed

Ouyang, Ann; Cerchiari, Alec E; Tang, Xinyan; Liebenberg, Ellen; Alliston, Tamara; Gartner, Zev J; Lotz, Jeffrey C

2017-01-01

Tissue engineering constructs to treat intervertebral disc degeneration must adapt to the hypoxic and inflammatory degenerative disc microenvironment. The objective of this study was to determine the effects of two key design factors, cell type and cell configuration, on the regenerative potential of nucleus pulposus cell (NPC) and mesenchymal stem cell (MSC) constructs. Anabolic and catabolic activity was quantified in constructs of varying cell type (NPCs, MSCs, and a 50:50 co-culture) and varying configuration (individual cells and micropellets). Anabolic and catabolic outcomes were both dependent on cell type. Gene expression of Agg and Col2A1, glycosaminoglycan (GAG) content, and aggrecan immunohistochemistry (IHC), were significantly higher in NPC-only and co-culture groups than in MSC-only groups, with NPC-only groups exhibiting the highest anabolic gene expression levels. However, NPC-only constructs also responded to inflammation and hypoxia with significant upregulation of catabolic genes (MMP-1, MMP-9, MMP-13, and ADAMTS-5). MSC-only groups were unaffected by degenerative media conditions, and co-culture with MSCs modulated catabolic induction of the NPCs. Culturing cells in a micropellet configuration dramatically reduced catabolic induction in co-culture and NPC-only groups. Co-culture micropellets, which take advantage of both cell type and configuration effects, had the most immunomodulatory response, with a significant decrease in MMP-13 and ADAMTS-5 expression in hypoxic and inflammatory media conditions. Co-culture micropellets were also found to self-organize into bilaminar formations with an MSC core and NPC outer layer. Further understanding of these cell type and configuration effects can improve tissue engineering designs. © 2016 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 35:61-73, 2017. © 2016 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society.

The use of open source bioinformatics tools to dissect transcriptomic data.

PubMed

Nitsche, Benjamin M; Ram, Arthur F J; Meyer, Vera

2012-01-01

Microarrays are a valuable technology to study fungal physiology on a transcriptomic level. Various microarray platforms are available comprising both single and two channel arrays. Despite different technologies, preprocessing of microarray data generally includes quality control, background correction, normalization, and summarization of probe level data. Subsequently, depending on the experimental design, diverse statistical analysis can be performed, including the identification of differentially expressed genes and the construction of gene coexpression networks.We describe how Bioconductor, a collection of open source and open development packages for the statistical programming language R, can be used for dissecting microarray data. We provide fundamental details that facilitate the process of getting started with R and Bioconductor. Using two publicly available microarray datasets from Aspergillus niger, we give detailed protocols on how to identify differentially expressed genes and how to construct gene coexpression networks.

Genome engineering for improved recombinant protein expression in Escherichia coli.

PubMed

Mahalik, Shubhashree; Sharma, Ashish K; Mukherjee, Krishna J

2014-12-19

A metabolic engineering perspective which views recombinant protein expression as a multistep pathway allows us to move beyond vector design and identify the downstream rate limiting steps in expression. In E.coli these are typically at the translational level and the supply of precursors in the form of energy, amino acids and nucleotides. Further recombinant protein production triggers a global cellular stress response which feedback inhibits both growth and product formation. Countering this requires a system level analysis followed by a rational host cell engineering to sustain expression for longer time periods. Another strategy to increase protein yields could be to divert the metabolic flux away from biomass formation and towards recombinant protein production. This would require a growth stoppage mechanism which does not affect the metabolic activity of the cell or the transcriptional or translational efficiencies. Finally cells have to be designed for efficient export to prevent buildup of proteins inside the cytoplasm and also simplify downstream processing. The rational and the high throughput strategies that can be used for the construction of such improved host cell platforms for recombinant protein expression is the focus of this review.

Upregulation of Endogenous HMOX1 Expression by a Computer-Designed Artificial Transcription Factor

PubMed Central

Guo, Hongfeng; Tian, Yi; Lu, Hai; Wei, Yong; Ying, Dajun

2010-01-01

Heme oxygenase-1 (HO-1) is well known as a cytoprotective factor. Research has revealed that it is a promising therapeutic target for cardiovascular diseases. In the current study, an HMOX1 (HO-1 gene) enhancer-specific artificial zinc-finger protein (AZP) was designed using bioinformatical methods. Then, an artificial transcription factor (ATF) was constructed based on the AZP. In the ATF, the p65 functional domain was used as the effector domain (ED), and a nuclear localization sequence (NLS) was also included. We next analyzed the affinity of the ATF to the HMOX1 enhancer and the effect of the ATF on endogenous HMOX1 expression. The results suggest that the ATF could effectively upregulate endogenous HMOX1 expression in ECV304 cells. With further research, the ATF could be developed as a potential drug for cardiovascular diseases. PMID:20706680

Computational design of a Zn2+ receptor that controls bacterial gene expression

NASA Astrophysics Data System (ADS)

Dwyer, M. A.; Looger, L. L.; Hellinga, H. W.

2003-09-01

The control of cellular physiology and gene expression in response to extracellular signals is a basic property of living systems. We have constructed a synthetic bacterial signal transduction pathway in which gene expression is controlled by extracellular Zn2+. In this system a computationally designed Zn2+-binding periplasmic receptor senses the extracellular solute and triggers a two-component signal transduction pathway via a chimeric transmembrane protein, resulting in transcriptional up-regulation of a -galactosidase reporter gene. The Zn2+-binding site in the designed receptor is based on a four-coordinate, tetrahedral primary coordination sphere consisting of histidines and glutamates. In addition, mutations were introduced in a secondary coordination sphere to satisfy the residual hydrogen-bonding potential of the histidines coordinated to the metal. The importance of the secondary shell interactions is demonstrated by their effect on metal affinity and selectivity, as well as protein stability. Three designed protein sequences, comprising two distinct metal-binding positions, were all shown to bind Zn2+ and to function in the cell-based assay, indicating the generality of the design methodology. These experiments demonstrate that biological systems can be manipulated with computationally designed proteins that have drastically altered ligand-binding specificities, thereby extending the repertoire of genetic control by extracellular signals.

Cytosolic expression of functional Fab fragments in Escherichia coli using a novel combination of dual SUMO expression cassette and EnBase® cultivation mode.

PubMed

Rezaie, F; Davami, F; Mansouri, K; Agha Amiri, S; Fazel, R; Mahdian, R; Davoudi, N; Enayati, S; Azizi, M; Khalaj, V

2017-05-08

The Escherichia coli expression system is highly effective in producing recombinant proteins. However, there are some limitations in this system, especially in obtaining correctly folded forms of some complex proteins such as Fab fragments. To improve the solubility and folding quality of Fab fragments, we have examined the effect of simultaneous application of a SUMO fusion tag, EnBase ® cultivation mode and a redox mutant strain in the E. coli expression system. A bicistronic gene construct was designed to express an antivascular endothelial growth factor (VEGF) Fab fragment as a model system. The construct contained a dual SUMO fusion gene fragment to encode SUMO-tagged heavy and light chains. While the expression of the construct in batch cultures of BL21 or SHuffle ® transformants produced insoluble and unfolded products, the induction of the transformants in EnBase ® medium resulted in soluble and correctly folded Fab fragment, reaching as high as 19% of the total protein in shuffle strain. The functional assays indicated that the biological activity of the target Fab is similar to the commercial anti-VEGF, Lucentis ® . This study demonstrated that the combination of SUMO fusion technology, EnBase ® cultivation system and recruiting a redox mutant of E. coli can efficiently enhance the solubility and productivity of recombinant Fab fragments. The presented strategy provides not only a novel method to produce soluble and active form of an anti-VEGF Fab but also may use in the efficient production of other antibody fragments. © 2017 The Society for Applied Microbiology.

Developing an instrument to measure effective factors on Clinical Learning.

PubMed

Dadgaran, Ideh; Shirazi, Mandana; Mohammadi, Aeen; Ravari, Ali

2016-07-01

Although nursing students spend a large part of their learning period in the clinical environment, clinical learning has not been perceived by its nature yet. To develop an instrument to measure effective factors on clinical learning in nursing students. This is a mixed methods study performed in 2 steps. First, the researchers defined "clinical learning" in nursing students through qualitative content analysis and designed items of the questionnaire based on semi-structured individual interviews with nursing students. Then, as the second step, psychometric properties of the questionnaire were evaluated using the face validity, content validity, construct validity, and internal consistency evaluated on 227 students from fourth or higher semesters. All the interviews were recorded and transcribed, and then, they were analyzed using Max Qualitative Data Analysis and all of qualitative data were analyzed using SPSS 14. To do the study, we constructed the preliminary questionnaire containing 102 expressions. After determination of face and content validities by qualitative and quantitative approaches, the expressions of the questionnaire were reduced to 45. To determine the construct validity, exploratory factor analysis was applied. The results indicated that the maximum variance percentage (40.55%) was defined by the first 3 factors while the rest of the total variance percentage (59.45%) was determined by the other 42 factors. Results of exploratory factor analysis of this questionnaire indicated the presence of 3 instructor-staff, students, and educational related factors. Finally, 41 expressions were kept in 3 factor groups. The α-Cronbach coefficient (0.93) confirmed the high internal consistency of the questionnaire. Results indicated that the prepared questionnaire was an efficient instrument in the study of the effective factors on clinical learning as viewed by nursing students since it involves 41 expressions and properties such as instrument design based on perception and experiences of the nursing students about effective factors on clinical learning, definition of facilitator and preventive factors of the clinical learning, simple scoring, suitable validity and reliability, and applicability in different occasions.

Constructing Chimeric Antigen for Precise Screening of HTLV-I Infection.

PubMed

Heydari Zarnagh, Hafez; Hassanpour, Kazem; Rasaee, Mohammad Javad

2015-08-01

Individual preparation of two human T-cell lymphotropic virus type I (HTLV-I) diagnostic GST fused peptides (MTA-1 and GD21) is time-consuming and expensive. The aim of this study was to design a novel single chimeric antigen (SCA) to obviate separate expression of proteins and reduce the cost of reagent preparation. Structural protein fragments, including immunodominant B cell linear epitopes, were selected and different SCAs were designed. Tertiary structure, epitope exposure, solubility and stability were calculated for each SCA and compared with each other. The synthetic DNA encoding the interested SCA was sub-cloned into pET32a expression vector, expressed as a soluble form in Escherichia coli BL21 (DE3) cells and purified under native condition using affinity chromatography. The SDS-PAGE results indicated that thioredoxin-fused SCA was successfully expressed as a soluble form in E. coli BL21 (DE3) cells. The results of ELISA confirmed that SCA reacted with anti-HTLV-I antibodies in a concentration-dependent manner. Our results indicated that the designed SCA may be a good candidate for the screening of HTLV-I carriers with antigen-antibody-based tests.

The Conjunction Fallacy: A Misunderstanding about Conjunction?

ERIC Educational Resources Information Center

Tentori, Katya; Bonini, Nicolao; Osherson, Daniel

2004-01-01

It is easy to construct pairs of sentences X, Y that lead many people to ascribe higher probability to the conjunction X-and-Y than to the conjuncts X, Y. Whether an error is thereby committed depends on reasoners' interpretation of the expressions "probability" and "and." We report two experiments designed to clarify the normative status of…

Rapid obtention of stable, bioluminescent tumor cell lines using a tCD2-luciferase chimeric construct

PubMed Central

2011-01-01

Background Bioluminescent tumor cell lines are experimental tools of major importance for cancer investigation, especially imaging of tumors in xenografted animals. Stable expression of exogenous luciferase in tumor cells combined to systemic injection of luciferin provides an excellent signal/background ratio for external optical imaging. Therefore, there is a need to rationalize and speed up the production of luciferase-positive tumor cell lines representative of multiple tumor phenotypes. For this aim we have designed a fusion gene linking the luciferase 2 protein to the c-terminus of a truncated form of the rat CD2 protein (tCD2-luc2). To allow simultaneous assessment of the wild-type luciferase 2 in a context of tCD2 co-expression, we have made a bicistronic construct for concomitant but separate expression of these two proteins (luc2-IRES-tCD2). Both the mono- and bi-cistronic constructs were transduced in lymphoid and epithelial cells using lentiviral vectors. Results The tCD2-luc2 chimera behaves as a type I membrane protein with surface presentation of CD2 epitopes. One of these epitopes reacts with the OX34, a widely spread, high affinity monoclonal antibody. Stably transfected cells are sorted by flow cytometry on the basis of OX34 staining. In vitro and, moreover, in xenografted tumors, the tCD2-luc2 chimera retains a substantial and stable luciferase activity, although not as high as the wild-type luciferase expressed from the luc2-IRES-tCD2 construct. Expression of the tCD2-luc2 chimera does not harm cell and tumor growth. Conclusion Lentiviral transduction of the chimeric tCD2-luc2 fusion gene allows selection of cell clones with stable luciferase expression in less than seven days without antibiotic selection. We believe that it will be helpful to increase the number of tumor cell lines available for in vivo imaging and assessment of novel therapeutic modalities. On a longer term, the tCD2-luc2 chimera has the potential to be expressed from multi-cassette vectors in combination with various inserts of interest. PMID:21435248

Generation of dTALEs and Libraries of Synthetic TALE-Activated Promoters for Engineering of Gene Regulatory Networks in Plants.

PubMed

Schreiber, Tom; Tissier, Alain

2017-01-01

Transcription factors with programmable DNA-binding specificity constitute valuable tools for the design of orthogonal gene regulatory networks for synthetic biology. Transcription activator-like effectors (TALEs), as natural transcription regulators, were used to design, build, and test libraries of synthetic TALE-activated promoters (STAPs) that show a broad range of expression levels in plants. In this chapter, we present protocols for the construction of artificial TALEs and corresponding STAPs.

Development of two bacterial artificial chromosome shuttle vectors for a recombination-based cloning and regulated expression of large genes in mammalian cells.

PubMed

Hong, Y K; Kim, D H; Beletskii, A; Lee, C; Memili, E; Strauss, W M

2001-04-01

Most conditional expression vectors designed for mammalian cells have been valuable systems for studying genes of interest by regulating their expressions. The available vectors, however, are reliable for the short-length cDNA clones and not optimal for relatively long fragments of genomic DNA or long cDNAs. Here, we report the construction of two bacterial artificial chromosome (BAC) vectors, capable of harboring large inserts and shuttling among Escherichia coli, yeast, and mammalian cells. These two vectors, pEYMT and pEYMI, contain conditional expression systems which are designed to be regulated by tetracycline and mouse interferons, respectively. To test the properties of the vectors, we cloned in both vectors the green fluorescence protein (GFP) through an in vitro ligation reaction and the 17.8-kb-long X-inactive-specific transcript (Xist) cDNA through homologous recombination in yeast. Subsequently, we characterized their regulated expression properties using real-time quantitative RT-PCR (TaqMan) and RNA-fluorescent in situ hybridization (FISH). We demonstrate that these two BAC vectors are good systems for recombination-based cloning and regulated expression of large genes in mammalian cells. Copyright 2001 Academic Press.

Design and construction of targeted AAVP vectors for mammalian cell transduction.

PubMed

Hajitou, Amin; Rangel, Roberto; Trepel, Martin; Soghomonyan, Suren; Gelovani, Juri G; Alauddin, Mian M; Pasqualini, Renata; Arap, Wadih

2007-01-01

Bacteriophage (phage) evolved as bacterial viruses, but can be adapted to transduce mammalian cells through ligand-directed targeting to a specific receptor. We have recently reported a new generation of hybrid prokaryotic-eukaryotic vectors, which are chimeras of genetic cis-elements of recombinant adeno-associated virus and phage (termed AAVP). This protocol describes the design and construction of ligand-directed AAVP vectors, production of AAVP particles and the methodology to transduce mammalian cells in vitro and to target tissues in vivo after systemic administration. Targeted AAVP particles are made in a two-step process. First, a ligand peptide of choice is displayed on the coat protein to generate a targeted backbone phage vector. Then, a recombinant AAV carrying a mammalian transgene cassette is inserted into an intergenomic region. High-titer suspensions (approximately 10(10)-10(11) transducing units per microl) can be produced within 3 days after vector construction. Transgene expression by targeted AAVP usually reaches maximum levels within 1 week.

Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae

PubMed Central

2017-01-01

Transferring the prokaryotic enzyme nitrogenase into a eukaryotic host with the final aim of developing N2 fixing cereal crops would revolutionize agricultural systems worldwide. Targeting it to mitochondria has potential advantages because of the organelle’s high O2 consumption and the presence of bacterial-type iron–sulfur cluster biosynthetic machinery. In this study, we constructed 96 strains of Saccharomyces cerevisiae in which transcriptional units comprising nine Azotobacter vinelandii nif genes (nifHDKUSMBEN) were integrated into the genome. Two combinatorial libraries of nif gene clusters were constructed: a library of mitochondrial leading sequences consisting of 24 clusters within four subsets of nif gene expression strength, and an expression library of 72 clusters with fixed mitochondrial leading sequences and nif expression levels assigned according to factorial design. In total, 29 promoters and 18 terminators were combined to adjust nif gene expression levels. Expression and mitochondrial targeting was confirmed at the protein level as immunoblot analysis showed that Nif proteins could be efficiently accumulated in mitochondria. NifDK tetramer formation, an essential step of nitrogenase assembly, was experimentally proven both in cell-free extracts and in purified NifDK preparations. This work represents a first step toward obtaining functional nitrogenase in the mitochondria of a eukaryotic cell. PMID:28221768

Establishment of expanded and streamlined pipeline of PITCh knock-in – a web-based design tool for MMEJ-mediated gene knock-in, PITCh designer, and the variations of PITCh, PITCh-TG and PITCh-KIKO

PubMed Central

Nakamae, Kazuki; Nishimura, Yuki; Takenaga, Mitsumasa; Sakamoto, Naoaki; Ide, Hiroshi; Sakuma, Tetsushi; Yamamoto, Takashi

2017-01-01

ABSTRACT The emerging genome editing technology has enabled the creation of gene knock-in cells easily, efficiently, and rapidly, which has dramatically accelerated research in the field of mammalian functional genomics, including in humans. We recently developed a microhomology-mediated end-joining-based gene knock-in method, termed the PITCh system, and presented various examples of its application. Since the PITCh system only requires very short microhomologies (up to 40 bp) and single-guide RNA target sites on the donor vector, the targeting construct can be rapidly prepared compared with the conventional targeting vector for homologous recombination-based knock-in. Here, we established a streamlined pipeline to design and perform PITCh knock-in to further expand the availability of this method by creating web-based design software, PITCh designer (http://www.mls.sci.hiroshima-u.ac.jp/smg/PITChdesigner/index.html), as well as presenting an experimental example of versatile gene cassette knock-in. PITCh designer can automatically design not only the appropriate microhomologies but also the primers to construct locus-specific donor vectors for PITCh knock-in. By using our newly established pipeline, a reporter cell line for monitoring endogenous gene expression, and transgenesis (TG) or knock-in/knockout (KIKO) cell line can be produced systematically. Using these new variations of PITCh, an exogenous promoter-driven gene cassette expressing fluorescent protein gene and drug resistance gene can be integrated into a safe harbor or a specific gene locus to create transgenic reporter cells (PITCh-TG) or knockout cells with reporter knock-in (PITCh-KIKO), respectively. PMID:28453368

Establishment of expanded and streamlined pipeline of PITCh knock-in - a web-based design tool for MMEJ-mediated gene knock-in, PITCh designer, and the variations of PITCh, PITCh-TG and PITCh-KIKO.

PubMed

Nakamae, Kazuki; Nishimura, Yuki; Takenaga, Mitsumasa; Nakade, Shota; Sakamoto, Naoaki; Ide, Hiroshi; Sakuma, Tetsushi; Yamamoto, Takashi

2017-05-04

The emerging genome editing technology has enabled the creation of gene knock-in cells easily, efficiently, and rapidly, which has dramatically accelerated research in the field of mammalian functional genomics, including in humans. We recently developed a microhomology-mediated end-joining-based gene knock-in method, termed the PITCh system, and presented various examples of its application. Since the PITCh system only requires very short microhomologies (up to 40 bp) and single-guide RNA target sites on the donor vector, the targeting construct can be rapidly prepared compared with the conventional targeting vector for homologous recombination-based knock-in. Here, we established a streamlined pipeline to design and perform PITCh knock-in to further expand the availability of this method by creating web-based design software, PITCh designer ( http://www.mls.sci.hiroshima-u.ac.jp/smg/PITChdesigner/index.html ), as well as presenting an experimental example of versatile gene cassette knock-in. PITCh designer can automatically design not only the appropriate microhomologies but also the primers to construct locus-specific donor vectors for PITCh knock-in. By using our newly established pipeline, a reporter cell line for monitoring endogenous gene expression, and transgenesis (TG) or knock-in/knockout (KIKO) cell line can be produced systematically. Using these new variations of PITCh, an exogenous promoter-driven gene cassette expressing fluorescent protein gene and drug resistance gene can be integrated into a safe harbor or a specific gene locus to create transgenic reporter cells (PITCh-TG) or knockout cells with reporter knock-in (PITCh-KIKO), respectively.

Development of an expression plasmid and its use in genetic manipulation of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (higher Basidiomycetes).

PubMed

Yu, Xuya; Ji, Sen-Lin; He, Yi-Long; Ren, Meng-Fei; Xu, Jun-Wei

2014-01-01

We report the construction of a plasmid, pJW-EXP, designed for the expression of homologous and heterologous genes in Ganoderma lucidum. pJW-EXP was generated from the plasmid pMD19-T by inserting the G. lucidum glyceraldehyde-3-phosphate dehydrogenase gene promoter, the G. lucidum iron-sulfur protein subunit of succinate dehydrogenase gene terminator and the homologous carboxin-resistance gene as selection marker. This expression plasmid can be efficiently transformed into Ganoderma through polyethylene glycol-mediated protoplast transformation. Southern blot analysis showed that most of the integrated DNA appeared as multiple copies in the genome. The applicability of the constructed plasmid was tested by expression of the truncated G. lucidum 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene that encodes the catalytic domain of HMGR. Overexpression of the truncated HMGR gene, which is a key gene in the biosynthetic pathway of the antitumor compounds, ganoderic acids, increased the transcription of the HMGR gene and enhanced ganoderic acid accumulation. pJW-EXP can serve as a useful tool in the genetic improvement and metabolic engineering of Ganoderma.

Production of an active feline interferon in the cocoon of transgenic silkworms using the fibroin H-chain expression system

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

Kurihara, H.; Sezutsu, H.; Tamura, T.

2007-04-20

We constructed the fibroin H-chain expression system to produce recombinant proteins in the cocoon of transgenic silkworms. Feline interferon (FeIFN) was used for production and to assess the quality of the product. Two types of FeIFN fusion protein, each with N- and C-terminal sequences of the fibroin H-chain, were designed to be secreted into the lumen of the posterior silk glands. The expression of the FeIFN/H-chain fusion gene was regulated by the fibroin H-chain promoter domain. The transgenic silkworms introduced these constructs with the piggyBac transposon-derived vector, which produced the normal sized cocoons containing each FeIFN/H-chain fusion protein. Although themore » native-protein produced by transgenic silkworms have almost no antiviral activity, the proteins after the treatment with PreScission protease to eliminate fibroin H-chain derived N- and C-terminal sequences from the products, had very high antiviral activity. This H-chain expression system, using transgenic silkworms, could be an alternative method to produce an active recombinant protein and silk-based biomaterials.« less

Lotus Base: An integrated information portal for the model legume Lotus japonicus

PubMed Central

Mun, Terry; Bachmann, Asger; Gupta, Vikas; Stougaard, Jens; Andersen, Stig U.

2016-01-01

Lotus japonicus is a well-characterized model legume widely used in the study of plant-microbe interactions. However, datasets from various Lotus studies are poorly integrated and lack interoperability. We recognize the need for a comprehensive repository that allows comprehensive and dynamic exploration of Lotus genomic and transcriptomic data. Equally important are user-friendly in-browser tools designed for data visualization and interpretation. Here, we present Lotus Base, which opens to the research community a large, established LORE1 insertion mutant population containing an excess of 120,000 lines, and serves the end-user tightly integrated data from Lotus, such as the reference genome, annotated proteins, and expression profiling data. We report the integration of expression data from the L. japonicus gene expression atlas project, and the development of tools to cluster and export such data, allowing users to construct, visualize, and annotate co-expression gene networks. Lotus Base takes advantage of modern advances in browser technology to deliver powerful data interpretation for biologists. Its modular construction and publicly available application programming interface enable developers to tap into the wealth of integrated Lotus data. Lotus Base is freely accessible at: https://lotus.au.dk. PMID:28008948

Designing a connectionist network supercomputer.

PubMed

Asanović, K; Beck, J; Feldman, J; Morgan, N; Wawrzynek, J

1993-12-01

This paper describes an effort at UC Berkeley and the International Computer Science Institute to develop a supercomputer for artificial neural network applications. Our perspective has been strongly influenced by earlier experiences with the construction and use of a simpler machine. In particular, we have observed Amdahl's Law in action in our designs and those of others. These observations inspire attention to many factors beyond fast multiply-accumulate arithmetic. We describe a number of these factors along with rough expressions for their influence and then give the applications targets, machine goals and the system architecture for the machine we are currently designing.

[Construction and functional identification of eukaryotic expression vector carrying Sprague-Dawley rat MSX-2 gene].

PubMed

Yang, Xian-Xian; Zhang, Mei; Yan, Zhao-Wen; Zhang, Ru-Hong; Mu, Xiong-Zheng

2008-01-01

To construct a high effective eukaryotic expressing plasmid PcDNA 3.1-MSX-2 encoding Sprague-Dawley rat MSX-2 gene for the further study of MSX-2 gene function. The full length SD rat MSX-2 gene was amplified by PCR, and the full length DNA was inserted in the PMD1 8-T vector. It was isolated by restriction enzyme digest with BamHI and Xhol, then ligated into the cloning site of the PcDNA3.1 expression plasmid. The positive recombinant was identified by PCR analysis, restriction endonudease analysis and sequence analysis. Expression of RNA and protein was detected by RT-PCR and Western blot analysis in PcDNA3.1-MSX-2 transfected HEK293 cells. Sequence analysis and restriction endonudease analysis of PcDNA3.1-MSX-2 demonstrated that the position and size of MSX-2 cDNA insertion were consistent with the design. RT-PCR and Western blot analysis showed specific expression of mRNA and protein of MSX-2 in the transfected HEK293 cells. The high effective eukaryotic expression plasmid PcDNA3.1-MSX-2 encoding Sprague-Dawley Rat MSX-2 gene which is related to craniofacial development can be successfully reconstructed. It may serve as the basis for the further study of MSX-2 gene function.

The design of free structure granular mappings: the use of the principle of justifiable granularity.

PubMed

Pedrycz, Witold; Al-Hmouz, Rami; Morfeq, Ali; Balamash, Abdullah

2013-12-01

The study introduces a concept of mappings realized in presence of information granules and offers a design framework supporting the formation of such mappings. Information granules are conceptually meaningful entities formed on a basis of a large number of experimental input–output numeric data available for the construction of the model. We develop a conceptually and algorithmically sound way of forming information granules. Considering the directional nature of the mapping to be formed, this directionality aspect needs to be taken into account when developing information granules. The property of directionality implies that while the information granules in the input space could be constructed with a great deal of flexibility, the information granules formed in the output space have to inherently relate to those built in the input space. The input space is granulated by running a clustering algorithm; for illustrative purposes, the focus here is on fuzzy clustering realized with the aid of the fuzzy C-means algorithm. The information granules in the output space are constructed with the aid of the principle of justifiable granularity (being one of the underlying fundamental conceptual pursuits of Granular Computing). The construct exhibits two important features. First, the constructed information granules are formed in the presence of information granules already constructed in the input space (and this realization is reflective of the direction of the mapping from the input to the output space). Second, the principle of justifiable granularity does not confine the realization of information granules to a single formalism such as fuzzy sets but helps form the granules expressed any required formalism of information granulation. The quality of the granular mapping (viz. the mapping realized for the information granules formed in the input and output spaces) is expressed in terms of the coverage criterion (articulating how well the experimental data are “covered” by information granules produced by the granular mapping for any input experimental data). Some parametric studies are reported by quantifying the performance of the granular mapping (expressed in terms of the coverage and specificity criteria) versus the values of a certain parameters utilized in the construction of output information granules through the principle of justifiable granularity. The plots of coverage–specificity dependency help determine a knee point and reach a sound compromise between these two conflicting requirements imposed on the quality of the granular mapping. Furthermore, quantified is the quality of the mapping with regard to the number of information granules (implying a certain granularity of the mapping). A series of experiments is reported as well.

In vitro engineering of fibrocartilage using CDMP1 induced dermal fibroblasts and polyglycolide.

PubMed

Zhao, Guiqing; Yin, Shuo; Liu, Guangpeng; Cen, Lian; Sun, Jian; Zhou, Heng; Liu, Wei; Cui, Lei; Cao, Yilin

2009-07-01

This study was designed to explore the feasibility of using cartilage-derived morphogenetic protein-1 (CDMP1) induced dermal fibroblasts (DFs) as seed cells and polyglycolide (PGA) as scaffold for fibrocartilage engineering. DFs isolated from canine were expanded and seeded on PGA scaffold to fabricate cell/scaffold constructs which were cultured with or without CDMP1. Proliferation and differentiation of DFs in different constructs were determined by DNA assay and glycosaminoglycan (GAG) production. Histological and immunohistochemical staining of the constructs after being in vitro cultured for 4 and 6 weeks were carried out to observe the fibrocartilage formation condition. The fibrocartilage-specific gene expression by cells in the constructs was analyzed by real-time PCR. It was shown that in the presence of CDMP1 the proliferation and GAG synthesis of DFs were significantly enhanced compared to those without CDMP1. Fibrocartilage-like tissue was formed in the CDMP1 induced construct after being cultured for 4 weeks, and it became more matured at 6 weeks as stronger staining for GAG and higher gene expression of collagen type II was observed. Since only weak staining for GAG and collagen type II was observed for the construct engineered without CDMP1, the induction effect on the fibrocartilage engineering can be ascertained when using DFs as seed cells. Furthermore, the potential of using DFs as seed cells to engineer fibrocartilage is substantiated and further study on using the engineered tissue to repair fibrocartilage defects is currently ongoing in our group.

Standards-Based Procedural Phenotyping: The Arden Syntax on i2b2.

PubMed

Mate, Sebastian; Castellanos, Ixchel; Ganslandt, Thomas; Prokosch, Hans-Ulrich; Kraus, Stefan

2017-01-01

Phenotyping, or the identification of patient cohorts, is a recurring challenge in medical informatics. While there are open source tools such as i2b2 that address this problem by providing user-friendly querying interfaces, these platforms lack semantic expressiveness to model complex phenotyping algorithms. The Arden Syntax provides procedural programming language construct, designed specifically for medical decision support and knowledge transfer. In this work, we investigate how language constructs of the Arden Syntax can be used for generic phenotyping. We implemented a prototypical tool to integrate i2b2 with an open source Arden execution environment. To demonstrate the applicability of our approach, we used the tool together with an Arden-based phenotyping algorithm to derive statistics about ICU-acquired hypernatremia. Finally, we discuss how the combination of i2b2's user-friendly cohort pre-selection and Arden's procedural expressiveness could benefit phenotyping.

Bacterial expression of self-assembling peptide hydrogelators

NASA Astrophysics Data System (ADS)

Sonmez, Cem

For tissue regeneration and drug delivery applications, various architectures are explored to serve as biomaterial tools. Via de novo design, functional peptide hydrogel materials have been developed as scaffolds for biomedical applications. The objective of this study is to investigate bacterial expression as an alternative method to chemical synthesis for the recombinant production of self-assembling peptides that can form rigid hydrogels under physiological conditions. The Schneider and Pochan Labs have designed and characterized a 20 amino acid beta-hairpin forming amphiphilic peptide containing a D-residue in its turn region (MAX1). As a result, this peptide must be prepared chemically. Peptide engineering, using the sequence of MAX1 as a template, afforded a small family of peptides for expression (EX peptides) that have different turn sequences consisting of natural amino acids and amenable to bacterial expression. Each sequence was initially chemically synthesized to quickly assess the material properties of its corresponding gel. One model peptide EX1, was chosen to start the bacterial expression studies. DNA constructs facilitating the expression of EX1 were designed in such that the peptide could be expressed with different fusion partners and subsequently cleaved by enzymatic or chemical means to afford the free peptide. Optimization studies were performed to increase the yield of pure peptide that ultimately allowed 50 mg of pure peptide to be harvested from one liter of culture, providing an alternate means to produce this hydrogel-forming peptide. Recombinant production of other self-assembling hairpins with different turn sequences was also successful using this optimized protocol. The studies demonstrate that new beta-hairpin self-assembling peptides that are amenable to bacterial production and form rigid hydrogels at physiological conditions can be designed and produced by fermentation in good yield at significantly reduced cost when compared to chemical synthesis.

The Center for Optimized Structural Studies (COSS) platform for automation in cloning, expression, and purification of single proteins and protein-protein complexes.

PubMed

Mlynek, Georg; Lehner, Anita; Neuhold, Jana; Leeb, Sarah; Kostan, Julius; Charnagalov, Alexej; Stolt-Bergner, Peggy; Djinović-Carugo, Kristina; Pinotsis, Nikos

2014-06-01

Expression in Escherichia coli represents the simplest and most cost effective means for the production of recombinant proteins. This is a routine task in structural biology and biochemistry where milligrams of the target protein are required in high purity and monodispersity. To achieve these criteria, the user often needs to screen several constructs in different expression and purification conditions in parallel. We describe a pipeline, implemented in the Center for Optimized Structural Studies, that enables the systematic screening of expression and purification conditions for recombinant proteins and relies on a series of logical decisions. We first use bioinformatics tools to design a series of protein fragments, which we clone in parallel, and subsequently screen in small scale for optimal expression and purification conditions. Based on a scoring system that assesses soluble expression, we then select the top ranking targets for large-scale purification. In the establishment of our pipeline, emphasis was put on streamlining the processes such that it can be easily but not necessarily automatized. In a typical run of about 2 weeks, we are able to prepare and perform small-scale expression screens for 20-100 different constructs followed by large-scale purification of at least 4-6 proteins. The major advantage of our approach is its flexibility, which allows for easy adoption, either partially or entirely, by any average hypothesis driven laboratory in a manual or robot-assisted manner.

Pydna: a simulation and documentation tool for DNA assembly strategies using python.

PubMed

Pereira, Filipa; Azevedo, Flávio; Carvalho, Ângela; Ribeiro, Gabriela F; Budde, Mark W; Johansson, Björn

2015-05-02

Recent advances in synthetic biology have provided tools to efficiently construct complex DNA molecules which are an important part of many molecular biology and biotechnology projects. The planning of such constructs has traditionally been done manually using a DNA sequence editor which becomes error-prone as scale and complexity of the construction increase. A human-readable formal description of cloning and assembly strategies, which also allows for automatic computer simulation and verification, would therefore be a valuable tool. We have developed pydna, an extensible, free and open source Python library for simulating basic molecular biology DNA unit operations such as restriction digestion, ligation, PCR, primer design, Gibson assembly and homologous recombination. A cloning strategy expressed as a pydna script provides a description that is complete, unambiguous and stable. Execution of the script automatically yields the sequence of the final molecule(s) and that of any intermediate constructs. Pydna has been designed to be understandable for biologists with limited programming skills by providing interfaces that are semantically similar to the description of molecular biology unit operations found in literature. Pydna simplifies both the planning and sharing of cloning strategies and is especially useful for complex or combinatorial DNA molecule construction. An important difference compared to existing tools with similar goals is the use of Python instead of a specifically constructed language, providing a simulation environment that is more flexible and extensible by the user.

Are Anxiety and Depression Just as Stable as Personality During Late Adolescence? Results From a Three-Year Longitudinal Latent Variable Study

PubMed Central

Prenoveau, Jason M.; Craske, Michelle G.; Zinbarg, Richard E.; Mineka, Susan; Rose, Raphael D.; Griffith, James W.

2012-01-01

Although considerable evidence shows that affective symptoms and personality traits demonstrate moderate to high relative stabilities during adolescence and early adulthood, there has been little work done to examine differential stability among these constructs or to study the manner in which the stability of these constructs is expressed. The present study used a three-year longitudinal design in an adolescent/young adult sample to examine the stability of depression symptoms, social phobia symptoms, specific phobia symptoms, neuroticism, and extraversion. When considering one-, two-, and three-year durations, anxiety and personality stabilities were generally similar and typically greater than the stability of depression. Comparison of various representations of a latent variable trait-state-occasion (TSO) model revealed that whereas the full TSO model was the best representation for depression, a trait stability model was the most parsimonious of the best-fitting models for the anxiety and personality constructs. Over three years, the percentages of variance explained by the trait component for the anxiety and personality constructs (73– 84%) were significantly greater than that explained by the trait component for depression (46%). These findings indicate that symptoms of depression are more episodic in nature, whereas symptoms of anxiety are more similar to personality variables in their expression of stability. PMID:21604827

A novel recombinant anti-epidermal growth factor receptor peptide vaccine capable of active immunization and reduction of tumor volume in a mouse model.

PubMed

Asadi-Ghalehni, Majid; Rasaee, Mohamad Javad; RajabiBazl, Masoumeh; Khosravani, Masood; Motaghinejad, Majid; Javanmardi, Masoud; Khalili, Saeed; Modjtahedi, Helmout; Sadroddiny, Esmaeil

2017-12-01

Over-expression of epidermal growth factor receptor (EGFR) has been reported in a number of human malignancies. Strong expression of this receptor has been associated with poor survival in many such patients. Active immunizations that elicit antibodies of the desired type could be an appealing alternative to conventional passive immunization. In this regard, a novel recombinant peptide vaccine capable of prophylactic and therapeutic effects was constructed. A novel fusion recombinant peptide base vaccine consisting of L2 domain of murine extra-cellular domain-EGFR and EGFR mimotope (EM-L2) was constructed and its prophylactic and therapeutic effects in a Lewis lung carcinoma mouse (C57/BL6) model evaluated. Constructed recombinant peptide vaccine is capable of reacting with anti-EGFR antibodies. Immunization of mice with EM-L2 peptide resulted in antibody production against EM-L2. The constructed recombinant peptide vaccine reduced tumor growth and increased the survival rate. Designing effective peptide vaccines could be an encouraging strategy in contemporary cancer immunotherapy. Investigating the efficacy of such cancer immunotherapy approaches may open exciting possibilities concerning hyperimmunization, leading to more promising effects on tumor regression and proliferation. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

Synthetic constructs in/for the environment: Managing the interplay between natural and engineered Biology

PubMed Central

Schmidt, Markus; de Lorenzo, Víctor

2012-01-01

The plausible release of deeply engineered or even entirely synthetic/artificial microorganisms raises the issue of their intentional (e.g. bioremediation) or accidental interaction with the Environment. Containment systems designed in the 1980s–1990s for limiting the spread of genetically engineered bacteria and their recombinant traits are still applicable to contemporary Synthetic Biology constructs. Yet, the ease of DNA synthesis and the uncertainty on how non-natural properties and strains could interplay with the existing biological word poses yet again the challenge of designing safe and efficacious firewalls to curtail possible interactions. Such barriers may include xeno-nucleic acids (XNAs) instead of DNA as information-bearing molecules, rewriting the genetic code to make it non-understandable by the existing gene expression machineries, and/or making growth dependent on xenobiotic chemicals. PMID:22710182

High levels of bioplastic are produced in fertile transplastomic tobacco plants engineered with a synthetic operon for the production of polyhydroxybutyrate.

PubMed

Bohmert-Tatarev, Karen; McAvoy, Susan; Daughtry, Sean; Peoples, Oliver P; Snell, Kristi D

2011-04-01

An optimized genetic construct for plastid transformation of tobacco (Nicotiana tabacum) for the production of the renewable, biodegradable plastic polyhydroxybutyrate (PHB) was designed using an operon extension strategy. Bacterial genes encoding the PHB pathway enzymes were selected for use in this construct based on their similarity to the codon usage and GC content of the tobacco plastome. Regulatory elements with limited homology to the host plastome yet known to yield high levels of plastidial recombinant protein production were used to enhance the expression of the transgenes. A partial transcriptional unit, containing genes of the PHB pathway and a selectable marker gene encoding spectinomycin resistance, was flanked at the 5' end by the host plant's psbA coding sequence and at the 3' end by the host plant's 3' psbA untranslated region. This design allowed insertion of the transgenes into the plastome as an extension of the psbA operon, rendering the addition of a promoter to drive the expression of the transgenes unnecessary. Transformation of the optimized construct into tobacco and subsequent spectinomycin selection of transgenic plants yielded T0 plants that were capable of producing up to 18.8% dry weight PHB in samples of leaf tissue. These plants were fertile and produced viable seed. T1 plants producing up to 17.3% dry weight PHB in samples of leaf tissue and 8.8% dry weight PHB in the total biomass of the plant were also isolated.

Formalization, equivalence and generalization of basic resonance electrical circuits

NASA Astrophysics Data System (ADS)

Penev, Dimitar; Arnaudov, Dimitar; Hinov, Nikolay

2017-12-01

In the work are presented basic resonance circuits, which are used in resonance energy converters. The following resonant circuits are considered: serial, serial with parallel load parallel capacitor, parallel and parallel with serial loaded inductance. For the circuits under consideration, expressions are generated for the frequencies of own oscillations and for the equivalence of the active power emitted in the load. Mathematical expressions are graphically constructed and verified using computer simulations. The results obtained are used in the model based design of resonant energy converters with DC or AC output. This guaranteed the output indicators of power electronic devices.

The Use of UML for Software Requirements Expression and Management

NASA Technical Reports Server (NTRS)

Murray, Alex; Clark, Ken

2015-01-01

It is common practice to write English-language "shall" statements to embody detailed software requirements in aerospace software applications. This paper explores the use of the UML language as a replacement for the English language for this purpose. Among the advantages offered by the Unified Modeling Language (UML) is a high degree of clarity and precision in the expression of domain concepts as well as architecture and design. Can this quality of UML be exploited for the definition of software requirements? While expressing logical behavior, interface characteristics, timeliness constraints, and other constraints on software using UML is commonly done and relatively straight-forward, achieving the additional aspects of the expression and management of software requirements that stakeholders expect, especially traceability, is far less so. These other characteristics, concerned with auditing and quality control, include the ability to trace a requirement to a parent requirement (which may well be an English "shall" statement), to trace a requirement to verification activities or scenarios which verify that requirement, and to trace a requirement to elements of the software design which implement that requirement. UML Use Cases, designed for capturing requirements, have not always been satisfactory. Some applications of them simply use the Use Case model element as a repository for English requirement statements. Other applications of Use Cases, in which Use Cases are incorporated into behavioral diagrams that successfully communicate the behaviors and constraints required of the software, do indeed take advantage of UML's clarity, but not in ways that support the traceability features mentioned above. Our approach uses the Stereotype construct of UML to precisely identify elements of UML constructs, especially behaviors such as State Machines and Activities, as requirements, and also to achieve the necessary mapping capabilities. We describe this approach in the context of a space-based software application currently under development at the Jet Propulsion Laboratory.

The rational design of a 'type 88' genetically stable peptide display vector in the filamentous bacteriophage fd.

PubMed

Enshell-Seijffers, D; Smelyanski, L; Gershoni, J M

2001-05-15

Filamentous bacteriophages are particularly efficient for the expression and display of combinatorial random peptides. Two phage proteins are often employed for peptide display: the infectivity protein, PIII, and the major coat protein, PVIII. The use of PVIII typically requires the expression of two pVIII genes: the wild-type and the recombinant pVIII gene, to generate mosaic phages. 'Type 88' vectors contain two pVIII genes in one phage genome. In this study a novel 'type 88' expression vector has been rationally designed and constructed. Two factors were taken into account: the insertion site and the genetic stability of the second pVIII gene. It was found that selective deletion of recombinant genes was encountered when inserts were cloned into either of the two non-coding regions of the phage genome. The deletions were independent of recA yet required a functional F-episome. Transcription was also found to be a positive factor for deletion. Taking the above into account led to the generation of a novel vector, designated fth1, which can be used to express recombinant peptides as pVIII chimeric proteins in mosaic bacteriophages. The fth1 vector is not only genetically stable but also of high copy number and produces high titers of recombinant phages.

RNAi Mediated curcin precursor gene silencing in Jatropha (Jatropha curcas L.).

PubMed

Patade, Vikas Yadav; Khatri, Deepti; Kumar, Kamal; Grover, Atul; Kumari, Maya; Gupta, Sanjay Mohan; Kumar, Devender; Nasim, Mohammed

2014-07-01

Curcin, a type I ribosomal inhibiting protein-RIP, encoded by curcin precursor gene, is a phytotoxin present in Jatropha (Jatropha curcas L.). Here, we report designing of RNAi construct for the curcin precursor gene and further its genetic transformation of Jatropha to reduce its transcript expression. Curcin precursor gene was first cloned from Jatropha strain DARL-2 and part of the gene sequence was cloned in sense and antisense orientation separated by an intron sequence in plant expression binary vector pRI101 AN. The construction of the RNAi vector was confirmed by double digestion and nucleotide sequencing. The vector was then mobilized into Agrobacterium tumefaciens strain GV 3101 and used for tissue culture independent in planta transformation protocol optimized for Jatropha. Germinating seeds were injured with a needle before infection with Agrobacterium and then transferred to sterilized sand medium. The seedlings were grown for 90 days and genomic DNA was isolated from leaves for transgenic confirmation based on real time PCR with NPT II specific dual labeled probe. Result of the transgenic confirmation analysis revealed presence of the gene silencing construct in ten out of 30 tested seedlings. Further, quantitative transcript expression analysis of the curcin precursor gene revealed reduction in the transcript abundance by more than 98% to undetectable level. The transgenic plants are being grown in containment for further studies on reduction in curcin protein content in Jatropha seeds.

A toolset of aequorin expression vectors for in planta studies of subcellular calcium concentrations in Arabidopsis thaliana

PubMed Central

Mehlmer, Norbert; Parvin, Nargis; Hurst, Charlotte H.; Knight, Marc R.; Teige, Markus; Vothknecht, Ute C.

2014-01-01

Calcium has long been acknowledged as one of the most important signalling components in plants. Many abiotic and biotic stimuli are transduced into a cellular response by temporal and spatial changes in cellular calcium concentration and the calcium-sensitive protein aequorin has been exploited as a genetically encoded calcium indicator for the measurement of calcium in planta. The objective of this work was to generate a compatible set of aequorin expression plasmids for the generation of transgenic plant lines to measure changes in calcium levels in different cellular subcompartments. Aequorin was fused to different targeting peptides or organellar proteins as a means to localize it to the cytosol, the nucleus, the plasma membrane, and the mitochondria. Furthermore, constructs were designed to localize aequorin in the stroma as well as the inner and outer surface of the chloroplast envelope membranes. The modular set-up of the plasmids also allows the easy replacement of targeting sequences to include other compartments. An additional YFP-fusion was included to verify the correct subcellular localization of all constructs by laser scanning confocal microscopy. For each construct, pBin19-based binary expression vectors driven by the 35S or UBI10 promoter were made for Agrobacterium-mediated transformation. Stable Arabidopsis lines were generated and initial tests of several lines confirmed their feasibility to measure calcium signals in vivo. PMID:22213817

Artificial genetic selection for an efficient translation initiation site for expression of human RACK1 gene in Escherichia coli

PubMed Central

Zhelyabovskaya, Olga B.; Berlin, Yuri A.; Birikh, Klara R.

2004-01-01

In bacterial expression systems, translation initiation is usually the rate limiting and the least predictable stage of protein synthesis. Efficiency of a translation initiation site can vary dramatically depending on the sequence context. This is why many standard expression vectors provide very poor expression levels of some genes. This notion persuaded us to develop an artificial genetic selection protocol, which allows one to find for a given target gene an individual efficient ribosome binding site from a random pool. In order to create Darwinian pressure necessary for the genetic selection, we designed a system based on translational coupling, in which microorganism survival in the presence of antibiotic depends on expression of the target gene, while putting no special requirements on this gene. Using this system we obtained superproducing constructs for the human protein RACK1 (receptor for activated C kinase). PMID:15034151

Execution of Multidisciplinary Design Optimization Approaches on Common Test Problems

NASA Technical Reports Server (NTRS)

Balling, R. J.; Wilkinson, C. A.

1997-01-01

A class of synthetic problems for testing multidisciplinary design optimization (MDO) approaches is presented. These test problems are easy to reproduce because all functions are given as closed-form mathematical expressions. They are constructed in such a way that the optimal value of all variables and the objective is unity. The test problems involve three disciplines and allow the user to specify the number of design variables, state variables, coupling functions, design constraints, controlling design constraints, and the strength of coupling. Several MDO approaches were executed on two sample synthetic test problems. These approaches included single-level optimization approaches, collaborative optimization approaches, and concurrent subspace optimization approaches. Execution results are presented, and the robustness and efficiency of these approaches an evaluated for these sample problems.

Generative Representations for Computer-Automated Evolutionary Design

NASA Technical Reports Server (NTRS)

Hornby, Gregory S.

2006-01-01

With the increasing computational power of computers, software design systems are progressing from being tools for architects and designers to express their ideas to tools capable of creating designs under human guidance. One of the main limitations for these computer-automated design systems is the representation with which they encode designs. If the representation cannot encode a certain design, then the design system cannot produce it. To be able to produce new types of designs, and not just optimize pre-defined parameterizations, evolutionary design systems must use generative representations. Generative representations are assembly procedures, or algorithms, for constructing a design thereby allowing for truly novel design solutions to be encoded. In addition, by enabling modularity, regularity and hierarchy, the level of sophistication that can be evolved is increased. We demonstrate the advantages of generative representations on two different design domains: the evolution of spacecraft antennas and the evolution of 3D objects.

Channeled Scaffolds for Engineering Myocardium with Mechanical Stimulation

PubMed Central

Zhang, Ting; Wan, Leo Q.; Xiong, Zhuo; Marsano, Anna; Maidhof, Robert; Park, Miri; Yan, Yongnian; Vunjak-Novakovic, Gordana

2011-01-01

The characteristics of the matrix (composition, structure, mechanical properties) and external culture environment (pulsatile perfusion, physical stimulation) are critically important for engineering functional myocardial tissue. We report the development of chitosan-collagen scaffolds with micro-pores and an array of parallel channels (~200 μm in diameter) that were specifically designed for cardiac tissue engineering with mechanical stimulation. The scaffolds were designed to have the structural and mechanical properties similar to those of the native human heart matrix. Scaffolds were seeded with neonatal rat heart cells and subjected to dynamic tensile stretch using a custom-designed bioreactor. The channels enhanced oxygen transport and facilitated the establishment of cell connections within the construct. The myocardial patches (14 mm in diameter, 1–2 mm thick) consisted of metabolically active cells and started to contract synchronously after 3 days of culture. Mechanical stimulation with high tensile stresses promoted cell alignment, elongation, and the expression of connexin-43 (Cx-43). This study confirms the importance of scaffold design and mechanical stimulation for the formation of contractile cardiac constructs. PMID:22081518

Channelled scaffolds for engineering myocardium with mechanical stimulation.

PubMed

Zhang, Ting; Wan, Leo Q; Xiong, Zhuo; Marsano, Anna; Maidhof, Robert; Park, Miri; Yan, Yongnian; Vunjak-Novakovic, Gordana

2012-10-01

The characteristics of the matrix (composition, structure, mechanical properties) and external culture environment (pulsatile perfusion, physical stimulation) of the heart are important characteristics in the engineering of functional myocardial tissue. This study reports on the development of chitosan-collagen scaffolds with micropores and an array of parallel channels (~ 200 µm in diameter) that were specifically designed for cardiac tissue engineering using mechanical stimulation. The scaffolds were designed to have similar structural and mechanical properties of those of native heart matrix. Scaffolds were seeded with neonatal rat heart cells and subjected to dynamic tensile stretch using a custom designed bioreactor. The channels enhanced oxygen transport and facilitated the establishment of cell connections within the construct. The myocardial patches (14 mm in diameter, 1-2 mm thick) consisted of metabolically active cells that began to contract synchronously after 3 days of culture. Mechanical stimulation with high tensile stress promoted cell alignment, elongation, and expression of connexin-43 (Cx-43). This study confirms the importance of scaffold design and mechanical stimulation for the formation of contractile cardiac constructs. Copyright © 2011 John Wiley & Sons, Ltd.

Optimization of a yeast RNA interference system for controlling gene expression and enabling rapid metabolic engineering.

PubMed

Crook, Nathan C; Schmitz, Alexander C; Alper, Hal S

2014-05-16

Reduction of endogenous gene expression is a fundamental operation of metabolic engineering, yet current methods for gene knockdown (i.e., genome editing) remain laborious and slow, especially in yeast. In contrast, RNA interference allows facile and tunable gene knockdown via a simple plasmid transformation step, enabling metabolic engineers to rapidly prototype knockdown strategies in multiple strains before expending significant cost to undertake genome editing. Although RNAi is naturally present in a myriad of eukaryotes, it has only been recently implemented in Saccharomyces cerevisiae as a heterologous pathway and so has not yet been optimized as a metabolic engineering tool. In this study, we elucidate a set of design principles for the construction of hairpin RNA expression cassettes in yeast and implement RNA interference to quickly identify routes for improvement of itaconic acid production in this organism. The approach developed here enables rapid prototyping of knockdown strategies and thus accelerates and reduces the cost of the design-build-test cycle in yeast.

A perspectivist approach to theory construction.

PubMed

McGuire, William J

2004-01-01

A perspectivist approach is taken to the theory-construction process in psychological research. This approach assumes that all hypotheses and theories are true, as all are false, depending on the perspective from which they are viewed, and that the purpose of research is to discover which are the crucial perspectives. Perspectivism assumes also that both the a priori conceptual phase of research and the a posteriori empirical phase have both discovery and testing functions. Topics discussed include how the perspectivist approach can improve methodology training and practice (particularly as regards theory construction); what researchers accept as theoretical explanations; the nature of mediational theories; how theories can be formalized, expressed in multiple modalities and for various scaling cases; and how experimental designs can be enriched by theory-guided mediational and interactional variables.

Human Monoclonal Antibodies as a Countermeasure Against Botulinum Toxins

DTIC Science & Technology

2012-11-30

official Department of the Army position, policy or decision, unless so designated by other documentation. 12. DISTRIBUTION AVAILIBILITY STATEMENT...cell origin.  1710  designates  B cells of PBMC origin (plates 1‐100 rabbit 74, 101‐200 rabbit 81), 1714 splenic origin (plates 1‐200  rabbit 74, 201‐400...domains are  designated  as A, B, or E with a bracket.  2.1.1 Characterization of E. coli expressed constructs and their refolding for BoNT reactivity

Flowcharting with D-charts

NASA Technical Reports Server (NTRS)

Meyer, D.

1985-01-01

A D-Chart is a style of flowchart using control symbols highly appropriate to modern structured programming languages. The intent of a D-Chart is to provide a clear and concise one-for-one mapping of control symbols to high-level language constructs for purposes of design and documentation. The notation lends itself to both high-level and code-level algorithmic description. The various issues that may arise when representing, in D-Chart style, algorithms expressed in the more popular high-level languages are addressed. In particular, the peculiarities of mapping control constructs for Ada, PASCAL, FORTRAN 77, C, PL/I, Jovial J73, HAL/S, and Algol are discussed.

Flowcharting with D-charts

NASA Technical Reports Server (NTRS)

Meyer, D. D.

1985-01-01

A D-Chart is a style of flowchart using control symbols highly appropriate to modern structured programming languages. The intent of a D-Chart is to provide a clear and concise one-for-one mapping of control symbols to high-level language constructs for purposes of design and documentation. The notation lends itself to both high-level and code-level algorithmic description. The various issues that may arise when representing, in D-Chart style, algorithms expressed in the more popular high-level languages are addressed. In particular, the peculiarities of mapping control constructs for Ada, PASCAL, FORTRAN 77, C, PL/I, Joviai J73, HAL/S, and Algol are discussed.

Identification of DEP domain-containing proteins by a machine learning method and experimental analysis of their expression in human HCC tissues

NASA Astrophysics Data System (ADS)

Liao, Zhijun; Wang, Xinrui; Zeng, Yeting; Zou, Quan

2016-12-01

The Dishevelled/EGL-10/Pleckstrin (DEP) domain-containing (DEPDC) proteins have seven members. However, whether this superfamily can be distinguished from other proteins based only on the amino acid sequences, remains unknown. Here, we describe a computational method to segregate DEPDCs and non-DEPDCs. First, we examined the Pfam numbers of the known DEPDCs and used the longest sequences for each Pfam to construct a phylogenetic tree. Subsequently, we extracted 188-dimensional (188D) and 20D features of DEPDCs and non-DEPDCs and classified them with random forest classifier. We also mined the motifs of human DEPDCs to find the related domains. Finally, we designed experimental verification methods of human DEPDC expression at the mRNA level in hepatocellular carcinoma (HCC) and adjacent normal tissues. The phylogenetic analysis showed that the DEPDCs superfamily can be divided into three clusters. Moreover, the 188D and 20D features can both be used to effectively distinguish the two protein types. Motif analysis revealed that the DEP and RhoGAP domain was common in human DEPDCs, human HCC and the adjacent tissues that widely expressed DEPDCs. However, their regulation was not identical. In conclusion, we successfully constructed a binary classifier for DEPDCs and experimentally verified their expression in human HCC tissues.

A systems-level approach for metabolic engineering of yeast cell factories.

PubMed

Kim, Il-Kwon; Roldão, António; Siewers, Verena; Nielsen, Jens

2012-03-01

The generation of novel yeast cell factories for production of high-value industrial biotechnological products relies on three metabolic engineering principles: design, construction, and analysis. In the last two decades, strong efforts have been put on developing faster and more efficient strategies and/or technologies for each one of these principles. For design and construction, three major strategies are described in this review: (1) rational metabolic engineering; (2) inverse metabolic engineering; and (3) evolutionary strategies. Independent of the selected strategy, the process of designing yeast strains involves five decision points: (1) choice of product, (2) choice of chassis, (3) identification of target genes, (4) regulating the expression level of target genes, and (5) network balancing of the target genes. At the construction level, several molecular biology tools have been developed through the concept of synthetic biology and applied for the generation of novel, engineered yeast strains. For comprehensive and quantitative analysis of constructed strains, systems biology tools are commonly used and using a multi-omics approach. Key information about the biological system can be revealed, for example, identification of genetic regulatory mechanisms and competitive pathways, thereby assisting the in silico design of metabolic engineering strategies for improving strain performance. Examples on how systems and synthetic biology brought yeast metabolic engineering closer to industrial biotechnology are described in this review, and these examples should demonstrate the potential of a systems-level approach for fast and efficient generation of yeast cell factories. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Phase Change Material Heat Sink for an ISS Flight Experiment

NASA Technical Reports Server (NTRS)

Quinn, Gregory; Stieber, Jesse; Sheth, Rubik; Ahlstrom, Thomas

2015-01-01

A flight experiment is being constructed to utilize the persistent microgravity environment of the International Space Station (ISS) to prove out operation of a microgravity compatible phase change material (PCM) heat sink. A PCM heat sink can help to reduce the overall mass and volume of future exploration spacecraft thermal control systems (TCS). The program is characterizing a new PCM heat sink that incorporates a novel phase management approach to prevent high pressures and structural deformation that often occur with PCM heat sinks undergoing cyclic operation in microgravity. The PCM unit was made using brazed aluminum construction with paraffin wax as the fusible material. It is designed to be installed into a propylene glycol and water cooling loop, with scaling consistent with the conceptual designs for the Orion Multipurpose Crew Vehicle. This paper reports on the construction of the PCM heat sink and on initial ground test results conducted at UTC Aerospace Systems prior to delivery to NASA. The prototype will be tested later on the ground and in orbit via a self-contained experiment package developed by NASA Johnson Space Center to operate in an ISS EXPRESS rack.

Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants.

PubMed

Lukan, Tjaša; Machens, Fabian; Coll, Anna; Baebler, Špela; Messerschmidt, Katrin; Gruden, Kristina

2018-01-01

Cloning multiple DNA fragments for delivery of several genes of interest into the plant genome is one of the main technological challenges in plant synthetic biology. Despite several modular assembly methods developed in recent years, the plant biotechnology community has not widely adopted them yet, probably due to the lack of appropriate vectors and software tools. Here we present Plant X-tender, an extension of the highly efficient, scar-free and sequence-independent multigene assembly strategy AssemblX, based on overlap-depended cloning methods and rare-cutting restriction enzymes. Plant X-tender consists of a set of plant expression vectors and the protocols for most efficient cloning into the novel vector set needed for plant expression and thus introduces advantages of AssemblX into plant synthetic biology. The novel vector set covers different backbones and selection markers to allow full design flexibility. We have included ccdB counterselection, thereby allowing the transfer of multigene constructs into the novel vector set in a straightforward and highly efficient way. Vectors are available as empty backbones and are fully flexible regarding the orientation of expression cassettes and addition of linkers between them, if required. We optimised the assembly and subcloning protocol by testing different scar-less assembly approaches: the noncommercial SLiCE and TAR methods and the commercial Gibson assembly and NEBuilder HiFi DNA assembly kits. Plant X-tender was applicable even in combination with low efficient homemade chemically competent or electrocompetent Escherichia coli. We have further validated the developed procedure for plant protein expression by cloning two cassettes into the newly developed vectors and subsequently transferred them to Nicotiana benthamiana in a transient expression setup. Thereby we show that multigene constructs can be delivered into plant cells in a streamlined and highly efficient way. Our results will support faster introduction of synthetic biology into plant science.

Plant X-tender: An extension of the AssemblX system for the assembly and expression of multigene constructs in plants

PubMed Central

Machens, Fabian; Coll, Anna; Baebler, Špela; Messerschmidt, Katrin; Gruden, Kristina

2018-01-01

Cloning multiple DNA fragments for delivery of several genes of interest into the plant genome is one of the main technological challenges in plant synthetic biology. Despite several modular assembly methods developed in recent years, the plant biotechnology community has not widely adopted them yet, probably due to the lack of appropriate vectors and software tools. Here we present Plant X-tender, an extension of the highly efficient, scar-free and sequence-independent multigene assembly strategy AssemblX, based on overlap-depended cloning methods and rare-cutting restriction enzymes. Plant X-tender consists of a set of plant expression vectors and the protocols for most efficient cloning into the novel vector set needed for plant expression and thus introduces advantages of AssemblX into plant synthetic biology. The novel vector set covers different backbones and selection markers to allow full design flexibility. We have included ccdB counterselection, thereby allowing the transfer of multigene constructs into the novel vector set in a straightforward and highly efficient way. Vectors are available as empty backbones and are fully flexible regarding the orientation of expression cassettes and addition of linkers between them, if required. We optimised the assembly and subcloning protocol by testing different scar-less assembly approaches: the noncommercial SLiCE and TAR methods and the commercial Gibson assembly and NEBuilder HiFi DNA assembly kits. Plant X-tender was applicable even in combination with low efficient homemade chemically competent or electrocompetent Escherichia coli. We have further validated the developed procedure for plant protein expression by cloning two cassettes into the newly developed vectors and subsequently transferred them to Nicotiana benthamiana in a transient expression setup. Thereby we show that multigene constructs can be delivered into plant cells in a streamlined and highly efficient way. Our results will support faster introduction of synthetic biology into plant science. PMID:29300787

A novel regulatory element (E77) isolated from CHO-K1 genomic DNA enhances stable gene expression in Chinese hamster ovary cells.

PubMed

Kang, Shin-Young; Kim, Yeon-Gu; Kang, Seunghee; Lee, Hong Weon; Lee, Eun Gyo

2016-05-01

Vectors flanked by regulatory DNA elements have been used to generate stable cell lines with high productivity and transgene stability; however, regulatory elements in Chinese hamster ovary (CHO) cells, which are the most widely used mammalian cells in biopharmaceutical production, are still poorly understood. We isolated a novel gene regulatory element from CHO-K1 cells, designated E77, which was found to enhance the stable expression of a transgene. A genomic library was constructed by combining CHO-K1 genomic DNA fragments with a CMV promoter-driven GFP expression vector, and the E77 element was isolated by screening. The incorporation of the E77 regulatory element resulted in the generation of an increased number of clones with high expression, thereby enhancing the expression level of the transgene in the stable transfectant cell pool. Interestingly, the E77 element was found to consist of two distinct fragments derived from different locations in the CHO genome shotgun sequence. High and stable transgene expression was obtained in transfected CHO cells by combining these fragments. Additionally, the function of E77 was found to be dependent on its site of insertion and specific orientation in the vector construct. Our findings demonstrate that stable gene expression mediated by the CMV promoter in CHO cells may be improved by the isolated novel gene regulatory element E77 identified in the present study. © 2016 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of PEG-induced water stress responsive transcripts using co-expression network in Eucalyptus grandis.

PubMed

Ghosh Dasgupta, Modhumita; Dharanishanthi, Veeramuthu

2017-09-05

Ecophysiological studies in Eucalyptus have shown that water is the principal factor limiting stem growth. Effect of water deficit conditions on physiological and biochemical parameters has been extensively reported in Eucalyptus. The present study was conducted to identify major polyethylene glycol induced water stress responsive transcripts in Eucalyptus grandis using gene co-expression network. A customized array representing 3359 water stress responsive genes was designed to document their expression in leaves of E. grandis cuttings subjected to -0.225MPa of PEG treatment. The differentially expressed transcripts were documented and significantly co-expressed transcripts were used for construction of network. The co-expression network was constructed with 915 nodes and 3454 edges with degree ranging from 2 to 45. Ninety four GO categories and 117 functional pathways were identified in the network. MCODE analysis generated 27 modules and module 6 with 479 nodes and 1005 edges was identified as the biologically relevant network. The major water responsive transcripts represented in the module included dehydrin, osmotin, LEA protein, expansin, arabinogalactans, heat shock proteins, major facilitator proteins, ARM repeat proteins, raffinose synthase, tonoplast intrinsic protein and transcription factors like DREB2A, ARF9, AGL24, UNE12, WLIM1 and MYB66, MYB70, MYB 55, MYB 16 and MYB 103. The coordinated analysis of gene expression patterns and coexpression networks developed in this study identified an array of transcripts that may regulate PEG induced water stress responses in E. grandis. Copyright © 2017 Elsevier B.V. All rights reserved.

Scaffolds for Artificial miRNA Expression in Animal Cells.

PubMed

Calloni, Raquel; Bonatto, Diego

2015-10-01

Artificial miRNAs (amiRNAs) are molecules that have been developed to promote gene silencing in a similar manner to naturally occurring miRNAs. amiRNAs are generally constructed by replacing the mature miRNA sequence in the pre-miRNA stem-loop with a sequence targeting a gene of interest. These molecules offer an interesting alternative to silencing approaches that are based on shRNAs and siRNAs because they present the same efficiency as these options and are less cytotoxic. amiRNAs have mostly been applied to gene knockdown in plants; they have been examined to a lesser extent in animal cells. Therefore, this article reviews the amiRNAs that have been developed for animal cells and focuses on the miRNA scaffolds that can already be applied to construct the artificial counterparts, as well as on the different approaches that have been described to promote amiRNA expression and silencing efficiency. Furthermore, the availability of amiRNA libraries and other tools that can be used to design and construct these molecules is briefly discussed, along with an overview of the therapeutic applications for which amiRNAs have already been evaluated.

[Construction and expression of fusion protein TRX-hJagged1 in E.coli BL21].

PubMed

Li, Guo-Hui; Fan, Yu-Zhen; Huang, Si-Yong; Liu, Qiang; Yin, Dan-Dan; Liu, Li; Chen, Ren-An; Hao, Miao-Wang; Liang, Ying-Min

2014-06-01

This study was purposed to construct prokaryotic expression vector and to investigate the expression of Notch ligand Jagged1 in E.coli. An expression vector pET-hJagged1 was constructed, which can be inserted in Jagged1 with different lengths, but the DSL domain of human Jagged1 should be contained. Then the recombinant plasmids were transformed into the competent cell of E.coli BL21, and the expression of the fusion protein was induced by IPTG. Fusion protein was purified from the supernatant of cell lysates via the Nickel affinity chromatography. The results showed that prokaryotic expression vectors pET-hJagged1 (Bgl II), pET-hJagged1 (Hind I) and pET-hJagged1 (Stu I) were successfully constructed, but only pET-hJagged1 (Stu I) could express the soluble TRX-hJagged1. The purified TRX-Jagged1 protein could be obtained via the Nickel affinity chromatography, and then confirmed by Western Blot. It is concluded that prokaryotic expression vector pET-hJagged1 is successfully constructed, but only pET-hJagged1 (Stu I) can express the soluble TRX-hJagged1 and the TRX-Jagged1 fusion protein is obtained through the prokaryotic expression system, which laid a solid foundation for further to explore the effects of Jagged1 in hematopoietic and lymphoid system.

Developing an instrument to measure effective factors on Clinical Learning

PubMed Central

DADGARAN, IDEH; SHIRAZI, MANDANA; MOHAMMADI, AEEN; RAVARI, ALI

2016-01-01

Introduction Although nursing students spend a large part of their learning period in the clinical environment, clinical learning has not been perceived by its nature yet. To develop an instrument to measure effective factors on clinical learning in nursing students. Methods This is a mixed methods study performed in 2 steps. First, the researchers defined “clinical learning” in nursing students through qualitative content analysis and designed items of the questionnaire based on semi-structured individual interviews with nursing students. Then, as the second step, psychometric properties of the questionnaire were evaluated using the face validity, content validity, construct validity, and internal consistency evaluated on 227 students from fourth or higher semesters. All the interviews were recorded and transcribed, and then, they were analyzed using Max Qualitative Data Analysis and all of qualitative data were analyzed using SPSS 14. Results To do the study, we constructed the preliminary questionnaire containing 102 expressions. After determination of face and content validities by qualitative and quantitative approaches, the expressions of the questionnaire were reduced to 45. To determine the construct validity, exploratory factor analysis was applied. The results indicated that the maximum variance percentage (40.55%) was defined by the first 3 factors while the rest of the total variance percentage (59.45%) was determined by the other 42 factors. Results of exploratory factor analysis of this questionnaire indicated the presence of 3 instructor-staff, students, and educational related factors. Finally, 41 expressions were kept in 3 factor groups. The α-Cronbach coefficient (0.93) confirmed the high internal consistency of the questionnaire. Conclusion Results indicated that the prepared questionnaire was an efficient instrument in the study of the effective factors on clinical learning as viewed by nursing students since it involves 41 expressions and properties such as instrument design based on perception and experiences of the nursing students about effective factors on clinical learning, definition of facilitator and preventive factors of the clinical learning, simple scoring, suitable validity and reliability, and applicability in different occasions. PMID:27382579

77 FR 1780 - Notice of Passenger Facility Charge (PFC) Approvals and Disapprovals

Federal Register 2010, 2011, 2012, 2013, 2014

2012-01-11

... development--design and construction. Site utilities--design and construction. Stormwater facilities--design and construction. Airside/apron--design and construction. Landside/roadway--design and construction. General aviation terminal/apron--design and construction. Airport beacon relocation--design and...

Design of a muscle cell-specific expression vector utilising human vascular smooth muscle alpha-actin regulatory elements.

PubMed

Keogh, M C; Chen, D; Schmitt, J F; Dennehy, U; Kakkar, V V; Lemoine, N R

1999-04-01

The facility to direct tissue-specific expression of therapeutic gene constructs is desirable for many gene therapy applications. We describe the creation of a muscle-selective expression vector which supports transcription in vascular smooth muscle, cardiac muscle and skeletal muscle, while it is essentially silent in other cell types such as endothelial cells, hepatocytes and fibroblasts. Specific transcriptional regulatory elements have been identified in the human vascular smooth muscle cell (VSMC) alpha-actin gene, and used to create an expression vector which directs the expression of genes in cis to muscle cells. The vector contains an enhancer element we have identified in the 5' flanking region of the human VSMC alpha-actin gene involved in mediating VSMC expression. Heterologous pairing experiments have shown that the enhancer does not interact with the basal transcription complex recruited at the minimal SV40 early promoter. Such a vector has direct application in the modulation of VSMC proliferation associated with intimal hyperplasia/restenosis.

Designer proton-channel transgenic algae for photobiological hydrogen production

DOEpatents

Lee, James Weifu [Knoxville, TN

2011-04-26

A designer proton-channel transgenic alga for photobiological hydrogen production that is specifically designed for production of molecular hydrogen (H.sub.2) through photosynthetic water splitting. The designer transgenic alga includes proton-conductive channels that are expressed to produce such uncoupler proteins in an amount sufficient to increase the algal H.sub.2 productivity. In one embodiment the designer proton-channel transgene is a nucleic acid construct (300) including a PCR forward primer (302), an externally inducible promoter (304), a transit targeting sequence (306), a designer proton-channel encoding sequence (308), a transcription and translation terminator (310), and a PCR reverse primer (312). In various embodiments, the designer proton-channel transgenic algae are used with a gas-separation system (500) and a gas-products-separation and utilization system (600) for photobiological H.sub.2 production.

De Novo Construction of Redox Active Proteins.

PubMed

Moser, C C; Sheehan, M M; Ennist, N M; Kodali, G; Bialas, C; Englander, M T; Discher, B M; Dutton, P L

2016-01-01

Relatively simple principles can be used to plan and construct de novo proteins that bind redox cofactors and participate in a range of electron-transfer reactions analogous to those seen in natural oxidoreductase proteins. These designed redox proteins are called maquettes. Hydrophobic/hydrophilic binary patterning of heptad repeats of amino acids linked together in a single-chain self-assemble into 4-alpha-helix bundles. These bundles form a robust and adaptable frame for uncovering the default properties of protein embedded cofactors independent of the complexities introduced by generations of natural selection and allow us to better understand what factors can be exploited by man or nature to manipulate the physical chemical properties of these cofactors. Anchoring of redox cofactors such as hemes, light active tetrapyrroles, FeS clusters, and flavins by His and Cys residues allow cofactors to be placed at positions in which electron-tunneling rates between cofactors within or between proteins can be predicted in advance. The modularity of heptad repeat designs facilitates the construction of electron-transfer chains and novel combinations of redox cofactors and new redox cofactor assisted functions. Developing de novo designs that can support cofactor incorporation upon expression in a cell is needed to support a synthetic biology advance that integrates with natural bioenergetic pathways. © 2016 Elsevier Inc. All rights reserved.

Co-expression of hepatitis C virus polytope-HBsAg and p19-silencing suppressor protein in tobacco leaves.

PubMed

Mohammadzadeh, Sara; Roohvand, Farzin; Memarnejadian, Arash; Jafari, Anis; Ajdary, Soheila; Salmanian, Ali-Hatef; Ehsani, Parastoo

2016-01-01

Plants transformed by virus-based vectors have emerged as promising tools to rapidly express large amounts and inexpensive antigens in transient condition. We studied the possibility of transient-expression of an HBsAg-fused polytopic construct (HCVpc) [containing H-2d and HLA-A2-restricted CD8+CTL-epitopic peptides of C (Core; aa 132-142), E6 (Envelope2; aa 614-622), N (NS3; aa 1406-1415), and E4 (Envelope2; aa 405-414) in tandem of CE6NE4] in tobacco (Nicotiana tabacum) leaves for the development of a plant-based HCV vaccine. A codon-optimized gene encoding the Kozak sequence, hexahistidine (6×His)-tag peptide, and HCVpc in tandem was designed, chemically synthesized, fused to HBsAg gene, and inserted into Potato virus X (PVX-GW) vector under the control of duplicated PVX coat protein promoter (CPP). The resulted recombinant plasmids (after confirmation by restriction and sequencing analyses) were transferred into Agrobacterium tumefaciens strain GV3101 and vacuum infiltrated into tobacco leaves. The effect of gene-silencing suppressor, p19 protein from tomato bushy stunt virus, on the expression yield of HCVpc-HBsAg was also evaluated by co-infiltration of a p19 expression vector. Codon-optimized gene increased adaptation index (CAI) value (from 0.61 to 0.92) in tobacco. The expression of the HCVpc-HBsAg was confirmed by western blot and HBsAg-based detection ELISA on total extractable proteins of tobacco leaves. The expression level of the fusion protein was significantly higher in p19 co-agroinfiltrated plants. The results indicated the possibility of expression of HCVpc-HBsAg constructs with proper protein conformations in tobacco for final application as a plant-derived HCV vaccine.

Optimized invertase expression and secretion cassette for improving Yarrowia lipolytica growth on sucrose for industrial applications.

PubMed

Lazar, Zbigniew; Rossignol, Tristan; Verbeke, Jonathan; Crutz-Le Coq, Anne-Marie; Nicaud, Jean-Marc; Robak, Małgorzata

2013-11-01

Yarrowia lipolytica requires the expression of a heterologous invertase to grow on a sucrose-based substrate. This work reports the construction of an optimized invertase expression cassette composed of Saccharomyces cerevisiae Suc2p secretion signal sequence followed by the SUC2 sequence and under the control of the strong Y. lipolytica pTEF promoter. This new construction allows a fast and optimal cleavage of sucrose into glucose and fructose and allows cells to reach the maximum growth rate. Contrary to pre-existing constructions, the expression of SUC2 is not sensitive to medium composition in this context. The strain JMY2593, expressing this new cassette with an optimized secretion signal sequence and a strong promoter, produces 4,519 U/l of extracellular invertase in bioreactor experiments compared to 597 U/l in a strain expressing the former invertase construction. The expression of this cassette strongly improved production of invertase and is suitable for simultaneously high production level of citric acid from sucrose-based media.

Recursively constructing analytic expressions for equilibrium distributions of stochastic biochemical reaction networks.

PubMed

Meng, X Flora; Baetica, Ania-Ariadna; Singhal, Vipul; Murray, Richard M

2017-05-01

Noise is often indispensable to key cellular activities, such as gene expression, necessitating the use of stochastic models to capture its dynamics. The chemical master equation (CME) is a commonly used stochastic model of Kolmogorov forward equations that describe how the probability distribution of a chemically reacting system varies with time. Finding analytic solutions to the CME can have benefits, such as expediting simulations of multiscale biochemical reaction networks and aiding the design of distributional responses. However, analytic solutions are rarely known. A recent method of computing analytic stationary solutions relies on gluing simple state spaces together recursively at one or two states. We explore the capabilities of this method and introduce algorithms to derive analytic stationary solutions to the CME. We first formally characterize state spaces that can be constructed by performing single-state gluing of paths, cycles or both sequentially. We then study stochastic biochemical reaction networks that consist of reversible, elementary reactions with two-dimensional state spaces. We also discuss extending the method to infinite state spaces and designing the stationary behaviour of stochastic biochemical reaction networks. Finally, we illustrate the aforementioned ideas using examples that include two interconnected transcriptional components and biochemical reactions with two-dimensional state spaces. © 2017 The Author(s).

Bioreactor-based bone tissue engineering: The influence of dynamic flow on osteoblast phenotypic expression and matrix mineralization

PubMed Central

Yu, Xiaojun; Botchwey, Edward A.; Levine, Elliot M.; Pollack, Solomon R.; Laurencin, Cato T.

2004-01-01

An important issue in tissue engineering concerns the possibility of limited tissue ingrowth in tissue-engineered constructs because of insufficient nutrient transport. We report a dynamic flow culture system using high-aspect-ratio vessel rotating bioreactors and 3D scaffolds for culturing rat calvarial osteoblast cells. 3D scaffolds were designed by mixing lighter-than-water (density, <1g/ml) and heavier-than-water (density, >1g/ml) microspheres of 85:15 poly(lactide-co-glycolide). We quantified the rate of 3D flow through the scaffolds by using a particle-tracking system, and the results suggest that motion trajectories and, therefore, the flow velocity around and through scaffolds in rotating bioreactors can be manipulated by varying the ratio of heavier-than-water to lighter-than-water microspheres. When rat primary calvarial cells were cultured on the scaffolds in bioreactors for 7 days, the 3D dynamic flow environment affected bone cell distribution and enhanced cell phenotypic expression and mineralized matrix synthesis within tissue-engineered constructs compared with static conditions. These studies provide a foundation for exploring the effects of dynamic flow on osteoblast function and provide important insight into the design and optimization of 3D scaffolds suitable in bioreactors for in vitro tissue engineering of bone. PMID:15277663

Recursively constructing analytic expressions for equilibrium distributions of stochastic biochemical reaction networks

PubMed Central

Baetica, Ania-Ariadna; Singhal, Vipul; Murray, Richard M.

2017-01-01

Noise is often indispensable to key cellular activities, such as gene expression, necessitating the use of stochastic models to capture its dynamics. The chemical master equation (CME) is a commonly used stochastic model of Kolmogorov forward equations that describe how the probability distribution of a chemically reacting system varies with time. Finding analytic solutions to the CME can have benefits, such as expediting simulations of multiscale biochemical reaction networks and aiding the design of distributional responses. However, analytic solutions are rarely known. A recent method of computing analytic stationary solutions relies on gluing simple state spaces together recursively at one or two states. We explore the capabilities of this method and introduce algorithms to derive analytic stationary solutions to the CME. We first formally characterize state spaces that can be constructed by performing single-state gluing of paths, cycles or both sequentially. We then study stochastic biochemical reaction networks that consist of reversible, elementary reactions with two-dimensional state spaces. We also discuss extending the method to infinite state spaces and designing the stationary behaviour of stochastic biochemical reaction networks. Finally, we illustrate the aforementioned ideas using examples that include two interconnected transcriptional components and biochemical reactions with two-dimensional state spaces. PMID:28566513

Feature-opinion pair identification of product reviews in Chinese: a domain ontology modeling method

NASA Astrophysics Data System (ADS)

Yin, Pei; Wang, Hongwei; Guo, Kaiqiang

2013-03-01

With the emergence of the new economy based on social media, a great amount of consumer feedback on particular products are conveyed through wide-spreading product online reviews, making opinion mining a growing interest for both academia and industry. According to the characteristic mode of expression in Chinese, this research proposes an ontology-based linguistic model to identify the basic appraisal expression in Chinese product reviews-"feature-opinion pair (FOP)." The product-oriented domain ontology is constructed automatically at first, then algorithms to identify FOP are designed by mapping product features and opinions to the conceptual space of the domain ontology, and finally comparative experiments are conducted to evaluate the model. Experimental results indicate that the performance of the proposed approach in this paper is efficient in obtaining a more accurate result compared to the state-of-art algorithms. Furthermore, through identifying and analyzing FOPs, the unstructured product reviews are converted into structured and machine-sensible expression, which provides valuable information for business application. This paper contributes to the related research in opinion mining by developing a solid foundation for further sentiment analysis at a fine-grained level and proposing a general way for automatic ontology construction.

Self-excising Cre/mutant lox marker recycling system for multiple gene integrations and consecutive gene deletions in Aspergillus oryzae.

PubMed

Zhang, Silai; Ban, Akihiko; Ebara, Naoki; Mizutani, Osamu; Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

2017-04-01

In this study, we developed a self-excising Cre/loxP-mediated marker recycling system with mutated lox sequences to introduce a number of biosynthetic genes into Aspergillus oryzae. To construct the self-excising marker cassette, both the selectable marker, the Aspergillus nidulans adeA gene, and the Cre recombinase gene (cre), conditionally expressed by the xylanase-encoding gene promoter, were designed to be located between the mutant lox sequences, lox66 and lox71. However, construction of the plasmid failed, possibly owing to a slight expression of cre downstream of the fungal gene promoter in Escherichia coli. Hence, to avoid the excision of the cassette in E. coli, a 71-bp intron of the A. oryzae xynG2 gene was inserted into the cre gene. The A. oryzae adeA deletion mutant was transformed with the resulting plasmid in the presence of glucose, and the transformants were cultured in medium containing xylose as the sole carbon source. PCR analysis of genomic DNA from resultant colonies revealed the excision of both the marker and Cre expression construct, indicating that the self-excising marker cassette was efficient at removing the selectable marker. Using the marker recycling system, hyperproduction of kojic acid could be achieved in A. oryzae by the introduction of two genes that encode oxidoreductase and transporter. Furthermore, we also constructed an alternative marker recycling cassette bearing the A. nidulans pyrithiamine resistant gene (ptrA) as a dominant selectable marker. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Design of chimeric expression elements that confer high-level gene activity in chromoplasts.

PubMed

Caroca, Rodrigo; Howell, Katharine A; Hasse, Claudia; Ruf, Stephanie; Bock, Ralph

2013-02-01

Non-green plastids, such as chromoplasts, generally have much lower activity of gene expression than chloroplasts in photosynthetically active tissues. Suppression of plastid genes in non-green tissues occurs through a complex interplay of transcriptional and translational control, with the contribution of regulation of transcript abundance versus translational activity being highly variable between genes. Here, we have investigated whether the low expression of the plastid genome in chromoplasts results from inherent limitations in gene expression capacity, or can be overcome by designing appropriate combinations of promoters and translation initiation signals in the 5' untranslated region (5'-UTR). We constructed chimeric expression elements that combine promoters and 5'-UTRs from plastid genes, which are suppressed during chloroplast-to-chromoplast conversion in Solanum lycopersicum (tomato) fruit ripening, either just at the translational level or just at the level of mRNA accumulation. These chimeric expression elements were introduced into the tomato plastid genome by stable chloroplast transformation. We report the identification of promoter-UTR combinations that confer high-level gene expression in chromoplasts of ripe tomato fruits, resulting in the accumulation of reporter protein GFP to up to 1% of total cellular protein. Our work demonstrates that non-green plastids are capable of expressing genes to high levels. Moreover, the chimeric cis-elements for chromoplasts developed here are widely applicable in basic and applied research using transplastomic methods. © 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.

Dual genetic selection of synthetic riboswitches in Escherichia coli.

PubMed

Nomura, Yoko; Yokobayashi, Yohei

2014-01-01

This chapter describes a genetic selection strategy to engineer synthetic riboswitches that can chemically regulate gene expression in Escherichia coli. Riboswitch libraries are constructed by randomizing the nucleotides that potentially comprise an expression platform and fused to the hybrid selection/screening marker tetA-gfpuv. Iterative ON and OFF selections are performed under appropriate conditions that favor the survival or the growth of the cells harboring the desired riboswitches. After the selection, rapid screening of individual riboswitch clones is performed by measuring GFPuv fluorescence without subcloning. This optimized dual genetic selection strategy can be used to rapidly develop synthetic riboswitches without detailed computational design or structural knowledge.

Construction of CRISPR Libraries for Functional Screening.

PubMed

Carstens, Carsten P; Felts, Katherine A; Johns, Sarah E

2018-01-01

Identification of gene function has been aided by the ability to generate targeted gene knockouts or transcriptional repression using the CRISPR/CAS9 system. Using pooled libraries of guide RNA expression vectors that direct CAS9 to a specific genomic site allows identification of genes that are either enriched or depleted in response to a selection scheme, thus linking the affected gene to the chosen phenotype. The quality of the data generated by the screening is dependent on the quality of the guide RNA delivery library with regards to error rates and especially evenness of distribution of the guides. Here, we describe a method for constructing complex plasmid libraries based on pooled designed oligomers with high representation and tight distributions. The procedure allows construction of plasmid libraries of >60,000 members with a 95th/5th percentile ratio of less than 3.5.

Production of heparanase constructs suitable for nuclear magnetic resonance and drug discovery studies.

PubMed

Mosulén, Silvia; Ortí, Leticia; Bas, Esperanza; Carbajo, Rodrigo J; Pineda-Lucena, Antonio

2011-02-01

Heparanase is an endo-β-D-glucosidase capable of specifically degrading heparan sulphate, one of the main components of the extracellular matrix. This 65 kDa polypeptide is implicated in cancer processes such as tumour formation, angiogenesis and metastasis, making it a very attractive target in antitumour treatments. Structure-based approaches to find inhibitors of heparanase have been historically hampered by the lack of success in crystallizing the protein. With the aim to undertake the NMR structural characterisation of heparanase, we have designed and produced, using recombinant methods, smaller constructs of heparanase containing the catalytically active glutamic acids and the two binding sites for heparan sulphate. An extensive range of expression and purification conditions were evaluated to alleviate the intrinsic low solubility and aggregation propensity of heparanase, allowing the obtention of the enzyme in milligram quantities, both unlabelled and ¹⁵N-labelled for NMR studies. Using the smallest of the designed constructs and applying NMR and SPR methodologies, we have demonstrated that known inhibitors of heparanase bind to this construct specifically and selectively with K(D) values in the range of those reported for human heparanase, validating it for future drug discovery projects focused on the identification of novel inhibitors of this enzyme. © 2010 Wiley Periodicals, Inc.

Mesh-based Monte Carlo code for fluorescence modeling in complex tissues with irregular boundaries

NASA Astrophysics Data System (ADS)

Wilson, Robert H.; Chen, Leng-Chun; Lloyd, William; Kuo, Shiuhyang; Marcelo, Cynthia; Feinberg, Stephen E.; Mycek, Mary-Ann

2011-07-01

There is a growing need for the development of computational models that can account for complex tissue morphology in simulations of photon propagation. We describe the development and validation of a user-friendly, MATLAB-based Monte Carlo code that uses analytically-defined surface meshes to model heterogeneous tissue geometry. The code can use information from non-linear optical microscopy images to discriminate the fluorescence photons (from endogenous or exogenous fluorophores) detected from different layers of complex turbid media. We present a specific application of modeling a layered human tissue-engineered construct (Ex Vivo Produced Oral Mucosa Equivalent, EVPOME) designed for use in repair of oral tissue following surgery. Second-harmonic generation microscopic imaging of an EVPOME construct (oral keratinocytes atop a scaffold coated with human type IV collagen) was employed to determine an approximate analytical expression for the complex shape of the interface between the two layers. This expression can then be inserted into the code to correct the simulated fluorescence for the effect of the irregular tissue geometry.

The Grammar of Exchange: A Comparative Study of Reciprocal Constructions Across Languages

PubMed Central

Majid, Asifa; Evans, Nicholas; Gaby, Alice; Levinson, Stephen C.

2010-01-01

Cultures are built on social exchange. Most languages have dedicated grammatical machinery for expressing this. To demonstrate that statistical methods can also be applied to grammatical meaning, we here ask whether the underlying meanings of these grammatical constructions are based on shared common concepts. To explore this, we designed video stimuli of reciprocated actions (e.g., “giving to each other”) and symmetrical states (e.g., “sitting next to each other”), and with the help of a team of linguists collected responses from 20 languages around the world. Statistical analyses revealed that many languages do, in fact, share a common conceptual core for reciprocal meanings but that this is not a universally expressed concept. The recurrent pattern of conceptual packaging found across languages is compatible with the view that there is a shared non-linguistic understanding of reciprocation. But, nevertheless, there are considerable differences between languages in the exact extensional patterns, highlighting that even in the domain of grammar semantics is highly language-specific. PMID:21713188

High Levels of Bioplastic Are Produced in Fertile Transplastomic Tobacco Plants Engineered with a Synthetic Operon for the Production of Polyhydroxybutyrate1[C][OA

PubMed Central

Bohmert-Tatarev, Karen; McAvoy, Susan; Daughtry, Sean; Peoples, Oliver P.; Snell, Kristi D.

2011-01-01

An optimized genetic construct for plastid transformation of tobacco (Nicotiana tabacum) for the production of the renewable, biodegradable plastic polyhydroxybutyrate (PHB) was designed using an operon extension strategy. Bacterial genes encoding the PHB pathway enzymes were selected for use in this construct based on their similarity to the codon usage and GC content of the tobacco plastome. Regulatory elements with limited homology to the host plastome yet known to yield high levels of plastidial recombinant protein production were used to enhance the expression of the transgenes. A partial transcriptional unit, containing genes of the PHB pathway and a selectable marker gene encoding spectinomycin resistance, was flanked at the 5′ end by the host plant’s psbA coding sequence and at the 3′ end by the host plant’s 3′ psbA untranslated region. This design allowed insertion of the transgenes into the plastome as an extension of the psbA operon, rendering the addition of a promoter to drive the expression of the transgenes unnecessary. Transformation of the optimized construct into tobacco and subsequent spectinomycin selection of transgenic plants yielded T0 plants that were capable of producing up to 18.8% dry weight PHB in samples of leaf tissue. These plants were fertile and produced viable seed. T1 plants producing up to 17.3% dry weight PHB in samples of leaf tissue and 8.8% dry weight PHB in the total biomass of the plant were also isolated. PMID:21325565

A part toolbox to tune genetic expression in Bacillus subtilis

PubMed Central

Guiziou, Sarah; Sauveplane, Vincent; Chang, Hung-Ju; Clerté, Caroline; Declerck, Nathalie; Jules, Matthieu; Bonnet, Jerome

2016-01-01

Libraries of well-characterised components regulating gene expression levels are essential to many synthetic biology applications. While widely available for the Gram-negative model bacterium Escherichia coli, such libraries are lacking for the Gram-positive model Bacillus subtilis, a key organism for basic research and biotechnological applications. Here, we engineered a genetic toolbox comprising libraries of promoters, Ribosome Binding Sites (RBS), and protein degradation tags to precisely tune gene expression in B. subtilis. We first designed a modular Expression Operating Unit (EOU) facilitating parts assembly and modifications and providing a standard genetic context for gene circuits implementation. We then selected native, constitutive promoters of B. subtilis and efficient RBS sequences from which we engineered three promoters and three RBS sequence libraries exhibiting ∼14 000-fold dynamic range in gene expression levels. We also designed a collection of SsrA proteolysis tags of variable strength. Finally, by using fluorescence fluctuation methods coupled with two-photon microscopy, we quantified the absolute concentration of GFP in a subset of strains from the library. Our complete promoters and RBS sequences library comprising over 135 constructs enables tuning of GFP concentration over five orders of magnitude, from 0.05 to 700 μM. This toolbox of regulatory components will support many research and engineering applications in B. subtilis. PMID:27402159

Improved genetic stability of recombinant yellow fever 17D virus expressing a lentiviral Gag gene fragment.

PubMed

de Santana, Marlon G Veloso; Neves, Patrícia C C; dos Santos, Juliana Ribeiro; Lima, Noemia S; dos Santos, Alexandre A C; Watkins, David I; Galler, Ricardo; Bonaldo, Myrna C

2014-03-01

We have previously designed a method to construct viable recombinant Yellow Fever (YF) 17D viruses expressing heterologous polypeptides including part of the Simian Immunodeficiency Virus (SIV) Gag protein. However, the expressed region, encompassing amino acid residues from 45 to 269, was genetically unstable. In this study, we improved the genetic stability of this recombinant YF 17D virus by introducing mutations in the IRES element localized at the 5' end of the SIV gag gene. The new stable recombinant virus elicited adaptive immune responses similar to those induced by the original recombinant virus. It is, therefore, possible to increase recombinant stability by removing functional motifs from the insert that may have deleterious effects on recombinant YF viral fitness. Copyright © 2014 Elsevier Inc. All rights reserved.

Metazoan tRNA introns generate stable circular RNAs in vivo.

PubMed

Lu, Zhipeng; Filonov, Grigory S; Noto, John J; Schmidt, Casey A; Hatkevich, Talia L; Wen, Ying; Jaffrey, Samie R; Matera, A Gregory

2015-09-01

We report the discovery of a class of abundant circular noncoding RNAs that are produced during metazoan tRNA splicing. These transcripts, termed tRNA intronic circular (tric)RNAs, are conserved features of animal transcriptomes. Biogenesis of tricRNAs requires anciently conserved tRNA sequence motifs and processing enzymes, and their expression is regulated in an age-dependent and tissue-specific manner. Furthermore, we exploited this biogenesis pathway to develop an in vivo expression system for generating "designer" circular RNAs in human cells. Reporter constructs expressing RNA aptamers such as Spinach and Broccoli can be used to follow the transcription and subcellular localization of tricRNAs in living cells. Owing to the superior stability of circular vs. linear RNA isoforms, this expression system has a wide range of potential applications, from basic research to pharmaceutical science. © 2015 Lu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Reliability and validity of a videotape method to describe expressive behavior in persons with Parkinson's disease.

PubMed

Lyons, Kathleen Doyle; Tickle-Degnen, Linda

2005-01-01

The ability to effectively communicate thoughts, feelings, and identity to others is an important aspect of occupational performance. The symptoms of Parkinson's disease can impair a person's ability to verbally and non-verbally communicate with others. In order to better understand issues of communication functioning for this population, research tools to describe expressive and communicative behavior during occupation and social interaction are needed. In this study, six persons with Parkinson's disease participated in individual, videotaped interviews focused on problem solving during daily activities. Three trained graduate students viewed edited clips from the videotapes and completed a rating scale of expressive behavior designed by the authors. Data support the reliability and construct validity of the behavioral rating scale, suggesting that measures of expressive behavior of persons with Parkinson's disease can be effectively derived using short segments of videotaped activity.

High-throughput deep screening and identification of four peripheral leucocyte microRNAs as novel potential combination biomarkers for preeclampsia

PubMed Central

Wang, Yonghong; Yang, Xukui; Yang, Yuanyuan; Wang, Wenjun; Zhao, Meiling; Liu, Huiqiang; Li, Dongyan; Hao, Min

2016-01-01

Objective: To identify the specific microRNA (miRNA) biomarkers of preeclampsia (PE), the miRNA profiles analysis were performed. Study Design: The blood samples were obtained from five PE patients and five normal healthy pregnant women. The small RNA profiles were analyzed to identify miRNA expression levels and find out miRNAs that may associate with PE. The quantitative reverse transcriptase–PCR (qRT-PCR) assay was used to validate differentially expressed peripheral leucocyte miRNAs in a new cohort. Result: The data analysis showed that 10 peripheral leucocyte miRNAs were significantly differently expressed in severe PE patients. Four differently expressed miRNAs were successfully validated using qRT-PCR method. Conclusion: We successfully constructed a model with high accuracy to predict PE. A combination of four peripheral leucocyte miRNAs has great potential to serve as diagnostic biomarkers of PE. PMID:26675000

BdCESA7, BdCESA8, and BdPMT utility promoter constructs for targeted expression to secondary cell-wall-forming cells of grasses

DOE PAGES

Petrik, Deborah L.; Cass, Cynthia L.; Padmakshan, Dharshana; ...

2016-02-04

Utility vectors with promoters that confer desired spatial and temporal expression patterns are useful tools for studying gene and cellular function and for industrial applications. To target the expression of DNA sequences of interest to cells forming plant secondary cell walls, which generate most of the vegetative biomass, upstream regulatory sequences of the Brachypodium distachyon lignin biosynthetic gene BdPMT and the cellulose synthase genes BdCESA7 and BdCESA8 were isolated and cloned into binary vectors designed for Agrobacterium-mediated transformation of monocots. Expression patterns were assessed using the β-glucuronidase gene GUSPlus and X-glucuronide staining. All three promoters showed strong expression levels inmore » stem tissue at the base of internodes where cell wall deposition is most active, in both vascular bundle xylem vessels and tracheids, and in interfascicular tissues, with expression less pronounced in developmentally older tissues. In leaves, BdCESA7 and BdCESA8 promoter-driven expression was strongest in leaf veins, leaf margins, and trichomes; relatively weaker and patchy expression was observed in the epidermis. BdPMT promoter-driven expression was similar to the BdCESA promoters expression patterns, including strong expression in trichomes. The intensity and extent of GUS staining varied considerably between transgenic lines, suggesting that positional effects influenced promoter activity. Introducing the BdPMT and BdCESA8 Open Reading Frames into BdPMT and BdCESA8 utility promoter binary vectors, respectively, and transforming those constructs into Brachypodium pmt and cesa8 loss-of-function mutants resulted in rescue of the corresponding mutant phenotypes. This work therefore validates the functionality of these utility promoter binary vectors for use in Brachypodium and likely other grass species. Lastly, the identification, in Bdcesa8-1 T-DNA mutant stems, of an 80% reduction in crystalline cellulose levels confirms that the BdCESA8 gene is a secondary-cell-wall-forming cellulose synthase.« less

BdCESA7, BdCESA8, and BdPMT utility promoter constructs for targeted expression to secondary cell-wall-forming cells of grasses

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

Petrik, Deborah L.; Cass, Cynthia L.; Padmakshan, Dharshana

Utility vectors with promoters that confer desired spatial and temporal expression patterns are useful tools for studying gene and cellular function and for industrial applications. To target the expression of DNA sequences of interest to cells forming plant secondary cell walls, which generate most of the vegetative biomass, upstream regulatory sequences of the Brachypodium distachyon lignin biosynthetic gene BdPMT and the cellulose synthase genes BdCESA7 and BdCESA8 were isolated and cloned into binary vectors designed for Agrobacterium-mediated transformation of monocots. Expression patterns were assessed using the β-glucuronidase gene GUSPlus and X-glucuronide staining. All three promoters showed strong expression levels inmore » stem tissue at the base of internodes where cell wall deposition is most active, in both vascular bundle xylem vessels and tracheids, and in interfascicular tissues, with expression less pronounced in developmentally older tissues. In leaves, BdCESA7 and BdCESA8 promoter-driven expression was strongest in leaf veins, leaf margins, and trichomes; relatively weaker and patchy expression was observed in the epidermis. BdPMT promoter-driven expression was similar to the BdCESA promoters expression patterns, including strong expression in trichomes. The intensity and extent of GUS staining varied considerably between transgenic lines, suggesting that positional effects influenced promoter activity. Introducing the BdPMT and BdCESA8 Open Reading Frames into BdPMT and BdCESA8 utility promoter binary vectors, respectively, and transforming those constructs into Brachypodium pmt and cesa8 loss-of-function mutants resulted in rescue of the corresponding mutant phenotypes. This work therefore validates the functionality of these utility promoter binary vectors for use in Brachypodium and likely other grass species. Lastly, the identification, in Bdcesa8-1 T-DNA mutant stems, of an 80% reduction in crystalline cellulose levels confirms that the BdCESA8 gene is a secondary-cell-wall-forming cellulose synthase.« less

Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in Mammalian Cells.

PubMed

Ede, Christopher; Chen, Ximin; Lin, Meng-Yin; Chen, Yvonne Y

2016-05-20

Inducible transcription systems play a crucial role in a wide array of synthetic biology circuits. However, the majority of inducible promoters are constructed from a limited set of tried-and-true promoter parts, which are susceptible to common shortcomings such as high basal expression levels (i.e., leakiness). To expand the toolbox for regulated mammalian gene expression and facilitate the construction of mammalian genetic circuits with precise functionality, we quantitatively characterized a panel of eight core promoters, including sequences with mammalian, viral, and synthetic origins. We demonstrate that this selection of core promoters can provide a wide range of basal gene expression levels and achieve a gradient of fold-inductions spanning 2 orders of magnitude. Furthermore, commonly used parts such as minimal CMV and minimal SV40 promoters were shown to achieve robust gene expression upon induction, but also suffer from high levels of leakiness. In contrast, a synthetic promoter, YB_TATA, was shown to combine low basal expression with high transcription rate in the induced state to achieve significantly higher fold-induction ratios compared to all other promoters tested. These behaviors remain consistent when the promoters are coupled to different genetic outputs and different response elements, as well as across different host-cell types and DNA copy numbers. We apply this quantitative understanding of core promoter properties to the successful engineering of human T cells that respond to antigen stimulation via chimeric antigen receptor signaling specifically under hypoxic environments. Results presented in this study can facilitate the design and calibration of future mammalian synthetic biology systems capable of precisely programmed functionality.

In vitro DNA SCRaMbLE.

PubMed

Wu, Yi; Zhu, Rui-Ying; Mitchell, Leslie A; Ma, Lu; Liu, Rui; Zhao, Meng; Jia, Bin; Xu, Hui; Li, Yun-Xiang; Yang, Zu-Ming; Ma, Yuan; Li, Xia; Liu, Hong; Liu, Duo; Xiao, Wen-Hai; Zhou, Xiao; Li, Bing-Zhi; Yuan, Ying-Jin; Boeke, Jef D

2018-05-22

The power of synthetic biology has enabled the expression of heterologous pathways in cells, as well as genome-scale synthesis projects. The complexity of biological networks makes rational de novo design a grand challenge. Introducing features that confer genetic flexibility is a powerful strategy for downstream engineering. Here we develop an in vitro method of DNA library construction based on structural variation to accomplish this goal. The "in vitro SCRaMbLE system" uses Cre recombinase mixed in a test tube with purified DNA encoding multiple loxPsym sites. Using a β-carotene pathway designed for expression in yeast as an example, we demonstrate top-down and bottom-up in vitro SCRaMbLE, enabling optimization of biosynthetic pathway flux via the rearrangement of relevant transcription units. We show that our system provides a straightforward way to correlate phenotype and genotype and is potentially amenable to biochemical optimization in ways that the in vivo system cannot achieve.

Biomimetic Artificial Epigenetic Code for Targeted Acetylation of Histones.

PubMed

Taniguchi, Junichi; Feng, Yihong; Pandian, Ganesh N; Hashiya, Fumitaka; Hidaka, Takuya; Hashiya, Kaori; Park, Soyoung; Bando, Toshikazu; Ito, Shinji; Sugiyama, Hiroshi

2018-06-13

While the central role of locus-specific acetylation of histone proteins in eukaryotic gene expression is well established, the availability of designer tools to regulate acetylation at particular nucleosome sites remains limited. Here, we develop a unique strategy to introduce acetylation by constructing a bifunctional molecule designated Bi-PIP. Bi-PIP has a P300/CBP-selective bromodomain inhibitor (Bi) as a P300/CBP recruiter and a pyrrole-imidazole polyamide (PIP) as a sequence-selective DNA binder. Biochemical assays verified that Bi-PIPs recruit P300 to the nucleosomes having their target DNA sequences and extensively accelerate acetylation. Bi-PIPs also activated transcription of genes that have corresponding cognate DNA sequences inside living cells. Our results demonstrate that Bi-PIPs could act as a synthetic programmable histone code of acetylation, which emulates the bromodomain-mediated natural propagation system of histone acetylation to activate gene expression in a sequence-selective manner.

An Independent Construct for Conditional Expression of Atonal Homolog-1

PubMed Central

Cheng, Yen-fu; Kinouchi, Hikaru; Bieber, Rebecca; Edge, Albert S.B.

2014-01-01

Abstract The mammalian homolog of the basic helix-loop-helix transcription factor atonal-1 (Atoh1 or Math1) is required for development of cochlear hair cells that function as the mechanosensory cells required for audition. Forced expression of Atoh1 in cochlear-supporting cells may provide a way to regenerate hair cells and provide for a therapy for hearing loss. Additionally, Atoh1 is an inhibitor of proliferation and has further clinical applications in anticancer therapies. The goal of these experiments was to improve the method for Atoh1 expression by engineering a genetic construct that may be used in future translational applications. To address the poor control of Atoh1 expression in standard gene expression systems where Atoh1 is expressed constitutively at abnormally elevated levels, our aim was to engineer an inducible system whereby Atoh1 was upregulated by an inducer and downregulated once the inducer was removed. A further aim was to engineer a single genetic construct that allowed for conditional expression of Atoh1 independent of secondary regulatory elements. Here we describe a stand-alone genetic construct that utilizes the tamoxifen sensitivity of a mutated estrogen receptor (ER) ligand-binding domain for the conditional expression of Atoh1. The Atoh1-ER-DsRed construct is translated into an ATOH1-ER-DSRED fusion protein that remains sequestered in the cytoplasm and therefore rendered inactive because it cannot enter the nucleus to activate Atoh1 signaling pathways. However, application of 4-hydroxytamoxifen results in translocation of the fusion protein to the nucleus, where it binds to the Atoh1 enhancer, upregulates transcription and translation of endogenous ATOH1 and activates downstream Atoh1 signaling such as upregulation of the hair cell protein MYOSIN 7A. Removal of tamoxifen reverses the upregulation of endogenous Atoh1 signaling. This construct serves as an independent genetic construct that allows for the conditional upregulation and downregulation of Atoh1, and may prove useful for manipulating Atoh1 expression in vivo. PMID:24066662

Expressing foreign genes in the pistil: a comparison of S-RNase constructs in different Nicotiana backgrounds.

PubMed

Murfett, J; McClure, B A

1998-06-01

Transgenic plant experiments have great potential for extending our understanding of the role of specific genes in controlling pollination. Often, the intent of such experiments is to over-express a gene and test for effects on pollination. We have examined the efficiency of six different S-RNase constructs in Nicotiana species and hybrids. Each construct contained the coding region, intron, and downstream sequences from the Nicotiana alata S(A2)-RNase gene. Among the six expression constructs, two utilized the cauliflower mosaic virus (CaMV) 35S promoter with duplicated enhancer, and four utilized promoters from genes expressed primarily in pistils. The latter included promoters from the tomato Chi2;1 and 9612 genes, a promoter from the N. alata S(A2)-RNase gene, and a promoter from the Brassica SLG-13 gene. Some or all of the constructs were tested in N. tabacum, N. plumbaginifolia, N. plumbaginifolia x SI N. alata S(C10)S(c10) hybrids, N. langsdorffii, and N. langsdorffii x SC N. alata hybrids. Stylar specific RNase activities and S(A2)-RNase transcript levels were determined in transformed plants. Constructs including the tomato Chi2;1 gene promoter or the Brassica SLG-13 promoter provided the highest levels of S(A2)-RNase expression. Transgene expression patterns were tightly regulated, the highest level of expression was observed in post-anthesis styles. Expression levels of the S(A2)-RNase transgenes was dependent on the genetic background of the host. Higher levels of S(A2)-RNase expression were observed in N. plumbaginifolia x SC N. alata hybrids than in N. plumbaginifolia.

The recombinant expression and activity detection of MAF-1 fusion protein.

PubMed

Fu, Ping; Wu, Jianwei; Gao, Song; Guo, Guo; Zhang, Yong; Liu, Jian

2015-10-01

This study establishes the recombinant expression system of MAF-1 (Musca domestica antifungal peptide-1) and demonstrates the antifungal activity of the expression product and shows the relationship between biological activity and structure. The gene segments on mature peptide part of MAF-1 were cloned, based on the primers designed according to the cDNA sequence of MAF-1. We constructed the recombinant prokaryotic expression plasmid using prokaryotic expression vector (pET-28a(+)) and converted it to the competent cell of BL21(DE3) to gain recombinant MAF-1 fusion protein with His tag sequence through purifying affinity chromatographic column of Ni-NTA. To conduct the Western Blotting test, recombinant MAF-1 fusion protein was used to produce the polyclonal antibody of rat. The antifungal activity of the expression product was detected using Candida albicans (ATCC10231) as the indicator. The MAF-1 recombinant fusion protein was purified to exhibit obvious antifungal activity, which lays the foundation for the further study of MAF-1 biological activity, the relationship between structure and function, as well as control of gene expression.

In silico design and expression of a novel fusion protein of HBHA and high antigenic region of FAP-P of Mycobacterium avium subsp. paratuberculosis in Pichia pastoris.

PubMed

Eraghi, Vida; Derakhshandeh, Abdollah; Hosseini, Arsalan; Motamedi-Boroojeni, Azar

2017-12-01

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's disease in ruminants and there has been a shift in the public health approach to MAP and human diseases like Crohn's disease. The prevention of infection by MAP in ruminants is thought to deter the high impact of economic losses in the level of dairy industry and possible spreading of this pathogen in dairy products. The present study was done to investigate the construction and expression of the soluble form of a novel fusion protein, consisting of Heparin-binding hemagglutinin (HBHA) and high antigenic region of Fibronectin Attachment Protein-P (FAP-P), in order to introduce as a Th1 inducer subunit vaccine against MAP. HBHA is a mycobacterial adhesin and it has been demonstrated that a HBHA-specific IFN-γ response, in latent M. tuberculosis infection, depends on the methylation of the antigen. Further, FAP-P induces Th1 polarization. Because methylation of HBHA was not performed in E. coli , Pichia pastoris was chosen as the host. The desired fusion protein had a similar 3D structure to that of HBHA with its native form and post-translational methylation in C-terminal. Hence, the uptake of the purified fusion protein will be done by M cells because of HBHA, and cell-mediated immunity will be induced because of both antigens. Eventually, successful construction and expression of the newly-designed chimeric protein under the mentioned conditions is reported in this article.

Construction Of Critical Thinking Skills Test Instrument Related The Concept On Sound Wave

NASA Astrophysics Data System (ADS)

Mabruroh, F.; Suhandi, A.

2017-02-01

This study aimed to construct test instrument of critical thinking skills of high school students related the concept on sound wave. This research using a mixed methods with sequential exploratory design, consists of: 1) a preliminary study; 2) design and review of test instruments. The form of test instruments in essay questions, consist of 18 questions that was divided into 5 indicators and 8 sub-indicators of the critical thinking skills expressed by Ennis, with questions that are qualitative and contextual. Phases of preliminary study include: a) policy studies; b) survey to the school; c) and literature studies. Phases of the design and review of test instruments consist of two steps, namely a draft design of test instruments include: a) analysis of the depth of teaching materials; b) the selection of indicators and sub-indicators of critical thinking skills; c) analysis of indicators and sub-indicators of critical thinking skills; d) implementation of indicators and sub-indicators of critical thinking skills; and e) making the descriptions about the test instrument. In the next phase of the review test instruments, consist of: a) writing about the test instrument; b) validity test by experts; and c) revision of test instruments based on the validator.

Protocell design through modular compartmentalization

PubMed Central

Miller, David; Booth, Paula J.; Seddon, John M.; Templer, Richard H.; Law, Robert V.; Woscholski, Rudiger; Ces, Oscar; Barter, Laura M. C.

2013-01-01

De novo synthetic biological design has the potential to significantly impact upon applications such as energy generation and nanofabrication. Current designs for constructing organisms from component parts are typically limited in scope, as they utilize a cut-and-paste ideology to create simple stepwise engineered protein-signalling pathways. We propose the addition of a new design element that segregates components into lipid-bound ‘proto-organelles’, which are interfaced with response elements and housed within a synthetic protocell. This design is inspired by living cells, which utilize multiple types of signalling molecules to facilitate communication between isolated compartments. This paper presents our design and validation of the components required for a simple multi-compartment protocell machine, for coupling a light transducer to a gene expression system. This represents a general design concept for the compartmentalization of different types of artificial cellular machinery and the utilization of non-protein signal molecules for signal transduction. PMID:23925982

Protocell design through modular compartmentalization.

PubMed

Miller, David; Booth, Paula J; Seddon, John M; Templer, Richard H; Law, Robert V; Woscholski, Rudiger; Ces, Oscar; Barter, Laura M C

2013-10-06

De novo synthetic biological design has the potential to significantly impact upon applications such as energy generation and nanofabrication. Current designs for constructing organisms from component parts are typically limited in scope, as they utilize a cut-and-paste ideology to create simple stepwise engineered protein-signalling pathways. We propose the addition of a new design element that segregates components into lipid-bound 'proto-organelles', which are interfaced with response elements and housed within a synthetic protocell. This design is inspired by living cells, which utilize multiple types of signalling molecules to facilitate communication between isolated compartments. This paper presents our design and validation of the components required for a simple multi-compartment protocell machine, for coupling a light transducer to a gene expression system. This represents a general design concept for the compartmentalization of different types of artificial cellular machinery and the utilization of non-protein signal molecules for signal transduction.

Co-expression of the Thermotoga neapolitana aglB gene with an upstream 3'-coding fragment of the malG gene improves enzymatic characteristics of recombinant AglB cyclomaltodextrinase.

PubMed

Lunina, Natalia A; Agafonova, Elena V; Chekanovskaya, Lyudmila A; Dvortsov, Igor A; Berezina, Oksana V; Shedova, Ekaterina N; Kostrov, Sergey V; Velikodvorskaya, Galina A

2007-07-01

A cluster of Thermotoga neapolitana genes participating in starch degradation includes the malG gene of sugar transport protein and the aglB gene of cyclomaltodextrinase. The start and stop codons of these genes share a common overlapping sequence, aTGAtg. Here, we compared properties of expression products of three different constructs with aglB from T. neapolitana. The first expression vector contained the aglB gene linked to an upstream 90-bp 3'-terminal region of the malG gene with the stop codon overlapping with the start codon of aglB. The second construct included the isolated coding sequence of aglB with two tandem potential start codons. The expression product of this construct in Escherichia coli had two tandem Met residues at its N terminus and was characterized by low thermostability and high tendency to aggregate. In contrast, co-expression of aglB and the 3'-terminal region of malG (the first construct) resulted in AglB with only one N-terminal Met residue and a much higher specific activity of cyclomaltodextrinase. Moreover, the enzyme expressed by such a construct was more thermostable and less prone to aggregation. The third construct was the same as the second one except that it contained only one ATG start codon. The product of its expression had kinetic and other properties similar to those of the enzyme with only one N-terminal Met residue.

Searching for Sexual Revolutions in India: Non-Governmental Organisation-Designed Sex Education Programmes as a Means towards Gender Equality and Sexual Empowerment in New Delhi, India

ERIC Educational Resources Information Center

Gabler, Mette

2012-01-01

At the foundation of most inequalities in expression of sexuality lie social constructions of gender. In this paper, sex education is considered as a possibility to challenge sexism and promote healthy and self-affirmative sex lives. In the past decade, the discourse of sex education in India has become a "battle of morality" where…

Automation of large scale transient protein expression in mammalian cells

PubMed Central

Zhao, Yuguang; Bishop, Benjamin; Clay, Jordan E.; Lu, Weixian; Jones, Margaret; Daenke, Susan; Siebold, Christian; Stuart, David I.; Yvonne Jones, E.; Radu Aricescu, A.

2011-01-01

Traditional mammalian expression systems rely on the time-consuming generation of stable cell lines; this is difficult to accommodate within a modern structural biology pipeline. Transient transfections are a fast, cost-effective solution, but require skilled cell culture scientists, making man-power a limiting factor in a setting where numerous samples are processed in parallel. Here we report a strategy employing a customised CompacT SelecT cell culture robot allowing the large-scale expression of multiple protein constructs in a transient format. Successful protocols have been designed for automated transient transfection of human embryonic kidney (HEK) 293T and 293S GnTI− cells in various flask formats. Protein yields obtained by this method were similar to those produced manually, with the added benefit of reproducibility, regardless of user. Automation of cell maintenance and transient transfection allows the expression of high quality recombinant protein in a completely sterile environment with limited support from a cell culture scientist. The reduction in human input has the added benefit of enabling continuous cell maintenance and protein production, features of particular importance to structural biology laboratories, which typically use large quantities of pure recombinant proteins, and often require rapid characterisation of a series of modified constructs. This automated method for large scale transient transfection is now offered as a Europe-wide service via the P-cube initiative. PMID:21571074

Paramyosin from the parasitic mite Sarcoptes scabiei: cDNA cloning and heterologous expression.

PubMed

Mattsson, J G; Ljunggren, E L; Bergström, K

2001-05-01

The burrowing mite Sarcoptes scabiei is the causative agent of the highly contagious disease sarcoptic mange or scabies. So far, there is no in vitro propagation system for S. scabiei available, and mites used for various purposes must be isolated from infected hosts. Lack of parasite-derived material has limited the possibilities to study several aspects of scabies, including pathogenesis and immunity. It has also hampered the development of high performance serological assays. We have now constructed an S. scabiei cDNA expression library with mRNA purified from mites isolated from red foxes. Immunoscreening of the library enabled us to clone a full-length cDNA coding for a 102.5 kDa protein. Sequence similarity searches identified the protein as a paramyosin. Recombinant S. scabiei paramyosin expressed in Escherichia coli was recognized by sera from dogs and swine infected with S. scabiei. We also designed a small paramyosin construct of about 17 kDa that included the N-terminal part, an evolutionary variable part of the helical core, and the C-terminal part of the molecule. The miniaturized protein was efficiently expressed in E. coli and was recognized by sera from immunized rabbits. These data demonstrate that the cDNA library can assist in the isolation of important S. scabiei antigens and that recombinant proteins can be useful for the study of scabies.

Functional Fluorescent Protein Insertions in Herpes Simplex Virus gB Report on gB Conformation before and after Execution of Membrane Fusion

PubMed Central

Gallagher, John R.; Atanasiu, Doina; Saw, Wan Ting; Paradisgarten, Matthew J.; Whitbeck, J. Charles; Eisenberg, Roselyn J.; Cohen, Gary H.

2014-01-01

Entry of herpes simplex virus (HSV) into a target cell requires complex interactions and conformational changes by viral glycoproteins gD, gH/gL, and gB. During viral entry, gB transitions from a prefusion to a postfusion conformation, driving fusion of the viral envelope with the host cell membrane. While the structure of postfusion gB is known, the prefusion conformation of gB remains elusive. As the prefusion conformation of gB is a critical target for neutralizing antibodies, we set out to describe its structure by making genetic insertions of fluorescent proteins (FP) throughout the gB ectodomain. We created gB constructs with FP insertions in each of the three globular domains of gB. Among 21 FP insertion constructs, we found 8 that allowed gB to remain membrane fusion competent. Due to the size of an FP, regions in gB that tolerate FP insertion must be solvent exposed. Two FP insertion mutants were cell-surface expressed but non-functional, while FP insertions located in the crown were not surface expressed. This is the first report of placing a fluorescent protein insertion within a structural domain of a functional viral fusion protein, and our results are consistent with a model of prefusion HSV gB constructed from the prefusion VSV G crystal structure. Additionally, we found that functional FP insertions from two different structural domains could be combined to create a functional form of gB labeled with both CFP and YFP. FRET was measured with this construct, and we found that when co-expressed with gH/gL, the FRET signal from gB was significantly different from the construct containing CFP alone, as well as gB found in syncytia, indicating that this construct and others of similar design are likely to be powerful tools to monitor the conformation of gB in any model system accessible to light microscopy. PMID:25233449

RNAi-Mediated Knockdown of IKK1 in Transgenic Mice Using a Transgenic Construct Containing the Human H1 Promoter

PubMed Central

Moreno-Maldonado, Rodolfo; Murillas, Rodolfo; Page, Angustias; Suarez-Cabrera, Cristian; Alameda, Josefa P.; Bravo, Ana; Casanova, M. Llanos

2014-01-01

Inhibition of gene expression through siRNAs is a tool increasingly used for the study of gene function in model systems, including transgenic mice. To achieve perdurable effects, the stable expression of siRNAs by an integrated transgenic construct is necessary. For transgenic siRNA expression, promoters transcribed by either RNApol II or III (such as U6 or H1 promoters) can be used. Relatively large amounts of small RNAs synthesis are achieved when using RNApol III promoters, which can be advantageous in knockdown experiments. To study the feasibility of H1 promoter-driven RNAi-expressing constructs for protein knockdown in transgenic mice, we chose IKK1 as the target gene. Our results indicate that constructs containing the H1 promoter are sensitive to the presence of prokaryotic sequences and to transgene position effects, similar to RNApol II promoters-driven constructs. We observed variable expression levels of transgenic siRNA among different tissues and animals and a reduction of up to 80% in IKK1 expression. Furthermore, IKK1 knockdown led to hair follicle alterations. In summary, we show that constructs directed by the H1 promoter can be used for knockdown of genes of interest in different organs and for the generation of animal models complementary to knockout and overexpression models. PMID:24523631

Report on renovation of "Shinagawa City Samezu Exercise Park " taking in the idea of Children

NASA Astrophysics Data System (ADS)

Ogawa, Susumu; Amma, Yoko

When constructing and renovating parks in Shinagawa city, we collect various needs from local citizens by briefing, workshop and public comment. However, only adult people usually come to workshop. And there are few expression by children who are main users of parks. Then we made plan of a new park with children who play major roles in their future as part of town development. This plan is shown in Shinagawa city basics design which instituted in April 2008. And during construction of it we held observation tour with children to get more familiar to their parks. After finishing construction, we are going to maintain together with them by planting flowers etc. In this way, we cultivate mind that adults and children conduct town development together by system in which children can continuously take part in each step of making parks.

Implementing Target Value Design.

PubMed

Alves, Thais da C L; Lichtig, Will; Rybkowski, Zofia K

2017-04-01

An alternative to the traditional way of designing projects is the process of target value design (TVD), which takes different departure points to start the design process. The TVD process starts with the client defining an allowable cost that needs to be met by the design and construction teams. An expected cost in the TVD process is defined through multiple interactions between multiple stakeholders who define wishes and others who define ways of achieving these wishes. Finally, a target cost is defined based on the expected profit the design and construction teams are expecting to make. TVD follows a series of continuous improvement efforts aimed at reaching the desired goals for the project and its associated target value cost. The process takes advantage of rapid cycles of suggestions, analyses, and implementation that starts with the definition of value for the client. In the traditional design process, the goal is to identify user preferences and find solutions that meet the needs of the client's expressed preferences. In the lean design process, the goal is to educate users about their values and advocate for a better facility over the long run; this way owners can help contractors and designers to identify better solutions. This article aims to inform the healthcare community about tools and techniques commonly used during the TVD process and how they can be used to educate and support project participants in developing better solutions to meet their needs now as well as in the future.

Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging

PubMed Central

Hsieh, Ya-Ju; Ke, Chien-Chih; Yeh, Skye Hsin-Hsien; Lin, Chien-Feng; Chen, Fu-Du; Lin, Kang-Ping; Chen, Ran-Chou; Liu, Ren-Shyan

2014-01-01

Multimodality imaging using noncytotoxic triple fusion (TF) reporter genes is an important application for cell-based tracking, drug screening, and therapy. The firefly luciferase (fl), monomeric red fluorescence protein (mrfp), and truncated herpes simplex virus type 1 thymidine kinase SR39 mutant (ttksr39) were fused together to create TF reporter gene constructs with different order. The enzymatic activities of TF protein in vitro and in vivo were determined by luciferase reporter assay, H-FEAU cellular uptake experiment, bioluminescence imaging, and micropositron emission tomography (microPET). The TF construct expressed in H1299 cells possesses luciferase activity and red fluorescence. The tTKSR39 activity is preserved in TF protein and mediates high levels of H-FEAU accumulation and significant cell death from ganciclovir (GCV) prodrug activation. In living animals, the luciferase and tTKSR39 activities of TF protein have also been successfully validated by multimodality imaging systems. The red fluorescence signal is relatively weak for in vivo imaging but may expedite FACS-based selection of TF reporter expressing cells. We have developed an optimized triple fusion reporter construct DsRedm-fl-ttksr39 for more effective and sensitive in vivo animal imaging using fluorescence, bioluminescence, and PET imaging modalities, which may facilitate different fields of biomedical research and applications. PMID:24809057

End Joining-Mediated Gene Expression in Mammalian Cells Using PCR-Amplified DNA Constructs that Contain Terminator in Front of Promoter.

PubMed

Nakamura, Mikiko; Suzuki, Ayako; Akada, Junko; Tomiyoshi, Keisuke; Hoshida, Hisashi; Akada, Rinji

2015-12-01

Mammalian gene expression constructs are generally prepared in a plasmid vector, in which a promoter and terminator are located upstream and downstream of a protein-coding sequence, respectively. In this study, we found that front terminator constructs-DNA constructs containing a terminator upstream of a promoter rather than downstream of a coding region-could sufficiently express proteins as a result of end joining of the introduced DNA fragment. By taking advantage of front terminator constructs, FLAG substitutions, and deletions were generated using mutagenesis primers to identify amino acids specifically recognized by commercial FLAG antibodies. A minimal epitope sequence for polyclonal FLAG antibody recognition was also identified. In addition, we analyzed the sequence of a C-terminal Ser-Lys-Leu peroxisome localization signal, and identified the key residues necessary for peroxisome targeting. Moreover, front terminator constructs of hepatitis B surface antigen were used for deletion analysis, leading to the identification of regions required for the particle formation. Collectively, these results indicate that front terminator constructs allow for easy manipulations of C-terminal protein-coding sequences, and suggest that direct gene expression with PCR-amplified DNA is useful for high-throughput protein analysis in mammalian cells.

Plant nitrogen regulatory P-PII genes

DOEpatents

Coruzzi, Gloria M.; Lam, Hon-Ming; Hsieh, Ming-Hsiun

2001-01-01

The present invention generally relates to plant nitrogen regulatory PII gene (hereinafter P-PII gene), a gene involved in regulating plant nitrogen metabolism. The invention provides P-PII nucleotide sequences, expression constructs comprising said nucleotide sequences, and host cells and plants having said constructs and, optionally expressing the P-PII gene from said constructs. The invention also provides substantially pure P-PII proteins. The P-PII nucleotide sequences and constructs of the

A Modular Plasmid Assembly Kit for Multigene Expression, Gene Silencing and Silencing Rescue in Plants

PubMed Central

Binder, Andreas; Lambert, Jayne; Morbitzer, Robert; Popp, Claudia; Ott, Thomas; Lahaye, Thomas; Parniske, Martin

2014-01-01

The Golden Gate (GG) modular assembly approach offers a standardized, inexpensive and reliable way to ligate multiple DNA fragments in a pre-defined order in a single-tube reaction. We developed a GG based toolkit for the flexible construction of binary plasmids for transgene expression in plants. Starting from a common set of modules, such as promoters, protein tags and transcribed regions of interest, synthetic genes are assembled, which can be further combined to multigene constructs. As an example, we created T-DNA constructs encoding multiple fluorescent proteins targeted to distinct cellular compartments (nucleus, cytosol, plastids) and demonstrated simultaneous expression of all genes in Nicotiana benthamiana, Lotus japonicus and Arabidopsis thaliana. We assembled an RNA interference (RNAi) module for the construction of intron-spliced hairpin RNA constructs and demonstrated silencing of GFP in N. benthamiana. By combination of the silencing construct together with a codon adapted rescue construct into one vector, our system facilitates genetic complementation and thus confirmation of the causative gene responsible for a given RNAi phenotype. As proof of principle, we silenced a destabilized GFP gene (dGFP) and restored GFP fluorescence by expression of a recoded version of dGFP, which was not targeted by the silencing construct. PMID:24551083

[Construction and expression analysis of the zebrafish heart-specific transgenetic vector based on Tol2 transposable element].

PubMed

Chen, Tingfang; Luo, Na; Xie, Huaping; Wu, Xiushan; Deng, Yun

2010-02-01

In an effort to generate a desired expression construct for making heart-specific expression transgenic zebrafish, a Tol2 plasmid, which can drive EGFP reporter gene specifically expressed in the heart, was modified using subcloning technology. An IRES fragment bearing multiple cloning site (MCS) was amplified directly from pIRES2-EGFP plasmid and was inserted between the CMLC2 promoter and EGFP fragment of the pDestTol2CG vector. This recombinant expression plasmid pTol2-CMLC2-IRES-EGFP can drive any interested gene specifically expressed in the zebrafish heart along with EGFP reporter gene. To test the effectiveness of this new expression plasmid, we constructed pTol2-CMLC2-RED-IRES-EGFP plasmid by inserting another reporter gene DsRed-Monome into MCS downstream of the CMLC2 promoter and injected this transgenic recombinant plasmid into one-cell stage embryos of zebrafish. Under fluorescence microscope, both the red fluorescence and the green fluorescence produced by pTol2-CMLC2-RED-IRES-EGFP were detected specifically in the heart tissue in the same expression pattern. This novel expression construct pTol2-CMLC2-IRES-EGFP will become an important tool for our research on identifying heart development candidate genes' function using zebrafish as a model.

Ectopic transgene expression in the retina of four transgenic mouse lines

PubMed Central

Gábriel, Robert; Erdélyi, Ferenc; Szabó, Gábor; Lawrence, J. Josh

2017-01-01

Retinal expression of transgenes was examined in four mouse lines. Two constructs were driven by the choline acetyltransferase (ChAT) promoter: green fluorescent protein conjugated to tau protein (tau-GFP) or cytosolic yellow fluorescent protein (YFP) generated through CRE recombinase-induced expression of Rosa26 (ChAT-CRE/ Rosa26YFP). Two other constructs targeted inhibitory interneurons: GABAergic horizontal and amacrine cells identified by glutamic acid decarboxylase (GAD65-GFP) or parvalbumin (PV) cells (PV-CRE/Rosa26YFP). Animals were transcardially perfused and retinal sections prepared. Antibodies against PV, calretinin (CALR), calbindin (CALB), and tyrosine hydroxylase (TH) were used to counterstain transgene-expressing cells. In PVxRosa and ChAT-tauGFP constructs, staining appeared in vertically oriented row of processes resembling Müller cells. In the ChATxRosa construct, populations of amacrine cells and neurons in the ganglion cell layer were labeled. Some cones also exhibited GFP fluorescence. CALR, PV and TH were found in none of these cells. Occasionally, we found GFP/ CALR and GFP/PV double-stained cells in the ganglion cell layer (GCL). In the GAD65-GFP construct, all layers of the neuroretina were labeled, except photoreceptors. Not all horizontal cells expressed GFP. We did not find GFP/TH double-labeled cells and GFP was rarely present in CALR-and CALB-containing cells. Many PV-positive neurons were also labeled for GFP, including small diameter amacrines. In the GCL, single labeling for GFP and PV was ascertained, as well as several CALR/PV double-stained neurons. In the GCL, cells triple labeled with GFP/CALR/ CALB were sparse. In conclusion, only one of the four transgenic constructs exhibited an expression pattern consistent with endogenous retinal protein expression, while the others strongly suggested ectopic gene expression. PMID:26563404

Representing Medical Knowledge in a Terminological Language is Difficult1

PubMed Central

Haimowits, Ira J.; Patil, Ramesh S.; Szolovits, Peter

1988-01-01

We report on an experiment to use a modern knowledge representation language, NIKL, to express the knowledge of a sophisticated medical reasoning program, ABEL. We are attempting to put the development of more capable medical programs on firmer representational grounds by moving from the ad hoc representations typical of current programs toward more principled representation languages now in use or under construction. Our experience with the project reported here suggests caution, however. Attempts at cleanliness and efficiency in the design of representation languages lead to a poverty of expressiveness that makes it difficult if not impossible to say in such languages what needs to be stated to support the application.

Indoor 3D Route Modeling Based On Estate Spatial Data

NASA Astrophysics Data System (ADS)

Zhang, H.; Wen, Y.; Jiang, J.; Huang, W.

2014-04-01

Indoor three-dimensional route model is essential for space intelligence navigation and emergency evacuation. This paper is motivated by the need of constructing indoor route model automatically and as far as possible. By comparing existing building data sources, this paper firstly explained the reason why the estate spatial management data is chosen as the data source. Then, an applicable method of construction three-dimensional route model in a building is introduced by establishing the mapping relationship between geographic entities and their topological expression. This data model is a weighted graph consist of "node" and "path" to express the spatial relationship and topological structure of a building components. The whole process of modelling internal space of a building is addressed by two key steps: (1) each single floor route model is constructed, including path extraction of corridor using Delaunay triangulation algorithm with constrained edge, fusion of room nodes into the path; (2) the single floor route model is connected with stairs and elevators and the multi-floor route model is eventually generated. In order to validate the method in this paper, a shopping mall called "Longjiang New City Plaza" in Nanjing is chosen as a case of study. And the whole building space is constructed according to the modelling method above. By integrating of existing path finding algorithm, the usability of this modelling method is verified, which shows the indoor three-dimensional route modelling method based on estate spatial data in this paper can support indoor route planning and evacuation route design very well.

Engineering customized TALE nucleases (TALENs) and TALE transcription factors by fast ligation-based automatable solid-phase high-throughput (FLASH) assembly.

PubMed

Reyon, Deepak; Maeder, Morgan L; Khayter, Cyd; Tsai, Shengdar Q; Foley, Jonathan E; Sander, Jeffry D; Joung, J Keith

2013-07-01

Customized DNA-binding domains made using transcription activator-like effector (TALE) repeats are rapidly growing in importance as widely applicable research tools. TALE nucleases (TALENs), composed of an engineered array of TALE repeats fused to the FokI nuclease domain, have been used successfully for directed genome editing in various organisms and cell types. TALE transcription factors (TALE-TFs), consisting of engineered TALE repeat arrays linked to a transcriptional regulatory domain, have been used to up- or downregulate expression of endogenous genes in human cells and plants. This unit describes a detailed protocol for the recently described fast ligation-based automatable solid-phase high-throughput (FLASH) assembly method. FLASH enables automated high-throughput construction of engineered TALE repeats using an automated liquid handling robot or manually using a multichannel pipet. Using the automated approach, a single researcher can construct up to 96 DNA fragments encoding TALE repeat arrays of various lengths in a single day, and then clone these to construct sequence-verified TALEN or TALE-TF expression plasmids in a week or less. Plasmids required for FLASH are available by request from the Joung lab (http://eGenome.org). This unit also describes improvements to the Zinc Finger and TALE Targeter (ZiFiT Targeter) web server (http://ZiFiT.partners.org) that facilitate the design and construction of FLASH TALE repeat arrays in high throughput. © 2013 by John Wiley & Sons, Inc.

Engineering Customized TALE Nucleases (TALENs) and TALE Transcription Factors by Fast Ligation-based Automatable Solid-phase High-throughput (FLASH) Assembly

PubMed Central

Reyon, Deepak; Maeder, Morgan L.; Khayter, Cyd; Tsai, Shengdar Q.; Foley, Jonathan E.; Sander, Jeffry D.; Joung, J. Keith

2013-01-01

Customized DNA-binding domains made using Transcription Activator-Like Effector (TALE) repeats are rapidly growing in importance as widely applicable research tools. TALE nucleases (TALENs), composed of an engineered array of TALE repeats fused to the FokI nuclease domain, have been used successfully for directed genome editing in multiple different organisms and cell types. TALE transcription factors (TALE-TFs), consisting of engineered TALE repeat arrays linked to a transcriptional regulatory domain, have been used to up- or down-regulate expression of endogenous genes in human cells and plants. Here we describe a detailed protocol for practicing the recently described Fast Ligation-based Automatable Solid-phase High-throughput (FLASH) assembly method. FLASH enables automated high-throughput construction of engineered TALE repeats using an automated liquid handling robot or manually using a multi-channel pipet. With the automated version of FLASH, a single researcher can construct up to 96 DNA fragments encoding various length TALE repeat arrays in one day and then clone these to construct sequence-verified TALEN or TALE-TF expression plasmids in one week or less. Plas-mids required to practice FLASH are available by request from the Joung Lab (http://www.jounglab.org/). We also describe here improvements to the Zinc Finger and TALE Targeter (ZiFiT Targeter) webserver (http://ZiFiTBeta.partners.org) that facilitate the design and construction of FLASH TALE repeat arrays in high-throughput. PMID:23821439

OSG-GEM: Gene Expression Matrix Construction Using the Open Science Grid.

PubMed

Poehlman, William L; Rynge, Mats; Branton, Chris; Balamurugan, D; Feltus, Frank A

2016-01-01

High-throughput DNA sequencing technology has revolutionized the study of gene expression while introducing significant computational challenges for biologists. These computational challenges include access to sufficient computer hardware and functional data processing workflows. Both these challenges are addressed with our scalable, open-source Pegasus workflow for processing high-throughput DNA sequence datasets into a gene expression matrix (GEM) using computational resources available to U.S.-based researchers on the Open Science Grid (OSG). We describe the usage of the workflow (OSG-GEM), discuss workflow design, inspect performance data, and assess accuracy in mapping paired-end sequencing reads to a reference genome. A target OSG-GEM user is proficient with the Linux command line and possesses basic bioinformatics experience. The user may run this workflow directly on the OSG or adapt it to novel computing environments.

OSG-GEM: Gene Expression Matrix Construction Using the Open Science Grid

PubMed Central

Poehlman, William L.; Rynge, Mats; Branton, Chris; Balamurugan, D.; Feltus, Frank A.

2016-01-01

High-throughput DNA sequencing technology has revolutionized the study of gene expression while introducing significant computational challenges for biologists. These computational challenges include access to sufficient computer hardware and functional data processing workflows. Both these challenges are addressed with our scalable, open-source Pegasus workflow for processing high-throughput DNA sequence datasets into a gene expression matrix (GEM) using computational resources available to U.S.-based researchers on the Open Science Grid (OSG). We describe the usage of the workflow (OSG-GEM), discuss workflow design, inspect performance data, and assess accuracy in mapping paired-end sequencing reads to a reference genome. A target OSG-GEM user is proficient with the Linux command line and possesses basic bioinformatics experience. The user may run this workflow directly on the OSG or adapt it to novel computing environments. PMID:27499617

The Role of Multiple Transcription Factors In Archaeal Gene Expression

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

Charles J. Daniels

2008-09-23

Since the inception of this research program, the project has focused on two central questions: What is the relationship between the 'eukaryal-like' transcription machinery of archaeal cells and its counterparts in eukaryal cells? And, how does the archaeal cell control gene expression using its mosaic of eukaryal core transcription machinery and its bacterial-like transcription regulatory proteins? During the grant period we have addressed these questions using a variety of in vivo approaches and have sought to specifically define the roles of the multiple TATA binding protein (TBP) and TFIIB-like (TFB) proteins in controlling gene expression in Haloferax volcanii. H. volcaniimore » was initially chosen as a model for the Archaea based on the availability of suitable genetic tools; however, later studies showed that all haloarchaea possessed multiple tbp and tfb genes, which led to the proposal that multiple TBP and TFB proteins may function in a manner similar to alternative sigma factors in bacterial cells. In vivo transcription and promoter analysis established a clear relationship between the promoter requirements of haloarchaeal genes and those of the eukaryal RNA polymerase II promoter. Studies on heat shock gene promoters, and the demonstration that specific tfb genes were induced by heat shock, provided the first indication that TFB proteins may direct expression of specific gene families. The construction of strains lacking tbp or tfb genes, coupled with the finding that many of these genes are differentially expressed under varying growth conditions, provided further support for this model. Genetic tools were also developed that led to the construction of insertion and deletion mutants, and a novel gene expression scheme was designed that allowed the controlled expression of these genes in vivo. More recent studies have used a whole genome array to examine the expression of these genes and we have established a linkage between the expression of specific tfb genes and the regulation of nitrogen metabolism and other global cellular responses.« less

Rationally designed, heterologous S. cerevisiae transcripts expose novel expression determinants

PubMed Central

Ben-Yehezkel, Tuval; Atar, Shimshi; Zur, Hadas; Diament, Alon; Goz, Eli; Marx, Tzipy; Cohen, Rafael; Dana, Alexandra; Feldman, Anna; Shapiro, Ehud; Tuller, Tamir

2015-01-01

Deducing generic causal relations between RNA transcript features and protein expression profiles from endogenous gene expression data remains a major unsolved problem in biology. The analysis of gene expression from heterologous genes contributes significantly to solving this problem, but has been heavily biased toward the study of the effect of 5′ transcript regions and to prokaryotes. Here, we employ a synthetic biology driven approach that systematically differentiates the effect of different regions of the transcript on gene expression up to 240 nucleotides into the ORF. This enabled us to discover new causal effects between features in previously unexplored regions of transcripts, and gene expression in natural regimes. We rationally designed, constructed, and analyzed 383 gene variants of the viral HRSVgp04 gene ORF, with multiple synonymous mutations at key positions along the transcript in the eukaryote S. cerevisiae. Our results show that a few silent mutations at the 5′UTR can have a dramatic effect of up to 15 fold change on protein levels, and that even synonymous mutations in positions more than 120 nucleotides downstream from the ORF 5′end can modulate protein levels up to 160%–300%. We demonstrate that the correlation between protein levels and folding energy increases with the significance of the level of selection of the latter in endogenous genes, reinforcing the notion that selection for folding strength in different parts of the ORF is related to translation regulation. Our measured protein abundance correlates notably(correlation up to r = 0.62 (p=0.0013)) with mean relative codon decoding times, based on ribosomal densities (Ribo-Seq) in endogenous genes, supporting the conjecture that translation elongation and adaptation to the tRNA pool can modify protein levels in a causal/direct manner. This report provides an improved understanding of transcript evolution, design principles of gene expression regulation, and suggests simple rules for engineering synthetic gene expression in eukaryotes. PMID:26176266

Rationally designed, heterologous S. cerevisiae transcripts expose novel expression determinants.

PubMed

Ben-Yehezkel, Tuval; Atar, Shimshi; Zur, Hadas; Diament, Alon; Goz, Eli; Marx, Tzipy; Cohen, Rafael; Dana, Alexandra; Feldman, Anna; Shapiro, Ehud; Tuller, Tamir

2015-01-01

Deducing generic causal relations between RNA transcript features and protein expression profiles from endogenous gene expression data remains a major unsolved problem in biology. The analysis of gene expression from heterologous genes contributes significantly to solving this problem, but has been heavily biased toward the study of the effect of 5' transcript regions and to prokaryotes. Here, we employ a synthetic biology driven approach that systematically differentiates the effect of different regions of the transcript on gene expression up to 240 nucleotides into the ORF. This enabled us to discover new causal effects between features in previously unexplored regions of transcripts, and gene expression in natural regimes. We rationally designed, constructed, and analyzed 383 gene variants of the viral HRSVgp04 gene ORF, with multiple synonymous mutations at key positions along the transcript in the eukaryote S. cerevisiae. Our results show that a few silent mutations at the 5'UTR can have a dramatic effect of up to 15 fold change on protein levels, and that even synonymous mutations in positions more than 120 nucleotides downstream from the ORF 5'end can modulate protein levels up to 160%-300%. We demonstrate that the correlation between protein levels and folding energy increases with the significance of the level of selection of the latter in endogenous genes, reinforcing the notion that selection for folding strength in different parts of the ORF is related to translation regulation. Our measured protein abundance correlates notably(correlation up to r = 0.62 (p=0.0013)) with mean relative codon decoding times, based on ribosomal densities (Ribo-Seq) in endogenous genes, supporting the conjecture that translation elongation and adaptation to the tRNA pool can modify protein levels in a causal/direct manner. This report provides an improved understanding of transcript evolution, design principles of gene expression regulation, and suggests simple rules for engineering synthetic gene expression in eukaryotes.

[Construction and expression of the targeting super-antigen EGF-SEA fusion gene].

PubMed

Xie, Yang; Peng, Shaoping; Liao, Zhiying; Liu, Jiafeng; Liu, Xuemei; Chen, Weifeng

2014-05-01

To construct expression vector for the SEA-EGF fusion gene. Clone the SEA gene and the EGF gene segment with PCR and RT-PCR independently, and connect this two genes by the bridge PCR. Insert the fusion gene EGF-SEA into the expression vector PET-44. Induced the secretion of the fusion protein SEA-EGF by the antileptic. The gene fragment encoding EGF and SEA mature peptide was successfully cloned. The fusion gene EGF-SEA was successfully constructed and was inserted into expression vector. The new recombinant expression vector for fusion gene EGF-SEA is specific for head and neck cancer, laid the foundation for the further study of fusion protein SEA-EGF targeting immune therapy in head and neck tumors.

Chemical Library Screening for Potential Therapeutics Using Novel Cell Based Models of ALS

DTIC Science & Technology

2017-06-01

Major Task 1, Subtask 1. Our first major activity in year 1 was the design and engineering of a custom inducible plasmid for expressing fluorescently...back to that construct. Other achievements during this first major activity derived from investigating why Broccoli RNA aptamer did not fold...These are outlined below in the CHANGES/PROBLEMS section. Major Task 1, Subtask 2. Our second major activity in year 1 was to generate human neural

Process design for microbial plastic factories: metabolic engineering of polyhydroxyalkanoates.

PubMed

Aldor, Ilana S; Keasling, Jay D

2003-10-01

Implementing several metabolic engineering strategies, either individually or in combination, it is possible to construct microbial plastic factories to produce a variety of polyhydroxyalkanoate (PHA) biopolymers with desirable structures and material properties. Approaches include external substrate manipulation, inhibitor addition, recombinant gene expression, host cell genome manipulation and, most recently, protein engineering of PHA biosynthetic enzymes. In addition, mathematical models and molecular methods can be used to elucidate metabolically engineered systems and to identify targets for performance improvement.

High-throughput plasmid construction using homologous recombination in yeast: its mechanisms and application to protein production for X-ray crystallography.

PubMed

Mizutani, Kimihiko

2015-01-01

Homologous recombination is a system for repairing the broken genomes of living organisms by connecting two DNA strands at their homologous sequences. Today, homologous recombination in yeast is used for plasmid construction as a substitute for traditional methods using restriction enzymes and ligases. This method has various advantages over the traditional method, including flexibility in the position of DNA insertion and ease of manipulation. Recently, the author of this review reported the construction of plasmids by homologous recombination in the methanol-utilizing yeast Pichia pastoris, which is known to be an excellent expression host for secretory proteins and membrane proteins. The method enabled high-throughput construction of expression systems of proteins using P. pastoris; the constructed expression systems were used to investigate the expression conditions of membrane proteins and to perform X-ray crystallography of secretory proteins. This review discusses the mechanisms and applications of homologous recombination, including the production of proteins for X-ray crystallography.

Transcriptome profiling analysis reveals biomarkers in colon cancer samples of various differentiation

PubMed Central

Yu, Tonghu; Zhang, Huaping; Qi, Hong

2018-01-01

The aim of the present study was to investigate more colon cancer-related genes in different stages. Gene expression profile E-GEOD-62932 was extracted for differentially expressed gene (DEG) screening. Series test of cluster analysis was used to obtain significant trending models. Based on the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases, functional and pathway enrichment analysis were processed and a pathway relation network was constructed. Gene co-expression network and gene signal network were constructed for common DEGs. The DEGs with the same trend were clustered and in total, 16 clusters with statistical significance were obtained. The screened DEGs were enriched into small molecule metabolic process and metabolic pathways. The pathway relation network was constructed with 57 nodes. A total of 328 common DEGs were obtained. Gene signal network was constructed with 71 nodes. Gene co-expression network was constructed with 161 nodes and 211 edges. ABCD3, CPT2, AGL and JAM2 are potential biomarkers for the diagnosis of colon cancer. PMID:29928385

Differentially Coexpressed Disease Gene Identification Based on Gene Coexpression Network.

PubMed

Jiang, Xue; Zhang, Han; Quan, Xiongwen

2016-01-01

Screening disease-related genes by analyzing gene expression data has become a popular theme. Traditional disease-related gene selection methods always focus on identifying differentially expressed gene between case samples and a control group. These traditional methods may not fully consider the changes of interactions between genes at different cell states and the dynamic processes of gene expression levels during the disease progression. However, in order to understand the mechanism of disease, it is important to explore the dynamic changes of interactions between genes in biological networks at different cell states. In this study, we designed a novel framework to identify disease-related genes and developed a differentially coexpressed disease-related gene identification method based on gene coexpression network (DCGN) to screen differentially coexpressed genes. We firstly constructed phase-specific gene coexpression network using time-series gene expression data and defined the conception of differential coexpression of genes in coexpression network. Then, we designed two metrics to measure the value of gene differential coexpression according to the change of local topological structures between different phase-specific networks. Finally, we conducted meta-analysis of gene differential coexpression based on the rank-product method. Experimental results demonstrated the feasibility and effectiveness of DCGN and the superior performance of DCGN over other popular disease-related gene selection methods through real-world gene expression data sets.

Monoclonal antibodies expression improvement in CHO cells by PiggyBac transposition regarding vectors ratios and design.

PubMed

Ahmadi, Samira; Davami, Fatemeh; Davoudi, Noushin; Nematpour, Fatemeh; Ahmadi, Maryam; Ebadat, Saeedeh; Azadmanesh, Kayhan; Barkhordari, Farzaneh; Mahboudi, Fereidoun

2017-01-01

Establishing stable Chinese Hamster Ovary (CHO) cells producing monoclonal antibodies (mAbs) usually pass through the random integration of vectors to the cell genome, which is sensitive to gene silencing. One approach to overcome this issue is to target a highly transcribed region in the genome. Transposons are useful devices to target active parts of genomes, and PiggyBac (PB) transposon can be considered as a good option. In the present study, three PB transposon donor vectors containing both heavy and light chains were constructed, one contained independent expression cassettes while the others utilized either an Internal Ribosome Entry Site (IRES) or 2A element to express mAb. Conventional cell pools were created by transferring donor vectors into the CHO cells, whereas transposon-based cells were generated by transfecting the cells with donor vectors with a companion of a transposase-encoding helper vector, with 1:2.5 helper/donor vectors ratio. To evaluate the influence of helper/donor vectors ratio on expression, the second transposon-based cell pools were generated with 1:5 helper/donor ratio. Expression levels in the transposon-based cells were two to five -folds more than those created by conventional method except for the IRES-mediated ones, in which the observed difference increased more than 100-fold. The results were dependent on both donor vector design and vectors ratios.

Monoclonal antibodies expression improvement in CHO cells by PiggyBac transposition regarding vectors ratios and design

PubMed Central

Ahmadi, Samira; Davami, Fatemeh; Davoudi, Noushin; Nematpour, Fatemeh; Ahmadi, Maryam; Ebadat, Saeedeh; Azadmanesh, Kayhan; Barkhordari, Farzaneh

2017-01-01

Establishing stable Chinese Hamster Ovary (CHO) cells producing monoclonal antibodies (mAbs) usually pass through the random integration of vectors to the cell genome, which is sensitive to gene silencing. One approach to overcome this issue is to target a highly transcribed region in the genome. Transposons are useful devices to target active parts of genomes, and PiggyBac (PB) transposon can be considered as a good option. In the present study, three PB transposon donor vectors containing both heavy and light chains were constructed, one contained independent expression cassettes while the others utilized either an Internal Ribosome Entry Site (IRES) or 2A element to express mAb. Conventional cell pools were created by transferring donor vectors into the CHO cells, whereas transposon-based cells were generated by transfecting the cells with donor vectors with a companion of a transposase-encoding helper vector, with 1:2.5 helper/donor vectors ratio. To evaluate the influence of helper/donor vectors ratio on expression, the second transposon-based cell pools were generated with 1:5 helper/donor ratio. Expression levels in the transposon-based cells were two to five -folds more than those created by conventional method except for the IRES-mediated ones, in which the observed difference increased more than 100-fold. The results were dependent on both donor vector design and vectors ratios. PMID:28662065

Promoter and Terminator Discovery and Engineering.

PubMed

Deaner, Matthew; Alper, Hal S

Control of gene expression is crucial to optimize metabolic pathways and synthetic gene networks. Promoters and terminators are stretches of DNA upstream and downstream (respectively) of genes that control both the rate at which the gene is transcribed and the rate at which mRNA is degraded. As a result, both of these elements control net protein expression from a synthetic construct. Thus, it is highly important to discover and engineer promoters and terminators with desired characteristics. This chapter highlights various approaches taken to catalogue these important synthetic elements. Specifically, early strategies have focused largely on semi-rational techniques such as saturation mutagenesis to diversify native promoters and terminators. Next, in an effort to reduce the length of the synthetic biology design cycle, efforts in the field have turned towards the rational design of synthetic promoters and terminators. In this vein, we cover recently developed methods such as hybrid engineering, high throughput characterization, and thermodynamic modeling which allow finer control in the rational design of novel promoters and terminators. Emphasis is placed on the methodologies used and this chapter showcases the utility of these methods across multiple host organisms.

A new maltose-inducible high-performance heterologous expression system in Bacillus subtilis.

PubMed

Yue, Jie; Fu, Gang; Zhang, Dawei; Wen, Jianping

2017-08-01

To improve heterologous proteins production, we constructed a maltose-inducible expression system in Bacillus subtilis. An expression system based on the promoter for maltose utilization constructed in B. subtilis. Successively, to improve the performance of the P malA -derived system, mutagenesis was employed by gradually shortening the length of P malA promoter and altering the spacing between the predicted MalR binding site and the -35 region. Furthermore, deletion of the maltose utilization genes (malL and yvdK) improved the P malA promoter activity. Finally, using this efficient maltose-inducible expression system, we enhanced the production of luciferase and D-aminoacylase, compared with the P hpaII system. A maltose-inducible expression system was constructed and evaluated. It could be used for high level expression of heterologous proteins production.

DNA recognition by synthetic constructs.

PubMed

Pazos, Elena; Mosquera, Jesús; Vázquez, M Eugenio; Mascareñas, José L

2011-09-05

The interaction of transcription factors with specific DNA sites is key for the regulation of gene expression. Despite the availability of a large body of structural data on protein-DNA complexes, we are still far from fully understanding the molecular and biophysical bases underlying such interactions. Therefore, the development of non-natural agents that can reproduce the DNA-recognition properties of natural transcription factors remains a major and challenging goal in chemical biology. In this review we summarize the basics of double-stranded DNA recognition by transcription factors, and describe recent developments in the design and preparation of synthetic DNA binders. We mainly focus on synthetic peptides that have been designed by following the DNA interaction of natural proteins, and we discuss how the tools of organic synthesis can be used to make artificial constructs equipped with functionalities that introduce additional properties to the recognition process, such as sensing and controllability. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rational Design of Evolutionarily Stable Microbial Kill Switches.

PubMed

Stirling, Finn; Bitzan, Lisa; O'Keefe, Samuel; Redfield, Elizabeth; Oliver, John W K; Way, Jeffrey; Silver, Pamela A

2017-11-16

The evolutionary stability of synthetic genetic circuits is key to both the understanding and application of genetic control elements. One useful but challenging situation is a switch between life and death depending on environment. Here are presented "essentializer" and "cryodeath" circuits, which act as kill switches in Escherichia coli. The essentializer element induces cell death upon the loss of a bi-stable cI/Cro memory switch. Cryodeath makes use of a cold-inducible promoter to express a toxin. We employ rational design and a toxin/antitoxin titering approach to produce and screen a small library of potential constructs, in order to select for constructs that are evolutionarily stable. Both kill switches were shown to maintain functionality in vitro for at least 140 generations. Additionally, cryodeath was shown to control the growth environment of a population, with an escape frequency of less than 1 in 10 5 after 10 days of growth in the mammalian gut. Copyright © 2017 Elsevier Inc. All rights reserved.

A Novel Terminator Primer and Enhancer Reagents for Direct Expression of PCR-Amplified Genes in Mammalian Cells.

PubMed

Nakamura, Mikiko; Suzuki, Ayako; Akada, Junko; Yarimizu, Tohru; Iwakiri, Ryo; Hoshida, Hisashi; Akada, Rinji

2015-08-01

Escherichia coli plasmids are commonly used for gene expression experiments in mammalian cells, while PCR-amplified DNAs are rarely used even though PCR is a much faster and easier method to construct recombinant DNAs. One difficulty may be the limited amount of DNA produced by PCR. For direct utilization of PCR-amplified DNA in transfection experiments, efficient transfection with a smaller amount of DNA should be attained. For this purpose, we investigated two enhancer reagents, polyethylene glycol and tRNA, for a chemical transfection method. The addition of the enhancers to a commercial transfection reagent individually and synergistically exhibited higher transfection efficiency applicable for several mammalian cell culture lines in a 96-well plate. By taking advantage of a simple transfection procedure using PCR-amplified DNA, SV40 and rabbit β-globin terminator lengths were minimized. The terminator length is short enough to design in oligonucleotides; thus, terminator primers can be used for the construction and analysis of numerous mutations, deletions, insertions, and tag-fusions at the 3'-terminus of any gene. The PCR-mediated gene manipulation with the terminator primers will transform gene expression by allowing for extremely simple and high-throughput experiments with small-scale, multi-well, and mammalian cell cultures.

Process for Assembly and Transformation into Saccharomyces cerevisiae of a Synthetic Yeast Artificial Chromosome Containing a Multigene Cassette to Express Enzymes That Enhance Xylose Utilization Designed for an Automated Platform.

PubMed

Hughes, Stephen R; Cox, Elby J; Bang, Sookie S; Pinkelman, Rebecca J; López-Núñez, Juan Carlos; Saha, Badal C; Qureshi, Nasib; Gibbons, William R; Fry, Michelle R; Moser, Bryan R; Bischoff, Kenneth M; Liu, Siqing; Sterner, David E; Butt, Tauseef R; Riedmuller, Steven B; Jones, Marjorie A; Riaño-Herrera, Néstor M

2015-12-01

A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system in yeast and to design an assembly process suitable for an automated platform. Expression of XI and XKS from the YAC was confirmed by Western blot and PCR analyses. The recombinant and wild-type strains showed similar growth on plates containing hexose sugars, but only recombinant grew on D-xylose and L-arabinose plates. In glucose fermentation, doubling time (4.6 h) and ethanol yield (0.44 g ethanol/g glucose) of recombinant were comparable to wild type (4.9 h and 0.44 g/g). In whole-corn hydrolysate, ethanol yield (0.55 g ethanol/g [glucose + xylose]) and xylose utilization (38%) for recombinant were higher than for wild type (0.47 g/g and 12%). In hydrolysate from spent coffee grounds, yield was 0.46 g ethanol/g (glucose + xylose), and xylose utilization was 93% for recombinant. These results indicate introducing a YAC expressing XI and XKS enhanced xylose utilization without affecting integrity of the host strain, and the process provides a potential platform for automated synthesis of a YAC for expression of multiple optimized genes to improve yeast strains. © 2015 Society for Laboratory Automation and Screening.

Finding gene regulatory network candidates using the gene expression knowledge base.

PubMed

Venkatesan, Aravind; Tripathi, Sushil; Sanz de Galdeano, Alejandro; Blondé, Ward; Lægreid, Astrid; Mironov, Vladimir; Kuiper, Martin

2014-12-10

Network-based approaches for the analysis of large-scale genomics data have become well established. Biological networks provide a knowledge scaffold against which the patterns and dynamics of 'omics' data can be interpreted. The background information required for the construction of such networks is often dispersed across a multitude of knowledge bases in a variety of formats. The seamless integration of this information is one of the main challenges in bioinformatics. The Semantic Web offers powerful technologies for the assembly of integrated knowledge bases that are computationally comprehensible, thereby providing a potentially powerful resource for constructing biological networks and network-based analysis. We have developed the Gene eXpression Knowledge Base (GeXKB), a semantic web technology based resource that contains integrated knowledge about gene expression regulation. To affirm the utility of GeXKB we demonstrate how this resource can be exploited for the identification of candidate regulatory network proteins. We present four use cases that were designed from a biological perspective in order to find candidate members relevant for the gastrin hormone signaling network model. We show how a combination of specific query definitions and additional selection criteria derived from gene expression data and prior knowledge concerning candidate proteins can be used to retrieve a set of proteins that constitute valid candidates for regulatory network extensions. Semantic web technologies provide the means for processing and integrating various heterogeneous information sources. The GeXKB offers biologists such an integrated knowledge resource, allowing them to address complex biological questions pertaining to gene expression. This work illustrates how GeXKB can be used in combination with gene expression results and literature information to identify new potential candidates that may be considered for extending a gene regulatory network.

[Construction and expression of the eukaryotic expression vector carrying HSV-1 gC glycoprotein gene].

PubMed

Dang, Yin-li; Yan, Yan; Zhang, Xiao-xiao; Li, Pu-yuan; Yu, Lan; Zhang, Lei; Zhang, Fang-lin; Xu, Zhi-kai; Wu, Xing-an

2011-05-01

To stably express herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) in Chinese hamster ovary cells (CHO-K1). The eukaryotic expression vector pCI-mCMV-gC-1-IRES-DHFR-L22R was constructed and transfected into CHO-K1 cells by Lipofectamine 2000. The transfected cells were selected by G418 and methotrexate (MTX). The expression of HSV-1 gC was analyzed by Slot blot. HSV-1 gC proteins were purified with His-Ni Sepharose and then detected by Western blot. The eukaryotic expression vector pCI-mCMV-gC-1-IRES-DHFR-L22R was constructed successfully. CHO-K1 cells stably expressing HSV-1 gC proteins were established and confirmed by Western blot. The HSV-1 gC proteins have been expressed successfully and have good bioactivity. The results make it possible for further study and clinical use of HSV-1 gC.

Three gene expression vector sets for concurrently expressing multiple genes in Saccharomyces cerevisiae.

PubMed

Ishii, Jun; Kondo, Takashi; Makino, Harumi; Ogura, Akira; Matsuda, Fumio; Kondo, Akihiko

2014-05-01

Yeast has the potential to be used in bulk-scale fermentative production of fuels and chemicals due to its tolerance for low pH and robustness for autolysis. However, expression of multiple external genes in one host yeast strain is considerably labor-intensive due to the lack of polycistronic transcription. To promote the metabolic engineering of yeast, we generated systematic and convenient genetic engineering tools to express multiple genes in Saccharomyces cerevisiae. We constructed a series of multi-copy and integration vector sets for concurrently expressing two or three genes in S. cerevisiae by embedding three classical promoters. The comparative expression capabilities of the constructed vectors were monitored with green fluorescent protein, and the concurrent expression of genes was monitored with three different fluorescent proteins. Our multiple gene expression tool will be helpful to the advanced construction of genetically engineered yeast strains in a variety of research fields other than metabolic engineering. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Use of expression constructs to dissect the functional domains of the CHS/beige protein: identification of multiple phenotypes.

PubMed

Ward, Diane McVey; Shiflett, Shelly L; Huynh, Dinh; Vaughn, Michael B; Prestwich, Glenn; Kaplan, Jerry

2003-06-01

The Chediak-Higashi Syndrome (CHS) and the orthologous murine disorder beige are characterized at the cellular level by the presence of giant lysosomes. The CHS1/Beige protein is a 3787 amino acid protein of unknown function. To determine functional domains of the CHS1/Beige protein, we generated truncated constructs of the gene/protein. These truncated proteins were transiently expressed in Cos-7 or HeLa cells and their effect on membrane trafficking was examined. Beige is apparently a cytosolic protein, as are most transiently expressed truncated Beige constructs. Expression of the Beige construct FM (amino acids 1-2037) in wild-type cells led to enlarged lysosomes. Similarly, expression of a 5.5-kb region (amino acids 2035-3787) of the carboxyl terminal of Beige (22B) also resulted in enlarged lysosomes. Expression of FM solely affected lysosome size, whereas expression of 22B led to alterations in lysosome size, changes in the Golgi and eventually cell death. The two constructs could be used to further dissect phenotypes resulting from loss of the Beige protein. CHS or beigej fibroblasts show an absence of nuclear staining using a monoclonal antibody directed against phosphatidylinositol 4,5 bisphosphate [PtdIns(4,5) P2]. Transformation of beige j fibroblasts with a YAC containing the full-length Beige gene resulted in the normalization of lysosome size and nuclear PtdIns(4,5)P2 staining. Expression of the carboxyl dominant negative construct 22B led to loss of nuclear PtdIns(4,5)P2 staining. Expression of the FM dominant negative clone did not alter nuclear PtdIns(4,5) P2 localization. These results suggest that the Beige protein interacts with at least two different partners and that the Beige protein affects cellular events, such as nuclear PtdIns(4,5)P2 localization, in addition to lysosome size.

Long-range transcriptional interference in E. coli used to construct a dual positive selection system for genetic switches

PubMed Central

Hoffmann, Stefan A.; Kruse, Sabrina M.; Arndt, Katja M.

2016-01-01

Abstract We have investigated transcriptional interference between convergent genes in E. coli and demonstrate substantial interference for inter-promoter distances of as far as 3 kb. Interference can be elicited by both strong σ70 dependent and T7 promoters. In the presented design, a strong promoter driving gene expression of a ‘forward’ gene interferes with the expression of a ‘reverse’ gene by a weak promoter. This arrangement allows inversely correlated gene expression without requiring further regulatory components. Thus, modulation of the activity of the strong promoter alters expression of both the forward and the reverse gene. We used this design to develop a dual selection system for conditional operator site binding, allowing positive selection both for binding and for non-binding to DNA. This study demonstrates the utility of this novel system using the Lac repressor as a model protein for conditional DNA binding, and spectinomycin and chloramphenicol resistance genes as positive selection markers in liquid culture. Randomized LacI libraries were created and subjected to subsequent dual selection, but mispairing IPTG and selection cues in respect to the wild-type LacI response, allowing the isolation of a LacI variant with a reversed IPTG response within three rounds of library generation and dual selection. PMID:26932362

Niche construction through phenological plasticity: life history dynamics and ecological consequences.

PubMed

Donohue, Kathleen

2005-04-01

The ability of an organism to alter the environment that it experiences has been termed 'niche construction'. Plants have several ways whereby they can determine the environment to which they are exposed at different life stages. This paper discusses three of these: plasticity in dispersal, flowering timing and germination timing. It reviews pathways through which niche construction alters evolutionary and ecological trajectories by altering the selective environment to which organisms are exposed, the phenotypic expression of plastic characters, and the expression of genetic variation. It provides examples whereby niche construction creates positive or negative feedbacks between phenotypes and environments, which in turn cause novel evolutionary constraints and novel life-history expression. Copyright New Phytologist (2005).

Characterization and expression analysis of a banana gene encoding 1-aminocyclopropane-1-carboxylate oxidase.

PubMed

Huang, P L; Do, Y Y; Huang, F C; Thay, T S; Chang, T W

1997-04-01

A cDNA encoding the banana 1-aminocyclopropane-1-carboxylate (ACC) oxidase has previously been isolated from a cDNA library that was constructed by extracting poly(A)+ RNA from peels of ripening banana. This cDNA, designated as pMAO2, has 1,199 bp and contains an open reading frame of 318 amino acids. In order to identify ripening-related promoters of the banana ACC oxidase gene, pMAO2 was used as a probe to screen a banana genomic library constructed in the lambda EMBL3 vector. The banana ACC oxidase MAO2 gene has four exons and three introns, with all of the boundaries between these introns and exons sharing a consensus dinucleotide sequence of GT-AG. The expression of MAO2 gene in banana begins after the onset of ripening (stage 2) and continuous into later stages of the ripening process. The accumulation of MAO2 mRNA can be induced by 1 microliter/l exogenous ethylene, and it reached steady state level when 100 microliters/l exogenous ethylene was present.

Construction of PR39 recombinant AAV under control of the HRE promoter and the effect of recombinant AAV on gene therapy of ischemic heart disease

PubMed Central

SUN, LIJUN; HAO, YUEWEN; NIE, XIAOWEI; ZHANG, XUEXIN; YANG, GUANGXIAO; WANG, QUANYING

2012-01-01

The objective of this study was to investigate the effect of the PR39 recombinant adeno-associated virus (AAV) controlled by the hypoxia-responsive element (HRE) on gene therapy of ischemic heart disease. The minimal HRE was artificially synthesized and the AAV vector controlled by HRE was introduced with NT4-TAT-His-PR39 to investigate the expression of AAV-PR39 in hypoxic vascular endothelial cells (VEC) of human umbilical vein (CRL-1730 cell line) and the angiogenesis-promoting effect in pigs with acute myocardial infraction (AMI). The minimal HRE/CMV was designed and artificially synthesized using the PCR method and cloned with the T vector cloning method. The pSS-HRE-CMV-NT4-6His-PR39-PolyA-AAV plasmid was constructed. Using the calcium phosphate precipitation method, HEK-293 cells were co-transfected with three plasmids to produce the recombinant virus. An equal volume of pSS-HRE-CMV-NT4-6His-PR39-PolyAAAV and enterovirus (EV, blank virus) was transfected into CRL-1730 cell lines, respectively. The immunohistochemical method was used to assay the expression of 6xHis in CRL-1730 cell lines and the expression of PR39 under hypoxia. Eighteen AMI miniature pigs were randomized into the experimental group (HRE-AAV-PR39 group), control group 1 (physical saline group) and control group 2 (EV group). The area of ischemia was assessed with conventional MRI and myocardium perfusion MRI. Pigs were sacrificed at preset time-points to obtain samples of ischemic myocardium. Morphological and pathological data were collected. According to data in the literature and databases, the minimal HRE was designed and synthesized with the PCR method. A large number of HREs were connected to modified pSSHGAAV (pSSV9int-/XbaI) vector followed by insertion of the NT4-6His-PR39 gene segment and, thus, the recombinant plasmid pSS-HRE-CMV-NT4-6His-PR39-PolyA-AAV was successfully constructed. The expression of 6xHis in CRL-1730 cells under the regulation of HRE was assayed using the immunohistochemical method and results showed that the expression was positive in the experimental group. Myocardium perfusion MRI displayed that the infracted area significantly decreased under the action of pSS-HRE-CMV-NT4-PR39-PolyA-AAV. The artificial minimal HRE in CRL-1730 cells effectively and rapidly regulates the expression of the downstream gene NT4-TAT-His-PR39 of the CMV promoter. Recombinant pSS-HRE-CMV-NT4-PR39-Poly-AAAV promotes neoangiogenesis in the ischemic area, reduces the area of infarction and improves heart function. PMID:23226731

Construction of PR39 recombinant AAV under control of the HRE promoter and the effect of recombinant AAV on gene therapy of ischemic heart disease.

PubMed

Sun, Lijun; Hao, Yuewen; Nie, Xiaowei; Zhang, Xuexin; Yang, Guangxiao; Wang, Quanying

2012-11-01

The objective of this study was to investigate the effect of the PR39 recombinant adeno-associated virus (AAV) controlled by the hypoxia-responsive element (HRE) on gene therapy of ischemic heart disease. The minimal HRE was artificially synthesized and the AAV vector controlled by HRE was introduced with NT4-TAT-His-PR39 to investigate the expression of AAV-PR39 in hypoxic vascular endothelial cells (VEC) of human umbilical vein (CRL-1730 cell line) and the angiogenesis-promoting effect in pigs with acute myocardial infraction (AMI). The minimal HRE/CMV was designed and artificially synthesized using the PCR method and cloned with the T vector cloning method. The pSS-HRE-CMV-NT4-6His-PR39-PolyA-AAV plasmid was constructed. Using the calcium phosphate precipitation method, HEK-293 cells were co-transfected with three plasmids to produce the recombinant virus. An equal volume of pSS-HRE-CMV-NT4-6His-PR39-PolyAAAV and enterovirus (EV, blank virus) was transfected into CRL-1730 cell lines, respectively. The immunohistochemical method was used to assay the expression of 6xHis in CRL-1730 cell lines and the expression of PR39 under hypoxia. Eighteen AMI miniature pigs were randomized into the experimental group (HRE-AAV-PR39 group), control group 1 (physical saline group) and control group 2 (EV group). The area of ischemia was assessed with conventional MRI and myocardium perfusion MRI. Pigs were sacrificed at preset time-points to obtain samples of ischemic myocardium. Morphological and pathological data were collected. According to data in the literature and databases, the minimal HRE was designed and synthesized with the PCR method. A large number of HREs were connected to modified pSSHGAAV (pSSV9int-/XbaI) vector followed by insertion of the NT4-6His-PR39 gene segment and, thus, the recombinant plasmid pSS-HRE-CMV-NT4-6His-PR39-PolyA-AAV was successfully constructed. The expression of 6xHis in CRL-1730 cells under the regulation of HRE was assayed using the immunohistochemical method and results showed that the expression was positive in the experimental group. Myocardium perfusion MRI displayed that the infracted area significantly decreased under the action of pSS-HRE-CMV-NT4-PR39-PolyA-AAV. The artificial minimal HRE in CRL-1730 cells effectively and rapidly regulates the expression of the downstream gene NT4-TAT-His-PR39 of the CMV promoter. Recombinant pSS-HRE-CMV-NT4-PR39-Poly-AAAV promotes neoangiogenesis in the ischemic area, reduces the area of infarction and improves heart function.

Exploring the ambiguities of masculinity in accounts of emotional distress in the military among young ex-servicemen.

PubMed

Green, Gill; Emslie, Carol; O'Neill, Dan; Hunt, Kate; Walker, Steven

2010-10-01

This paper examines the experiences and perspectives of ex-military servicemen in the UK. It focuses specifically on the complex links between emotional distress and various constructions of 'masculinity' in a military context. Aspects of military culture that exacerbate vulnerability and also those that are protective to mental health are identified and discussed with reference to the theoretical constructs relating to hegemonic masculinity. A qualitative research design using semi-structured interviews provided in-depth accounts of the experiences of 20 ex-servicemen aged 23-44, all but one of whom were in the Army. We found that in a military setting hegemonic masculinity is embedded in the construction of a soldier identity and expression of emotion may be seen as inappropriate. As a result soldiers often lack a language with which to express distress (in a context in which they may witness extremely distressing events), which may result in delays in recognising and treating mental health problems. However, constructions of masculinity in this setting to some degree also promote a caring, sharing ethos based on strong inter-dependent bonds. A young soldier who can cope with the stresses of military life 'becomes a man', adopts a masculine/soldier identity and is well-placed to benefit from these protective factors, notably the camaraderie that is part of service life. In this manner a caring ethos in which some admissions of weakness may be permissible is situated within hegemonic masculinity. This seeming paradox between hyper masculinity and caring masculinities appears to be embedded within military culture, perhaps reflecting the flexibility and ambiguity inherent in constructions of hegemonic masculinity. Copyright © 2010 Elsevier Ltd. All rights reserved.

The application of domain-driven design in NMS

NASA Astrophysics Data System (ADS)

Zhang, Jinsong; Chen, Yan; Qin, Shengjun

2011-12-01

In the traditional design approach of data-model-driven, system analysis and design phases are often separated which makes the demand information can not be expressed explicitly. The method is also easy to lead developer to the process-oriented programming, making codes between the modules or between hierarchies disordered. So it is hard to meet requirement of system scalability. The paper proposes a software hiberarchy based on rich domain model according to domain-driven design named FHRDM, then the Webwork + Spring + Hibernate (WSH) framework is determined. Domain-driven design aims to construct a domain model which not only meets the demand of the field where the software exists but also meets the need of software development. In this way, problems in Navigational Maritime System (NMS) development like big system business volumes, difficulty of requirement elicitation, high development costs and long development cycle can be resolved successfully.

Construction of heat-inducible expression vector of Corynebacterium glutamicum and C. ammoniagenes: fusion of lambda operator with promoters isolated from C. ammoniagenes.

PubMed

Park, Jong-Uk; Jo, Jae-Hyung; Kim, Young-Ji; Chung, So-Sun; Lee, Jin-Ho; Lee, Hyune Hwan

2008-04-01

The heat-inducible expression vectors for Corynebacterium glutamicum and C. ammoniagenes were constructed by using the lambdaOL1 and the cryptic promoters, CJ1 and CJ4 that express genes constitutively in C. ammoniagenes.. Although the promoters were isolated from C. ammoniagenes, CJ1 and CJ4 were also active in C. glutamicum. To construct vectors, the OL1 from the lambdaPL promoter was isolated and fused to the CJ1 and CJ4 promoters by recombinant PCR. The resulting artificial promoters, CJ1O and CJ4O, which have one lambdaOL1, and CJ1OX2, which has two successive lambdaOL1, were fused to the green fluorescent protein (GFP) gene followed by subcloning into pCES208. The expression of GFP in the corynebacteria harboring the vectors was regulated successfully by the temperature sensitive cI857 repressor. Among them, C. ammoniagenes harboring plasmid pCJ1OX2G containing GFP fused to CJ1OX2 showed more GFP than the other ones and the expression was tightly regulated by the repressor. To construct the generally applicable expression vector using the plasmid pCJ1OX2G, the His-tag, enterokinase (EK) moiety, and the MCS were inserted in front of the GFP gene. Using the vector, the expression of pyrR from C. glutamicum was tried by temperature shift-up. The results indicated that the constructed vectors (pCeHEMG) can be successfully used in the expression and regulation of foreign genes in corynebacteria.

Design optimization of steel frames using an enhanced firefly algorithm

NASA Astrophysics Data System (ADS)

Carbas, Serdar

2016-12-01

Mathematical modelling of real-world-sized steel frames under the Load and Resistance Factor Design-American Institute of Steel Construction (LRFD-AISC) steel design code provisions, where the steel profiles for the members are selected from a table of steel sections, turns out to be a discrete nonlinear programming problem. Finding the optimum design of such design optimization problems using classical optimization techniques is difficult. Metaheuristic algorithms provide an alternative way of solving such problems. The firefly algorithm (FFA) belongs to the swarm intelligence group of metaheuristics. The standard FFA has the drawback of being caught up in local optima in large-sized steel frame design problems. This study attempts to enhance the performance of the FFA by suggesting two new expressions for the attractiveness and randomness parameters of the algorithm. Two real-world-sized design examples are designed by the enhanced FFA and its performance is compared with standard FFA as well as with particle swarm and cuckoo search algorithms.

Quantification of construction waste prevented by BIM-based design validation: Case studies in South Korea.

PubMed

Won, Jongsung; Cheng, Jack C P; Lee, Ghang

2016-03-01

Waste generated in construction and demolition processes comprised around 50% of the solid waste in South Korea in 2013. Many cases show that design validation based on building information modeling (BIM) is an effective means to reduce the amount of construction waste since construction waste is mainly generated due to improper design and unexpected changes in the design and construction phases. However, the amount of construction waste that could be avoided by adopting BIM-based design validation has been unknown. This paper aims to estimate the amount of construction waste prevented by a BIM-based design validation process based on the amount of construction waste that might be generated due to design errors. Two project cases in South Korea were studied in this paper, with 381 and 136 design errors detected, respectively during the BIM-based design validation. Each design error was categorized according to its cause and the likelihood of detection before construction. The case studies show that BIM-based design validation could prevent 4.3-15.2% of construction waste that might have been generated without using BIM. Copyright © 2015 Elsevier Ltd. All rights reserved.

Analysis of C. elegans VIG-1 expression.

PubMed

Shin, Kyoung-Hwa; Choi, Boram; Park, Yang-Seo; Cho, Nam Jeong

2008-12-31

Double-stranded RNA (dsRNA) induces gene silencing in a sequence-specific manner by a process known as RNA interference (RNAi). The RNA-induced silencing complex (RISC) is a multi-subunit ribonucleoprotein complex that plays a key role in RNAi. VIG (Vasa intronic gene) has been identified as a component of Drosophila RISC; however, the role VIG plays in regulating RNAi is poorly understood. Here, we examined the spatial and temporal expression patterns of VIG-1, the C. elegans ortholog of Drosophila VIG, using a vig-1::gfp fusion construct. This construct contains the 908-bp region immediately upstream of vig-1 gene translation initiation site. Analysis by confocal microscopy demonstrated GFP-VIG-1 expression in a number of tissues including the pharynx, body wall muscle, hypodermis, intestine, reproductive system, and nervous system at the larval and adult stages. Furthermore, western blot analysis showed that VIG-1 is present in each developmental stage examined. To investigate regulatory sequences for vig-1 gene expression, we generated constructs containing deletions in the upstream region. It was determined that the GFP expression pattern of a deletion construct (delta-908 to -597) was generally similar to that of the non-deletion construct. In contrast, removal of a larger segment (delta-908 to -191) resulted in the loss of GFP expression in most cell types. Collectively, these results indicate that the 406-bp upstream region (-596 to -191) contains essential regulatory sequences required for VIG-1 expression.

Inducible repression of multiple expansin genes leads to growth suppression during leaf development.

PubMed

Goh, Hoe-Han; Sloan, Jennifer; Dorca-Fornell, Carmen; Fleming, Andrew

2012-08-01

Expansins are cell wall proteins implicated in the control of plant growth via loosening of the extracellular matrix. They are encoded by a large gene family, and data linked to loss of single gene function to support a role of expansins in leaf growth remain limited. Here, we provide a quantitative growth analysis of transgenics containing an inducible artificial microRNA construct designed to down-regulate the expression of a number of expansin genes that an expression analysis indicated are expressed during the development of Arabidopsis (Arabidopsis thaliana) leaf 6. The results support the hypothesis that expansins are required for leaf growth and show that decreased expansin gene expression leads to a more marked repression of growth during the later stage of leaf development. In addition, a histological analysis of leaves in which expansin gene expression was suppressed indicates that, despite smaller leaves, mean cell size was increased. These data provide functional evidence for a role of expansins in leaf growth, indicate the importance of tissue/organ developmental context for the outcome of altered expansin gene expression, and highlight the separation of the outcome of expansin gene expression at the cellular and organ levels.

76 FR 70807 - Notice of Passenger Facility Charge (PFC) Approvals and Disapprovals

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-15

... (design and construction). Enplane road structural improvements (design and construction). Landside signage improvements (design and construction). Taxiway B-2 extension and taxiway B-1 rehabilitation (design and construction). Elevator and escalator safety code compliance improvements (design and...

[Prokaryotic expression, purification and antigenicity identification of recombinant human survivin protein].

PubMed

Yin, Xiaotao; Wang, Wei; Tian, Renli; Xu, Yuanji; Yan, Jinqi; Zhang, Wei; Gao, Jiangping; Yu, Jiyun

2013-08-01

To construct a prokaryotic expression plasmid pET28a-survivin, optimize the recombinant protein expression conditions in E.coli, and purify the survivin recombinant protein and identify its antigenicity. Survivin cDNA segment was amplified by PCR and cloned into prokaryotic expression vector pET28a(+) to construct the recombinant expression vector pET28a-survivin. The expression vector was transformed into BL21 (DE3) and the fusion protein survivin/His was induced by IPTG. The fusion protein was purified through Ni affinity chromatography. The antigenicity of the purified survivin protein was identified by Western blotting and ELISA. The recombinant expression vector was verified successfully by BamHI and HindIII. The fusion protein induced by IPTG was obtained with Mr; about 24 000. The purity of the purified protein reached 90% by SDS-PAGE analysis. And the antigenicity of the survivin protein was validated by Western blotting and ELISA. The prokaryotic expression plasmid pET28a-survivin was successfully constructed and the survivin protein was expressed and purified in E.coli. The antigenicity of the purified survivin protein was demonstrated desirable.

Creating homogenous strain distribution within 3D cell-encapsulated constructs using a simple and cost-effective uniaxial tensile bioreactor: Design and validation study.

PubMed

Subramanian, Gayathri; Elsaadany, Mostafa; Bialorucki, Callan; Yildirim-Ayan, Eda

2017-08-01

Mechanical loading bioreactors capable of applying uniaxial tensile strains are emerging to be a valuable tool to investigate physiologically relevant cellular signaling pathways and biochemical expression. In this study, we have introduced a simple and cost-effective uniaxial tensile strain bioreactor for the application of precise and homogenous uniaxial strains to 3D cell-encapsulated collagen constructs at physiological loading strains (0-12%) and frequencies (0.01-1 Hz). The bioreactor employs silicone-based loading chambers specifically designed to stretch constructs without direct gripping to minimize stress concentration at the ends of the construct and preserve its integrity. The loading chambers are driven by a versatile stepper motor ball-screw actuation system to produce stretching of the constructs. Mechanical characterization of the bioreactor performed through Finite Element Analysis demonstrated that the constructs experienced predominantly uniaxial tensile strain in the longitudinal direction. The strains produced were found to be homogenous over a 15 × 4 × 2 mm region of the construct equivalent to around 60% of the effective region of characterization. The strain values were also shown to be consistent and reproducible during cyclic loading regimes. Biological characterization confirmed the ability of the bioreactor to promote cell viability, proliferation, and matrix organization of cell-encapsulated collagen constructs. This easy-to-use uniaxial tensile strain bioreactor can be employed for studying morphological, structural, and functional responses of cell-embedded matrix systems in response to physiological loading of musculoskeletal tissues. It also holds promise for tissue-engineered strategies that involve delivery of mechanically stimulated cells at the site of injury through a biological carrier to develop a clinically useful therapy for tissue healing. Biotechnol. Bioeng. 2017;114: 1878-1887. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Production of red-flowered plants by genetic engineering of multiple flavonoid biosynthetic genes.

PubMed

Nakatsuka, Takashi; Abe, Yoshiko; Kakizaki, Yuko; Yamamura, Saburo; Nishihara, Masahiro

2007-11-01

Orange- to red-colored flowers are difficult to produce by conventional breeding techniques in some floricultural plants. This is due to the deficiency in the formation of pelargonidin, which confers orange to red colors, in their flowers. Previous researchers have reported that brick-red colored flowers can be produced by introducing a foreign dihydroflavonol 4-reductase (DFR) with different substrate specificity in Petunia hybrida, which does not accumulate pelargonidin pigments naturally. However, because these experiments used dihydrokaempferol (DHK)-accumulated mutants as transformation hosts, this strategy cannot be applied directly to other floricultural plants. Thus in this study, we attempted to produce red-flowered plants by suppressing two endogenous genes and expressing one foreign gene using tobacco as a model plant. We used a chimeric RNAi construct for suppression of two genes (flavonol synthase [FLS] and flavonoid 3'-hydroxylase [F3'H]) and expression of the gerbera DFR gene in order to accumulate pelargonidin pigments in tobacco flowers. We successfully produced red-flowered tobacco plants containing high amounts of additional pelargonidin as confirmed by HPLC analysis. The flavonol content was reduced in the transgenic plants as expected, although complete inhibition was not achieved. Expression analysis also showed that reduction of the two-targeted genes and expression of the foreign gene occurred simultaneously. These results demonstrate that flower color modification can be achieved by multiple gene regulation without use of mutants if the vector constructs are designed resourcefully.

Expression of an Mg2+-Dependent HIV-1 RNase H Construct for Drug Screening▿†

PubMed Central

Farias, Richard V.; Vargas, Deborah A.; Castillo, Andres E.; Valenzuela, Beatriz; Coté, Marie L.; Roth, Monica J.; Leon, Oscar

2011-01-01

A single polypeptide of the HIV-1 reverse transcriptase that reconstituted Mg2+-dependent RNase H activity has been made. Using molecular modeling, the construct was designed to encode the p51 subunit joined by a linker to the thumb (T), connection (C), and RNase H (R) domains of p66. This p51-G-TCR construct was purified from the soluble fraction of an Escherichia coli strain, MIC2067(DE3), lacking endogenous RNase HI and HII. The p51-G-TCR RNase H construct displayed Mg2+-dependent activity using a fluorescent nonspecific assay and showed the same cleavage pattern as HIV-1 reverse transcriptase (RT) on substrates that mimic the tRNA removal required for second-strand transfer reactions. The mutant E706Q (E478Q in RT) was purified under similar conditions and was not active. The RNase H of the p51-G-TCR RNase H construct and wild type HIV-1 RT had similar Kms for an RNA-DNA hybrid substrate and showed similar inhibition kinetics to two known inhibitors of the HIV-1 RT RNase H. PMID:21768506

Monitoring the Error Rate of Modern Methods of Construction Based on Wood

NASA Astrophysics Data System (ADS)

Švajlenka, Jozef; Kozlovská, Mária

2017-06-01

A range of new and innovative construction systems, currently developed, represent modern methods of construction (MMC), which has the ambition to improve the performance parameters of buildings throughout their life cycle. Regarding the implementation modern methods of construction in Slovakia, assembled buildings based on wood seem to be the most preferred construction system. In the study, presented in the paper, were searched already built and lived-in wood based family houses. The residents' attitudes to such type of buildings in the context with declared designing and qualitative parameters of efficiency and sustainability are overlooked. The methodology of the research study is based on the socio-economic survey carried out during the years 2015 - 2017 within the Slovak Republic. Due to the large extent of data collected through questionnaire, only selected parts of the survey results are evaluated and discussed in the paper. This paper is aimed at evaluating the quality of buildings expressed in a view of users of existing wooden buildings. Research indicates some defects, which can be eliminated in the next production process. Research indicates, that some defects occur, so the production process quality should be improved in the future development.

Online Analytical Processing (OLAP): A Fast and Effective Data Mining Tool for Gene Expression Databases

PubMed Central

2005-01-01

Gene expression databases contain a wealth of information, but current data mining tools are limited in their speed and effectiveness in extracting meaningful biological knowledge from them. Online analytical processing (OLAP) can be used as a supplement to cluster analysis for fast and effective data mining of gene expression databases. We used Analysis Services 2000, a product that ships with SQLServer2000, to construct an OLAP cube that was used to mine a time series experiment designed to identify genes associated with resistance of soybean to the soybean cyst nematode, a devastating pest of soybean. The data for these experiments is stored in the soybean genomics and microarray database (SGMD). A number of candidate resistance genes and pathways were found. Compared to traditional cluster analysis of gene expression data, OLAP was more effective and faster in finding biologically meaningful information. OLAP is available from a number of vendors and can work with any relational database management system through OLE DB. PMID:16046824

Quantitative characterization of genetic parts and circuits for plant synthetic biology.

PubMed

Schaumberg, Katherine A; Antunes, Mauricio S; Kassaw, Tessema K; Xu, Wenlong; Zalewski, Christopher S; Medford, June I; Prasad, Ashok

2016-01-01

Plant synthetic biology promises immense technological benefits, including the potential development of a sustainable bio-based economy through the predictive design of synthetic gene circuits. Such circuits are built from quantitatively characterized genetic parts; however, this characterization is a significant obstacle in work with plants because of the time required for stable transformation. We describe a method for rapid quantitative characterization of genetic plant parts using transient expression in protoplasts and dual luciferase outputs. We observed experimental variability in transient-expression assays and developed a mathematical model to describe, as well as statistical normalization methods to account for, this variability, which allowed us to extract quantitative parameters. We characterized >120 synthetic parts in Arabidopsis and validated our method by comparing transient expression with expression in stably transformed plants. We also tested >100 synthetic parts in sorghum (Sorghum bicolor) protoplasts, and the results showed that our method works in diverse plant groups. Our approach enables the construction of tunable gene circuits in complex eukaryotic organisms.

Uses for lunar crawler transporters

NASA Astrophysics Data System (ADS)

Kaden, Richard A.

This article discusses state-of-the-art crawler transporters and expresses the need for additional research and development for lunar crawlers. The thrust of the paper illustrates how the basic crawler technology has progressed to a point where extremely large modules can be shop fabricated and move to some distant location at a considerable savings. Also, extremely heavy loads may be lifted by large crawler cranes and placed in designed locations. The Transi-Lift Crawler crane with its traveling counterweight is an attractive concept for lunar construction.

The experimental study of genetic engineering human neural stem cells mediated by lentivirus to express multigene.

PubMed

Cai, Pei-qiang; Tang, Xun; Lin, Yue-qiu; Martin, Oudega; Sun, Guang-yun; Xu, Lin; Yang, Yun-kang; Zhou, Tian-hua

2006-02-01

To explore the feasibility to construct genetic engineering human neural stem cells (hNSCs) mediated by lentivirus to express multigene in order to provide a graft source for further studies of spinal cord injury (SCI). Human neural stem cells from the brain cortex of human abortus were isolated and cultured, then gene was modified by lentivirus to express both green fluorescence protein (GFP) and rat neurotrophin-3 (NT-3); the transgenic expression was detected by the methods of fluorescence microscope, dorsal root ganglion of fetal rats and slot blot. Genetic engineering hNSCs were successfully constructed. All of the genetic engineering hNSCs which expressed bright green fluorescence were observed under the fluorescence microscope. The conditioned medium of transgenic hNSCs could induce neurite flourishing outgrowth from dorsal root ganglion (DRG). The genetic engineering hNSCs expressed high level NT-3 which could be detected by using slot blot. Genetic engineering hNSCs mediated by lentivirus can be constructed to express multigene successfully.

Synthetic Biology Toolbox for Controlling Gene Expression in the Cyanobacterium Synechococcus sp. strain PCC 7002

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

Markley, Andrew L.; Begemann, Matthew B.; Clarke, Ryan E.

The application of synthetic biology requires characterized tools to precisely control gene expression. This toolbox of genetic parts previously did not exist for the industrially promising cyanobacterium, Synechococcus sp. strain PCC 7002. To address this gap, two orthogonal constitutive promoter libraries, one based on a cyanobacterial promoter and the other ported from Escherichia coli, were built and tested in PCC 7002. The libraries demonstrated 3 and 2.5 log dynamic ranges, respectively, but correlated poorly with E. coli expression levels. These promoter libraries were then combined to create and optimize a series of IPTG inducible cassettes. The resultant induction system hadmore » a 48-fold dynamic range and was shown to out-perform P trc constructs. Finally, a RBS library was designed and tested in PCC 7002. The presented synthetic biology toolbox will enable accelerated engineering of PCC 7002.« less

Synthetic Biology Toolbox for Controlling Gene Expression in the Cyanobacterium Synechococcus sp. strain PCC 7002

DOE PAGES

Markley, Andrew L.; Begemann, Matthew B.; Clarke, Ryan E.; ...

2014-09-12

The application of synthetic biology requires characterized tools to precisely control gene expression. This toolbox of genetic parts previously did not exist for the industrially promising cyanobacterium, Synechococcus sp. strain PCC 7002. To address this gap, two orthogonal constitutive promoter libraries, one based on a cyanobacterial promoter and the other ported from Escherichia coli, were built and tested in PCC 7002. The libraries demonstrated 3 and 2.5 log dynamic ranges, respectively, but correlated poorly with E. coli expression levels. These promoter libraries were then combined to create and optimize a series of IPTG inducible cassettes. The resultant induction system hadmore » a 48-fold dynamic range and was shown to out-perform P trc constructs. Finally, a RBS library was designed and tested in PCC 7002. The presented synthetic biology toolbox will enable accelerated engineering of PCC 7002.« less

Is synthetic biology mechanical biology?

PubMed

Holm, Sune

2015-12-01

A widespread and influential characterization of synthetic biology emphasizes that synthetic biology is the application of engineering principles to living systems. Furthermore, there is a strong tendency to express the engineering approach to organisms in terms of what seems to be an ontological claim: organisms are machines. In the paper I investigate the ontological and heuristic significance of the machine analogy in synthetic biology. I argue that the use of the machine analogy and the aim of producing rationally designed organisms does not necessarily imply a commitment to mechanical biology. The ideal of applying engineering principles to biology is best understood as expressing recognition of the machine-unlikeness of natural organisms and the limits of human cognition. The paper suggests an interpretation of the identification of organisms with machines in synthetic biology according to which it expresses a strategy for representing, understanding, and constructing living systems that are more machine-like than natural organisms.

Semantic technologies in a decision support system

NASA Astrophysics Data System (ADS)

Wasielewska, K.; Ganzha, M.; Paprzycki, M.; Bǎdicǎ, C.; Ivanovic, M.; Lirkov, I.

2015-10-01

The aim of our work is to design a decision support system based on ontological representation of domain(s) and semantic technologies. Specifically, we consider the case when Grid / Cloud user describes his/her requirements regarding a "resource" as a class expression from an ontology, while the instances of (the same) ontology represent available resources. The goal is to help the user to find the best option with respect to his/her requirements, while remembering that user's knowledge may be "limited." In this context, we discuss multiple approaches based on semantic data processing, which involve different "forms" of user interaction with the system. Specifically, we consider: (a) ontological matchmaking based on SPARQL queries and class expression, (b) graph-based semantic closeness of instances representing user requirements (constructed from the class expression) and available resources, and (c) multicriterial analysis based on the AHP method, which utilizes expert domain knowledge (also ontologically represented).

In Planta Synthesis of Designer-Length Tobacco Mosaic Virus-Based Nano-Rods That Can Be Used to Fabricate Nano-Wires.

PubMed

Saunders, Keith; Lomonossoff, George P

2017-01-01

We have utilized plant-based transient expression to produce tobacco mosaic virus (TMV)-based nano-rods of predetermined lengths. This is achieved by expressing RNAs containing the TMV origin of assembly sequence (OAS) and the sequence of the TMV coat protein either on the same RNA molecule or on two separate constructs. We show that the length of the resulting nano-rods is dependent upon the length of the RNA that possesses the OAS element. By expressing a version of the TMV coat protein that incorporates a metal-binding peptide at its C-terminus in the presence of RNA containing the OAS we have been able to produce nano-rods of predetermined length that are coated with cobalt-platinum. These nano-rods have the properties of defined-length nano-wires that make them ideal for many developing bionanotechnological processes.

In Planta Synthesis of Designer-Length Tobacco Mosaic Virus-Based Nano-Rods That Can Be Used to Fabricate Nano-Wires

PubMed Central

Saunders, Keith; Lomonossoff, George P.

2017-01-01

We have utilized plant-based transient expression to produce tobacco mosaic virus (TMV)-based nano-rods of predetermined lengths. This is achieved by expressing RNAs containing the TMV origin of assembly sequence (OAS) and the sequence of the TMV coat protein either on the same RNA molecule or on two separate constructs. We show that the length of the resulting nano-rods is dependent upon the length of the RNA that possesses the OAS element. By expressing a version of the TMV coat protein that incorporates a metal-binding peptide at its C-terminus in the presence of RNA containing the OAS we have been able to produce nano-rods of predetermined length that are coated with cobalt-platinum. These nano-rods have the properties of defined-length nano-wires that make them ideal for many developing bionanotechnological processes. PMID:28878782

Reprint of "versatile and stable vectors for efficient gene expression in Ralstonia eutropha H16".

PubMed

Gruber, Steffen; Hagen, Jeremias; Schwab, Helmut; Koefinger, Petra

2014-12-20

The Gram-negative β-proteobacterium Ralstonia eutropha H16 is primarily known for polyhydroxybutyrate (PHB) production and its ability to grow chemolithoautotrophically by using CO2 and H2 as sole carbon and energy sources. The majority of metabolic engineering and heterologous expression studies conducted so far rely on a small number of suitable expression systems. Particularly the plasmid based expression systems already developed for the use in R. eutropha H16 suffer from high segregational instability and plasmid loss after a short time of fermentation. In order to develop efficient and highly stable plasmid expression vectors for the use in R. eutropha H16, a new plasmid design was created including the RP4 partitioning system, as well as various promoters and origins of replication. The application of minireplicons derived from broad-host-range plasmids RSF1010, pBBR1, RP4 and pSa for the construction of expression vectors and the use of numerous, versatile promoters extend the range of feasible expression levels considerably. In particular, the use of promoters derived from the bacteriophage T5 was described for the first time in this work, characterizing the j5 promoter as the strongest promoter yet to be applied in R. eutropha H16. Moreover, the implementation of the RP4 partition sequence in plasmid design increased plasmid stability significantly and enables fermentations with marginal plasmid loss of recombinant R. eutropha H16 for at least 96h. The utility of the new vector family in R. eutropha H16 is demonstrated by providing expression data with different model proteins and consequently further raises the value of this organism as cell factory for biotechnological applications including protein and metabolite production. Copyright © 2014 Elsevier B.V. All rights reserved.

Versatile and stable vectors for efficient gene expression in Ralstonia eutropha H16.

PubMed

Gruber, Steffen; Hagen, Jeremias; Schwab, Helmut; Koefinger, Petra

2014-09-30

The Gram-negative β-proteobacterium Ralstonia eutropha H16 is primarily known for polyhydroxybutyrate (PHB) production and its ability to grow chemolithoautotrophically by using CO2 and H2 as sole carbon and energy sources. The majority of metabolic engineering and heterologous expression studies conducted so far rely on a small number of suitable expression systems. Particularly the plasmid based expression systems already developed for the use in R. eutropha H16 suffer from high segregational instability and plasmid loss after a short time of fermentation. In order to develop efficient and highly stable plasmid expression vectors for the use in R. eutropha H16, a new plasmid design was created including the RP4 partitioning system, as well as various promoters and origins of replication. The application of minireplicons derived from broad-host-range plasmids RSF1010, pBBR1, RP4 and pSa for the construction of expression vectors and the use of numerous, versatile promoters extend the range of feasible expression levels considerably. In particular, the use of promoters derived from the bacteriophage T5 was described for the first time in this work, characterizing the j5 promoter as the strongest promoter yet to be applied in R. eutropha H16. Moreover, the implementation of the RP4 partition sequence in plasmid design increased plasmid stability significantly and enables fermentations with marginal plasmid loss of recombinant R. eutropha H16 for at least 96 h. The utility of the new vector family in R. eutropha H16 is demonstrated by providing expression data with different model proteins and consequently further raises the value of this organism as cell factory for biotechnological applications including protein and metabolite production. Copyright © 2014 Elsevier B.V. All rights reserved.

Development of a Cyclic Strain Bioreactor for Mechanical Enhancement and Assessment of Bioengineered Myocardial Constructs

PubMed Central

Salazar, Betsy H.; Cashion, Avery T.; Dennis, Robert G.; Birla, Ravi K.

2015-01-01

Purpose The purpose of this study was to develop enabling bioreactor technologies using a novel voice coil actuator system for investigating the effects of periodic strain on cardiac patches fabricated with rat cardiomyocytes. Methods The bioengineered muscle constructs used in this study were formed by culturing rat neonatal primary cardiac cells on a fibrin gel. The physical design of the bioreactor was initially conceived using Solidworks to test clearances and perform structural strain analysis. Once the software design phase was completed the bioreactor was assembled using a combination of commercially available, custom machined, and 3-D printed parts. We utilized the bioreactor to evaluate the effect of a 4-hour stretch protocol on the contractile properties of the tissue after which immunohistological assessment of the tissue was also performed. Results An increase in contractile force was observed after the strain protocol of 10% stretch at 1Hz, with no significant increase observed in the control group. Additionally, an increase in cardiac myofibril alignment, connexin 43 expression, and collagen type I distribution were noted. Conclusion In this study we demonstrated the effectiveness of a new bioreactor design to improve contractility of engineered cardiac muscle tissue. PMID:26577484

Development of a Cyclic Strain Bioreactor for Mechanical Enhancement and Assessment of Bioengineered Myocardial Constructs.

PubMed

Salazar, Betsy H; Cashion, Avery T; Dennis, Robert G; Birla, Ravi K

2015-12-01

The purpose of this study was to develop enabling bioreactor technologies using a novel voice coil actuator system for investigating the effects of periodic strain on cardiac patches fabricated with rat cardiomyocytes. The bioengineered muscle constructs used in this study were formed by culturing rat neonatal primary cardiac cells on a fibrin gel. The physical design of the bioreactor was initially conceived using Solidworks to test clearances and perform structural strain analysis. Once the software design phase was completed the bioreactor was assembled using a combination of commercially available, custom machined, and 3-D printed parts. We utilized the bioreactor to evaluate the effect of a 4-h stretch protocol on the contractile properties of the tissue after which immunohistological assessment of the tissue was also performed. An increase in contractile force was observed after the strain protocol of 10% stretch at 1 Hz, with no significant increase observed in the control group. Additionally, an increase in cardiac myofibril alignment, connexin 43 expression, and collagen type I distribution were noted. In this study we demonstrated the effectiveness of a new bioreactor design to improve contractility of engineered cardiac muscle tissue.

A domain-specific design architecture for composite material design and aircraft part redesign

NASA Technical Reports Server (NTRS)

Punch, W. F., III; Keller, K. J.; Bond, W.; Sticklen, J.

1992-01-01

Advanced composites have been targeted as a 'leapfrog' technology that would provide a unique global competitive position for U.S. industry. Composites are unique in the requirements for an integrated approach to designing, manufacturing, and marketing of products developed utilizing the new materials of construction. Numerous studies extending across the entire economic spectrum of the United States from aerospace to military to durable goods have identified composites as a 'key' technology. In general there have been two approaches to composite construction: build models of a given composite materials, then determine characteristics of the material via numerical simulation and empirical testing; and experience-directed construction of fabrication plans for building composites with given properties. The first route sets a goal to capture basic understanding of a device (the composite) by use of a rigorous mathematical model; the second attempts to capture the expertise about the process of fabricating a composite (to date) at a surface level typically expressed in a rule based system. From an AI perspective, these two research lines are attacking distinctly different problems, and both tracks have current limitations. The mathematical modeling approach has yielded a wealth of data but a large number of simplifying assumptions are needed to make numerical simulation tractable. Likewise, although surface level expertise about how to build a particular composite may yield important results, recent trends in the KBS area are towards augmenting surface level problem solving with deeper level knowledge. Many of the relative advantages of composites, e.g., the strength:weight ratio, is most prominent when the entire component is designed as a unitary piece. The bottleneck in undertaking such unitary design lies in the difficulty of the re-design task. Designing the fabrication protocols for a complex-shaped, thick section composite are currently very difficult. It is in fact this difficulty that our research will address.

[Clone, construct, expression and verification of lactoferricin B gene and several sequence mutations in yeast].

PubMed

Feng, Yong-qian; Zha, Xiao-jun; Zhai, Chao-yang

2007-07-01

To construct the eucaryotic recombinant plasmid of pYES2/LactoferricinB expressing in yeast of S. cerevisiae, of which the expressed protein antibacterial activity was verified in preliminary. By self-template PCR method, the gene of Lactoferricin B and its several sequence mutations were amplified with the parts of the pre-synthesized single chains. And then Lactoferricin B gene and its mutants were cloned into the vector of pYES2 to construct the recombined expression plasmid pYES2/Lactoferricin B etc. extracted and used to transform the yeast S. cerevisiae. The expressions of proteins were determined after induced by galactose. The expression proteins were collected and purified by hydronium-exchange column, and the bacterial inhibited test was applied to identify the protein antibacterial activities. The PCR amplifying and DNA sequencing tests indicated that the purpose plasmid contained the Lactoferricin B gene and several mutations. The induced target proteins were confirmed by SDS-PAGE electrophoresis and mass spectrum test. The protein antibacterial activities of mutations were verified in preliminary. The recombined plasmid pYES2/Lactoferricin B etc. are successfully constructed and induced to express in yeast cell of S. cerevisiae; the obtained recombined protein of Lactoferricin B provides a basis for further research work on the biological function and antibacterial activity.

[Prokaryotic expression and immunological activity of human neutrophil gelatinase associated lipocalin].

PubMed

Wu, Jianwei; Cai, Lei; Qian, Wei; Jiao, Liyuan; Li, Jiangfeng; Song, Xiaoli; Wang, Jihua

2015-07-01

To construct a prokaryotic expression vector of human neutrophil gelatinase associated lipocalin (NGAL) and identify the bioactivity of the fusion protein. The cDNA of human NGAL obtained from GenBank was linked to a cloning vector to construct the prokaryotic expression vector pCold-NGAL. Then the vector was transformed into E.coli BL21(DE3) plysS. Under the optimal induction condition, the recombinant NGAL (rNGAL) was expressed and purified by Ni Sepharose 6 Fast Flow affinity chromatography. The purity and activity of the rNGAL were respectively identified by SDS-PAGE and Western blotting combined with NGAL reagent (Latex enhanced immunoturbidimetry). Restriction enzyme digestion and nucleotide sequencing proved that the expression vector pCold-NGAL was successfully constructed. Under the optimal induction condition that we determined, the rNGAL was expressed in soluble form in E.coli BL21(DE3) plysS. The relative molecular mass of the rNGAL was 25 000, and its purity was more than 98.0%. Furthermore, Western blotting and immunoturbidimetry indicated that the rNGAL reacted with NGAL mAb specifically. Human rNGAL of high purity and bioactivity was successfully constructed in E.coli BL21(DE3) plysS using the expression vector pCold-NGAL.

Generation of a Tet-On Expression System to Study Transactivation Ability of Tax-2.

PubMed

Bignami, Fabio; Sozzi, Riccardo Alessio; Pilotti, Elisabetta

2017-01-01

HTLV Tax proteins (Tax-1 and Tax-2) are known to be able to transactivate several host cellular genes involved in complex molecular pathways. Here, we describe a stable and regulated high-level expression model based on Tet-On system, to study the capacity of Tax-2 to transactivate host genes. In particular, the Jurkat Tet-On cell line suitable for evaluating the ability of Tax-2 to stimulate transactivation of a specific host gene, CCL3L1 (C-C motif chemokine ligand 3 like 1 gene), was selected. Then, a plasmid expressing tax-2 gene under control of a tetracycline-response element was constructed. To avoid the production of a fusion protein between the report gene and the inserted gene, a bidirectional plasmid was designed. Maximum expression and fast response time were achieved by using nucleofection technology as transfection method. After developing an optimized protocol for efficiently transferring tax-2 gene in Jurkat Tet-On cellular model and exposing transfected cells to Dox (doxycycline, a tetracycline derivate), a kinetics of tax-2 expression through TaqMan Real-time PCR assay was determined.

Constructing Media Artifacts in a Social Constructivist Environment to Enhance Students' Environmental Awareness and Activism

NASA Astrophysics Data System (ADS)

Karahan, Engin; Roehrig, Gillian

2015-02-01

Current science education reforms and policy documents highlight the importance of environmental awareness and perceived need for activism. As "environmental problems are socially constructed in terms of their conceptualized effects on individuals, groups, other living things and systems research based on constructivist principles provides not only a coherent framework in which to theorize about learning, but also a context for understanding socially constructed issues" (Palmer and Suggate in Res Pap Educ 19(2), 2004, p. 208). This research study investigated the impacts of the learning processes structured based on the theories of constructionism and social constructivism on students' environmental awareness and perceived need for activism. Students constructed multimedia artifacts expressing their knowledge, attitudes, awareness, and activism about environmental issues through a constructionist design process. In addition, a social networking site was designed and used to promote social interaction among students. Twenty-two high school environmental science students participated in this study. A convergent mixed methods design was implemented to allow for the triangulation of methods by directly comparing and contrasting quantitative results with qualitative findings for corroboration and validation purposes. Using a mixed method approach, quantitative findings are supported with qualitative data (student video projects, writing prompts, blog entries, video projects of the students, observational field notes, and reflective journals) including spontaneous responses in both synchronous and asynchronous conversations on the social network to provide a better understanding of the change in students' environmental awareness and perceived need for activism. The findings of the study indicated that students' environmental awareness and perceived need for activism were improved at different scales (personal, community, global) throughout the constructionist and social constructivist learning processes.

Construction of siRNA/miRNA expression vectors based on a one-step PCR process

PubMed Central

Xu, Jun; Zeng, Jie Qiong; Wan, Gang; Hu, Gui Bin; Yan, Hong; Ma, Li Xin

2009-01-01

Background RNA interference (RNAi) has become a powerful means for silencing target gene expression in mammalian cells and is envisioned to be useful in therapeutic approaches to human disease. In recent years, high-throughput, genome-wide screening of siRNA/miRNA libraries has emerged as a desirable approach. Current methods for constructing siRNA/miRNA expression vectors require the synthesis of long oligonucleotides, which is costly and suffers from mutation problems. Results Here we report an ingenious method to solve traditional problems associated with construction of siRNA/miRNA expression vectors. We synthesized shorter primers (< 50 nucleotides) to generate a linear expression structure by PCR. The PCR products were directly transformed into chemically competent E. coli and converted to functional vectors in vivo via homologous recombination. The positive clones could be easily screened under UV light. Using this method we successfully constructed over 500 functional siRNA/miRNA expression vectors. Sequencing of the vectors confirmed a high accuracy rate. Conclusion This novel, convenient, low-cost and highly efficient approach may be useful for high-throughput assays of RNAi libraries. PMID:19490634

Synthetic Gene Expression Circuits for Designing Precision Tools in Oncology

PubMed Central

Re, Angela

2017-01-01

Precision medicine in oncology needs to enhance its capabilities to match diagnostic and therapeutic technologies to individual patients. Synthetic biology streamlines the design and construction of functionalized devices through standardization and rational engineering of basic biological elements decoupled from their natural context. Remarkable improvements have opened the prospects for the availability of synthetic devices of enhanced mechanism clarity, robustness, sensitivity, as well as scalability and portability, which might bring new capabilities in precision cancer medicine implementations. In this review, we begin by presenting a brief overview of some of the major advances in the engineering of synthetic genetic circuits aimed to the control of gene expression and operating at the transcriptional, post-transcriptional/translational, and post-translational levels. We then focus on engineering synthetic circuits as an enabling methodology for the successful establishment of precision technologies in oncology. We describe significant advancements in our capabilities to tailor synthetic genetic circuits to specific applications in tumor diagnosis, tumor cell- and gene-based therapy, and drug delivery. PMID:28894736

Type 3 fimbriae and biofilm formation are regulated by the transcriptional regulators MrkHI in Klebsiella pneumoniae.

PubMed

Johnson, Jeremiah G; Murphy, Caitlin N; Sippy, Jean; Johnson, Tylor J; Clegg, Steven

2011-07-01

Klebsiella pneumoniae is an opportunistic pathogen which frequently causes hospital-acquired urinary and respiratory tract infections. K. pneumoniae may establish these infections in vivo following adherence, using the type 3 fimbriae, to indwelling devices coated with extracellular matrix components. Using a colony immunoblot screen, we identified transposon insertion mutants which were deficient for type 3 fimbrial surface production. One of these mutants possessed a transposon insertion within a gene, designated mrkI, encoding a putative transcriptional regulator. A site-directed mutant of this gene was constructed and shown to be deficient for fimbrial surface expression under aerobic conditions. MrkI mutants have a significantly decreased ability to form biofilms on both abiotic and extracellular matrix-coated surfaces. This gene was found to be cotranscribed with a gene predicted to encode a PilZ domain-containing protein, designated MrkH. This protein was found to bind cyclic-di-GMP (c-di-GMP) and regulate type 3 fimbrial expression.

Type 3 Fimbriae and Biofilm Formation Are Regulated by the Transcriptional Regulators MrkHI in Klebsiella pneumoniae▿

PubMed Central

Johnson, Jeremiah G.; Murphy, Caitlin N.; Sippy, Jean; Johnson, Tylor J.; Clegg, Steven

2011-01-01

Klebsiella pneumoniae is an opportunistic pathogen which frequently causes hospital-acquired urinary and respiratory tract infections. K. pneumoniae may establish these infections in vivo following adherence, using the type 3 fimbriae, to indwelling devices coated with extracellular matrix components. Using a colony immunoblot screen, we identified transposon insertion mutants which were deficient for type 3 fimbrial surface production. One of these mutants possessed a transposon insertion within a gene, designated mrkI, encoding a putative transcriptional regulator. A site-directed mutant of this gene was constructed and shown to be deficient for fimbrial surface expression under aerobic conditions. MrkI mutants have a significantly decreased ability to form biofilms on both abiotic and extracellular matrix-coated surfaces. This gene was found to be cotranscribed with a gene predicted to encode a PilZ domain-containing protein, designated MrkH. This protein was found to bind cyclic-di-GMP (c-di-GMP) and regulate type 3 fimbrial expression. PMID:21571997

A controlled double-duration inducible gene expression system for cartilage tissue engineering.

PubMed

Ma, Ying; Li, Junxiang; Yao, Yi; Wei, Daixu; Wang, Rui; Wu, Qiong

2016-05-25

Cartilage engineering that combines competent seeding cells and a compatible scaffold is increasingly gaining popularity and is potentially useful for the treatment of various bone and cartilage diseases. Intensive efforts have been made by researchers to improve the viability and functionality of seeding cells of engineered constructs that are implanted into damaged cartilage. Here, we designed an integrative system combining gene engineering and the controlled-release concept to solve the problems of both seeding cell viability and functionality through precisely regulating the anti-apoptotic gene bcl-2 in the short-term and the chondrogenic master regulator Sox9 in the long-term. Both in vitro and in vivo experiments demonstrated that our system enhances the cell viability and chondrogenic effects of the engineered scaffold after introduction of the system while restricting anti-apoptotic gene expression to only the early stage, thereby preventing potential oncogenic and overdose effects. Our system was designed to be modular and can also be readily adapted to other tissue engineering applications with minor modification.

Development of a Prediction Model Based on RBF Neural Network for Sheet Metal Fixture Locating Layout Design and Optimization.

PubMed

Wang, Zhongqi; Yang, Bo; Kang, Yonggang; Yang, Yuan

2016-01-01

Fixture plays an important part in constraining excessive sheet metal part deformation at machining, assembly, and measuring stages during the whole manufacturing process. However, it is still a difficult and nontrivial task to design and optimize sheet metal fixture locating layout at present because there is always no direct and explicit expression describing sheet metal fixture locating layout and responding deformation. To that end, an RBF neural network prediction model is proposed in this paper to assist design and optimization of sheet metal fixture locating layout. The RBF neural network model is constructed by training data set selected by uniform sampling and finite element simulation analysis. Finally, a case study is conducted to verify the proposed method.

Development of a Prediction Model Based on RBF Neural Network for Sheet Metal Fixture Locating Layout Design and Optimization

PubMed Central

Wang, Zhongqi; Yang, Bo; Kang, Yonggang; Yang, Yuan

2016-01-01

Fixture plays an important part in constraining excessive sheet metal part deformation at machining, assembly, and measuring stages during the whole manufacturing process. However, it is still a difficult and nontrivial task to design and optimize sheet metal fixture locating layout at present because there is always no direct and explicit expression describing sheet metal fixture locating layout and responding deformation. To that end, an RBF neural network prediction model is proposed in this paper to assist design and optimization of sheet metal fixture locating layout. The RBF neural network model is constructed by training data set selected by uniform sampling and finite element simulation analysis. Finally, a case study is conducted to verify the proposed method. PMID:27127499

[Construction and expression of a eukaryotic expression vector containing human CR2-Fc fusion protein].

PubMed

Li, Xinxin; Wu, Zhihao; Zhang, Chuanfu; Jia, Leili; Song, Hongbin; Xu, Yuanyong

2014-01-01

To construct a eukaryotic expression vector containing human complement receptor 2 (CR2)-Fc and express the CR2-Fc fusion protein in Chinese hamster ovary (CHO) cells. The extracellular domain of human CR2 and IgG1 Fc were respectively amplified, ligated and inserted into the eukaryotic expression vector PCI-neo. After verified by restriction enzyme digestion and sequencing, the recombinant plasmid was transfected into CHO K1 cells. The ones with stable expression of the fusion protein were obtained by means of G418 selection. The expression of the CR2-Fc fusion protein was detected and confirmed by SDS-PAGE and Western blotting. Restriction enzyme digestion and sequencing demonstrated that the recombinant plasmid was valid. SDS-PAGE showed that relative molecular mass (Mr;) of the purified product was consistent with the expected value. Western blotting further proved the single band at the same position. We constructed the eukaryotic expression vector of CR2-Fc/PCI-neo successfully. The obtained fusion protein was active and can be used for the further study of the role in HIV control.

Generation of genetic constructs that simultaneously express several shRNAs.

PubMed

Kretova, Olga V; Alembekov, Ildar R; Tchurikov, Nickolai A

2012-05-01

RNAi has potential as an antiviral gene therapy strategy. Cassette constructs simultaneously expressing several siRNAs could prove to be the most efficient technique in developing gene therapy approaches for highly mutable viruses such as HIV-1. Here we describe a rapid and cost-saving protocol to generate cassettes that simultaneously express three siRNAs for repression of HIV-1 and CCR5 transcripts. siRNA biological activity was tested in a non-viral system, and exhibited both efficiency and specificity. Our results suggest this protocol can be used to rapidly generate cassette constructs for antiviral gene therapy applications.

High Expression of Cry1Ac Protein in Cotton (Gossypium hirsutum) by Combining Independent Transgenic Events that Target the Protein to Cytoplasm and Plastids.

PubMed

Singh, Amarjeet Kumar; Paritosh, Kumar; Kant, Uma; Burma, Pradeep Kumar; Pental, Deepak

2016-01-01

Transgenic cotton was developed using two constructs containing a truncated and codon-modified cry1Ac gene (1,848 bp), which was originally characterized from Bacillus thuringiensis subspecies kurstaki strain HD73 that encodes a toxin highly effective against many lepidopteran pests. In Construct I, the cry1Ac gene was cloned under FMVde, a strong constitutively expressing promoter, to express the encoded protein in the cytoplasm. In Construct II, the encoded protein was directed to the plastids using a transit peptide taken from the cotton rbcSIb gene. Genetic transformation experiments with Construct I resulted in a single copy insertion event in which the Cry1Ac protein expression level was 2-2.5 times greater than in the Bacillus thuringiensis cotton event Mon 531, which is currently used in varieties and hybrids grown extensively in India and elsewhere. Another high expression event was selected from transgenics developed with Construct II. The Cry protein expression resulting from this event was observed only in the green plant parts. No transgenic protein expression was observed in the non-green parts, including roots, seeds and non-green floral tissues. Thus, leucoplasts may lack the mechanism to allow entry of a protein tagged with the transit peptide from a protein that is only synthesized in tissues containing mature plastids. Combining the two events through sexual crossing led to near additive levels of the toxin at 4-5 times the level currently used in the field. The two high expression events and their combination will allow for effective resistance management against lepidopteran insect pests, particularly Helicoverpa armigera, using a high dosage strategy.

Anger expression among Danish cyclists and drivers: A comparison based on mode specific anger expression inventories.

PubMed

Møller, M; Haustein, S

2017-11-01

Based on the short form of the driving anger expression inventory (DAX-short, 15-item), the present study developed an adapted version of the DAX for cyclists (CAX, 14 items). The data basis was an online survey of 2000 inhabitants of Denmark. A principle component analysis on the translated DAX-short confirmed the 4-factor solution of the original study differentiating between (1) personal physical aggressive expression, (2) use of a vehicle to express anger, (3) verbal aggressive expression and (4) adaptive/constructive expression. In case of cycling, the factor "use of a vehicle to express anger" only included one item and was left out. Based on the results, reliable subscales were developed. Drivers scored higher in verbal aggressive expression than cyclists, while there was no significant difference in constructive expression. The subscales for drivers and cyclists showed significant relations to age, gender, self-reported aggressive behaviours and traffic fines: Women scored for instance lower in physical expression, while older people scored higher in constructive expression. The effect of age and gender on anger expression among drivers and cyclists remained significant when controlling for exposure and other factors in linear regression analyses. These analyses also showed a relationship between a positive attitude towards driving and higher levels of anger expression among drivers, while this was not the case for cyclists. Copyright © 2017 Elsevier Ltd. All rights reserved.

ATP interacts with the CPVT mutation-associated central domain of the cardiac ryanodine receptor.

PubMed

Blayney, Lynda; Beck, Konrad; MacDonald, Ewan; D'Cruz, Leon; Nomikos, Michail; Griffiths, Julia; Thanassoulas, Angelos; Nounesis, George; Lai, F Anthony

2013-10-01

This study was designed to determine whether the cardiac ryanodine receptor (RyR2) central domain, a region associated with catecholamine polymorphic ventricular tachycardia (CPVT) mutations, interacts with the RyR2 regulators, ATP and the FK506-binding protein 12.6 (FKBP12.6). Wild-type (WT) RyR2 central domain constructs (G(2236)to G(2491)) and those containing the CPVT mutations P2328S and N2386I, were expressed as recombinant proteins. Folding and stability of the proteins were examined by circular dichroism (CD) spectroscopy and guanidine hydrochloride chemical denaturation. The far-UV CD spectra showed a soluble stably-folded protein with WT and mutant proteins exhibiting a similar secondary structure. Chemical denaturation analysis also confirmed a stable protein for both WT and mutant constructs with similar two-state unfolding. ATP and caffeine binding was measured by fluorescence spectroscopy. Both ATP and caffeine bound with an EC50 of ~200-400μM, and the affinity was the same for WT and mutant constructs. Sequence alignment with other ATP binding proteins indicated the RyR2 central domain contains the signature of an ATP binding pocket. Interaction of the central domain with FKBP12.6 was tested by glutaraldehyde cross-linking and no association was found. The RyR2 central domain, expressed as a 'correctly' folded recombinant protein, bound ATP in accord with bioinformatics evidence of conserved ATP binding sequence motifs. An interaction with FKBP12.6 was not evident. CPVT mutations did not disrupt the secondary structure nor binding to ATP. Part of the RyR2 central domain CPVT mutation cluster, can be expressed independently with retention of ATP binding. Copyright © 2013 Elsevier B.V. All rights reserved.

Cloning and expression of synthetic genes encoding angiotensin-I converting enzyme (ACE)-inhibitory bioactive peptides in Bifidobacterium pseudocatenulatum.

PubMed

Losurdo, Luca; Quintieri, Laura; Caputo, Leonardo; Gallerani, Raffaele; Mayo, Baltasar; De Leo, Francesca

2013-03-01

A wide range of biopeptides potentially able to lower blood pressure through inhibition of the angiotensin-I converting enzyme (ACE) is produced in fermented foods by proteolytic starter cultures. This work applies a procedure based on recombinant DNA technologies for the synthesis and expression of three ACE-inhibitory peptides using a probiotic cell factory. ACE-inhibitory genes and their pro-active precursors were designed, synthesized by PCR, and cloned in Escherichia coli; after which, they were cloned into the pAM1 E. coli-bifidobacteria shuttle vector. After E. coli transformation, constructs carrying the six recombinant clones were electrotransferred into the Bifidobacterium pseudocatenulatum M115 probiotic strain. Interestingly, five of the six constructs proved to be stable. Their expression was confirmed by reverse transcription PCR. Furthermore, transformed strains displayed ACE-inhibitory activity linearly correlated to increasing amounts of cell-free cellular lysates. In particular, 50 μg of lysates from constructs pAM1-Pro-BP3 and pAM1-BP2 showed a 50% higher ACE-inhibitory activity than that of the controls. As a comparison, addition of 50 ng of Pro-BP1 and Pro-BP3 synthetic peptides to 50 μg of cell-free extracts of B. pseudocatenulatum M115 wild-type strain showed an average of 67% of ACE inhibition; this allowed estimating the amount of the peptides produced by the transformants. Engineering of bifidobacteria for the production of biopeptides is envisioned as a promising cell factory model system. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Iterative design of peptide-based hydrogels and the effect of network electrostatics on primary chondrocyte behavior

PubMed Central

Sinthuvanich, Chomdao; Haines-Butterick, Lisa A.; Nagy, Katelyn J.; Schneider, Joel P.

2012-01-01

Iterative peptide design was used to generate two peptide-based hydrogels to study the effect of network electrostatics on primary chondrocyte behavior. MAX8 and HLT2 peptides have formal charge states of +7 and +5 per monomer, respectively. These peptides undergo triggered folding and self-assembly to afford hydrogel networks having similar rheological behavior and local network morphologies, yet different electrostatic character. Each gel can be used to directly encapsulate and syringe-deliver cells. The influence of network electrostatics on cell viability after encapsulation and delivery, extracellular matrix deposition, gene expression, and the bulk mechanical properties of the gel-cell constructs as a function of culture time was assessed. The less electropositive HLT2 gel provides a microenvironment more conducive to chondrocyte encapsulation, delivery, and phenotype maintenance. Cell viability was higher for this gel and although a moderate number of cells dedifferentiated to a fibroblast-like phenotype, many retained their chondrocytic behavior. As a result, gel-cell constructs prepared with HLT2, cultured under static in vitro conditions, contained more GAG and type II collagen resulting in mechanically superior constructs. Chondrocytes delivered in the more electropositive MAX8 gel experienced a greater degree of cell death during encapsulation and delivery and the remaining viable cells were less prone to maintain their phenotype. As a result, MAX8 gel-cell constructs had fewer cells, of which a limited number were capable of laying down cartilage-specific ECM. PMID:22841922

Iterative design of peptide-based hydrogels and the effect of network electrostatics on primary chondrocyte behavior.

PubMed

Sinthuvanich, Chomdao; Haines-Butterick, Lisa A; Nagy, Katelyn J; Schneider, Joel P

2012-10-01

Iterative peptide design was used to generate two peptide-based hydrogels to study the effect of network electrostatics on primary chondrocyte behavior. MAX8 and HLT2 peptides have formal charge states of +7 and +5 per monomer, respectively. These peptides undergo triggered folding and self-assembly to afford hydrogel networks having similar rheological behavior and local network morphologies, yet different electrostatic character. Each gel can be used to directly encapsulate and syringe-deliver cells. The influence of network electrostatics on cell viability after encapsulation and delivery, extracellular matrix deposition, gene expression, and the bulk mechanical properties of the gel-cell constructs as a function of culture time was assessed. The less electropositive HLT2 gel provides a microenvironment more conducive to chondrocyte encapsulation, delivery, and phenotype maintenance. Cell viability was higher for this gel and although a moderate number of cells dedifferentiated to a fibroblast-like phenotype, many retained their chondrocytic behavior. As a result, gel-cell constructs prepared with HLT2, cultured under static in vitro conditions, contained more GAG and type II collagen resulting in mechanically superior constructs. Chondrocytes delivered in the more electropositive MAX8 gel experienced a greater degree of cell death during encapsulation and delivery and the remaining viable cells were less prone to maintain their phenotype. As a result, MAX8 gel-cell constructs had fewer cells, of which a limited number were capable of laying down cartilage-specific ECM. Published by Elsevier Ltd.

Maximizing capture of gene co-expression relationships through pre-clustering of input expression samples: an Arabidopsis case study.

PubMed

Feltus, F Alex; Ficklin, Stephen P; Gibson, Scott M; Smith, Melissa C

2013-06-05

In genomics, highly relevant gene interaction (co-expression) networks have been constructed by finding significant pair-wise correlations between genes in expression datasets. These networks are then mined to elucidate biological function at the polygenic level. In some cases networks may be constructed from input samples that measure gene expression under a variety of different conditions, such as for different genotypes, environments, disease states and tissues. When large sets of samples are obtained from public repositories it is often unmanageable to associate samples into condition-specific groups, and combining samples from various conditions has a negative effect on network size. A fixed significance threshold is often applied also limiting the size of the final network. Therefore, we propose pre-clustering of input expression samples to approximate condition-specific grouping of samples and individual network construction of each group as a means for dynamic significance thresholding. The net effect is increase sensitivity thus maximizing the total co-expression relationships in the final co-expression network compendium. A total of 86 Arabidopsis thaliana co-expression networks were constructed after k-means partitioning of 7,105 publicly available ATH1 Affymetrix microarray samples. We term each pre-sorted network a Gene Interaction Layer (GIL). Random Matrix Theory (RMT), an un-supervised thresholding method, was used to threshold each of the 86 networks independently, effectively providing a dynamic (non-global) threshold for the network. The overall gene count across all GILs reached 19,588 genes (94.7% measured gene coverage) and 558,022 unique co-expression relationships. In comparison, network construction without pre-sorting of input samples yielded only 3,297 genes (15.9%) and 129,134 relationships. in the global network. Here we show that pre-clustering of microarray samples helps approximate condition-specific networks and allows for dynamic thresholding using un-supervised methods. Because RMT ensures only highly significant interactions are kept, the GIL compendium consists of 558,022 unique high quality A. thaliana co-expression relationships across almost all of the measurable genes on the ATH1 array. For A. thaliana, these networks represent the largest compendium to date of significant gene co-expression relationships, and are a means to explore complex pathway, polygenic, and pleiotropic relationships for this focal model plant. The networks can be explored at sysbio.genome.clemson.edu. Finally, this method is applicable to any large expression profile collection for any organism and is best suited where a knowledge-independent network construction method is desired.

Maximizing capture of gene co-expression relationships through pre-clustering of input expression samples: an Arabidopsis case study

PubMed Central

2013-01-01

Background In genomics, highly relevant gene interaction (co-expression) networks have been constructed by finding significant pair-wise correlations between genes in expression datasets. These networks are then mined to elucidate biological function at the polygenic level. In some cases networks may be constructed from input samples that measure gene expression under a variety of different conditions, such as for different genotypes, environments, disease states and tissues. When large sets of samples are obtained from public repositories it is often unmanageable to associate samples into condition-specific groups, and combining samples from various conditions has a negative effect on network size. A fixed significance threshold is often applied also limiting the size of the final network. Therefore, we propose pre-clustering of input expression samples to approximate condition-specific grouping of samples and individual network construction of each group as a means for dynamic significance thresholding. The net effect is increase sensitivity thus maximizing the total co-expression relationships in the final co-expression network compendium. Results A total of 86 Arabidopsis thaliana co-expression networks were constructed after k-means partitioning of 7,105 publicly available ATH1 Affymetrix microarray samples. We term each pre-sorted network a Gene Interaction Layer (GIL). Random Matrix Theory (RMT), an un-supervised thresholding method, was used to threshold each of the 86 networks independently, effectively providing a dynamic (non-global) threshold for the network. The overall gene count across all GILs reached 19,588 genes (94.7% measured gene coverage) and 558,022 unique co-expression relationships. In comparison, network construction without pre-sorting of input samples yielded only 3,297 genes (15.9%) and 129,134 relationships. in the global network. Conclusions Here we show that pre-clustering of microarray samples helps approximate condition-specific networks and allows for dynamic thresholding using un-supervised methods. Because RMT ensures only highly significant interactions are kept, the GIL compendium consists of 558,022 unique high quality A. thaliana co-expression relationships across almost all of the measurable genes on the ATH1 array. For A. thaliana, these networks represent the largest compendium to date of significant gene co-expression relationships, and are a means to explore complex pathway, polygenic, and pleiotropic relationships for this focal model plant. The networks can be explored at sysbio.genome.clemson.edu. Finally, this method is applicable to any large expression profile collection for any organism and is best suited where a knowledge-independent network construction method is desired. PMID:23738693

Beta-lactamase targeted enzyme activatable photosensitizers for antimicrobial PDT

NASA Astrophysics Data System (ADS)

Zheng, Xiang; Verma, Sarika; Sallum, Ulysses W.; Hasan, Tayyaba

2009-06-01

Photodynamic therapy (PDT) as a treatment modality for infectious disease has shown promise. However, most of the antimicrobial photosensitizers (PS) non-preferentially accumulate in both bacteria and host tissues, causing host tissue phototoxicity during treatment. We have developed a new antimicrobial PDT strategy which exploits beta-lactam resistance mechanism, one of the major drug-resistance bacteria evolved, to achieve enhanced target specificity with limited host damage. Our strategy comprises a prodrug construct with a PS and a quencher linked by beta-lactam ring, resulting in a diminished phototoxicity. This construct, beta-lactamase enzyme-activated-photosensitizer (beta-LEAP), can only be activated in the presence of both light and bacteria, and remains inactive elsewhere such as mammalian tissue. Beta-LEAP construct had shown specific cleavage by purified beta-lactamase and by beta-lactamase over-expressing methicillin resistant Staphylococcus aureus (MRSA). Specific photodynamic toxicity was observed towards MRSA, while dark and light toxicity were equivalent to reference strains. The prodrug design, synthesis and photophysical properties will be discussed.

Development and manufacture of an investigational human living dermal equivalent (ICX-SKN).

PubMed

Flasza, Marzena; Kemp, Paul; Shering, David; Qiao, Jizeng; Marshall, Damian; Bokta, Ausha; Johnson, Penny A

2007-11-01

To design and manufacture an investigational living skin graft replacement (ICX-SKN) that is able to incorporate into the host, providing healing by primary intent without the need for a second intervention. The ICX-SKN skin graft replacement has been designed as an allogeneic dermal substitute comprising an extracellular matrix composed largely of human collagen and human dermal fibroblast cells (HDFs). ICX-SKN is first formed by casting a provisional matrix of fibrin, into which HDFs are seeded. Through a process of maturation, HDFs are induced to lay down collagen and other extracellular matrix materials and, as the construct matures, the original fibrin is largely replaced by collagen, which provides tensile strength and flexibility to the construct. In order to design a product and manufacturing system that lends itself to large-scale production the process was developed as a discontinuous process consisting of four stages: 1. batch casting and maturation of the initial construct (pSKN), 2. freeze-drying of pSKN to produce a second intermediate (dSKN), 3. sterilization by gamma-irradiation of dSKN to produce a third intermediate (sSKN), and finally, 4. repopulation of sSKN by fresh HDFs to produce the final product, ICX-SKN skin graft replacement. Preliminary characterization of ICX-SKN and its application in a preclinical model are described. The 7-week maturation period resulted in a construct (pSKN) with robust handling properties, which was composed mainly of human collagen I. Following development of a process for freeze-drying and subsequent sterilization, the matrix was successfully repopulated with fresh HDFs. In addition, it was demonstrated that human keratinocytes attached and differentiated on the matrix. Application of human keratinocytes to the repopulated constructs (ICX-SKN) resulted in expression of markers of basement membranes that was largely dependent on the presence of living HDFs on the constructs. ICX-SKN graft replacements applied to excision wounds in mice healed and were rapidly re-epithelialized. ICX-SKN has been developed as a platform product that can be used as a skin graft replacement and the process by which it is manufactured has been designed for the product to be available to the end-user off-the-shelf and for ease-of-use in practice.

The discovery of zinc fingers and their development for practical applications in gene regulation and genome manipulation.

PubMed

Klug, Aaron

2010-02-01

A long-standing goal of molecular biologists has been to construct DNA-binding proteins for the control of gene expression. The classical Cys2His2 (C2H2) zinc finger design is ideally suited for such purposes. Discriminating between closely related DNA sequences both in vitro and in vivo, this naturally occurring design was adopted for engineering zinc finger proteins (ZFPs) to target genes specifically. Zinc fingers were discovered in 1985, arising from the interpretation of our biochemical studies on the interaction of the Xenopus protein transcription factor IIIA (TFIIIA) with 5S RNA. Subsequent structural studies revealed its three-dimensional structure and its interaction with DNA. Each finger constitutes a self-contained domain stabilized by a zinc (Zn) ion ligated to a pair of cysteines and a pair of histidines and also by an inner structural hydrophobic core. This discovery showed not only a new protein fold but also a novel principle of DNA recognition. Whereas other DNA-binding proteins generally make use of the 2-fold symmetry of the double helix, functioning as homo- or heterodimers, zinc fingers can be linked linearly in tandem to recognize nucleic acid sequences of varying lengths. This modular design offers a large number of combinatorial possibilities for the specific recognition of DNA (or RNA). It is therefore not surprising that the zinc finger is found widespread in nature, including 3% of the genes of the human genome. The zinc finger design can be used to construct DNA-binding proteins for specific intervention in gene expression. By fusing selected zinc finger peptides to repression or activation domains, genes can be selectively switched off or on by targeting the peptide to the desired gene target. It was also suggested that by combining an appropriate zinc finger peptide with other effector or functional domains, e.g. from nucleases or integrases to form chimaeric proteins, genomes could be modified or manipulated. The first example of the power of the method was published in 1994 when a three-finger protein was constructed to block the expression of a human oncogene transformed into a mouse cell line. The same paper also described how a reporter gene was activated by targeting an inserted 9-base pair (bp) sequence, which acts as the promoter. Thus, by fusing zinc finger peptides to repression or activation domains, genes can be selectively switched off or on. It was also suggested that, by combining zinc fingers with other effector or functional domains, e.g. from nucleases or integrases, to form chimaeric proteins, genomes could be manipulated or modified. Several applications of such engineered ZFPs are described here, including some of therapeutic importance, and also their adaptation for breeding improved crop plants.

Enhanced synergistic anti-Lewis lung carcinoma effect of a DNA vaccine harboring a MUC1-VEGFR2 fusion gene used with GM-CSF as an adjuvant.

PubMed

Ruan, Junzhong; Duan, Yong; Li, Fugen; Wang, Zitong

2017-01-01

In order to achieve a synergistic effect on anti-tumour and anti-angiogenesis activity, we designed and constructed a DNA vaccine that expresses MUC1and VEGFR2 in the same reading frame. The aim of this study was to investigate the anti-tumour activity of this DNA vaccine. Furthermore, we also investigated the enhanced synergistic anti-Lewis lung carcinoma effect of this DNA vaccine by using GM-CSF as an adjuvant. A series of DNA plasmids encoding MUC1, VEGFR2, GM-CSF, and their conjugates were constructed and injected into mice intramuscularly (i.m.) followed by an electric pulse. The humoral and cellular immune responses after immunization were detected by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot (ELISPOT), respectively. To evaluate the anti-tumour efficacy of these plasmids, murine models with MUC1-expressing tumours were generated. After injection into the tumour-bearing mouse model, the plasmid carrying the fusion gene of MUC1 and VEGFR2 showed stronger inhibition of tumour growth than the plasmid expressing MUC1 or VEGFR2 alone, which indicated that MUC1 and VEGFR2 could exert a synergistic anti-tumour effect. Furthermore, mice vaccinated with the combination of the GM-CSF expressing plasmid and the plasmid carrying the fusion gene of MUC1 and VEGFR2 showed an increased inhibition in the growth of MUC1-expressing tumours and prolonged mouse survival. These observations emphasize the potential of the synergistic anti-tumour and anti-angiogenesis strategy used in DNA vaccines, and the potential of the GM-CSF gene as an adjuvant for DNA vaccines, which could represent a promising approach for tumour immunotherapy. © 2016 John Wiley & Sons Australia, Ltd.

The effect of eukaryotic expression vectors and adjuvants on DNA vaccines in chickens using an avian influenza model.

PubMed

Suarez, D L; Schultz-Cherry, S

2000-01-01

Vaccination of poultry with naked plasmid DNA has been successfully demonstrated with several different poultry pathogens, but the technology needs to be further developed before it can be practically implemented. Many different methods can conceivably enhance the efficacy of DNA vaccines, and this report examines the use of different eukaryotic expression vectors with different promoters and different adjuvants to express the influenza hemagglutinin protein. Four different promoters in five different plasmids were used to express the hemagglutinin protein of an H5 avian influenza virus, including two different immediate early cytomegaloviruses (CMVs), Rous sarcoma virus, chicken actin, and simian virus 40 promoters. All five constructs expressed detectable hemagglutinin protein in cell culture, but the pCI-neo HA plasmid with the CMV promoter provided the best response in chickens when vaccinated intramuscularly at 1 day of age on the basis of antibody titer and survivability after challenge with a highly pathogenic avian influenza virus at 6 wk postinoculation. A beneficial response was observed in birds boostered at 3 wk of age, in birds given larger amounts of DNA, and with the use of multiple injection sites to administer the vaccine. With the use of the pCI-neo construct, the effects of different adjuvants designed to increase the uptake of plasmid DNA, including 25% sucrose, diethylaminoethyl dextran, calcium phosphate, polybrene, and two different cationic liposomes, were examined. Both liposomes tested enhanced antibody titers as compared with the positive controls, but the other chemical adjuvants decreased the antibody response as compared with the control chickens that received just the plasmid alone. The results observed are promising for continued studies, but continued improvements in vaccine response and reduced costs are necessary before the technology can be commercially developed.

Expression of Anthocyanins and Proanthocyanidins after Transformation of Alfalfa with Maize Lc12

PubMed Central

Ray, Heather; Yu, Min; Auser, Patricia; Blahut-Beatty, Laureen; McKersie, Brian; Bowley, Steve; Westcott, Neil; Coulman, Bruce; Lloyd, Alan; Gruber, Margaret Y.

2003-01-01

Three anthocyanin regulatory genes of maize (Zea mays; Lc, B-Peru, and C1) were introduced into alfalfa (Medicago sativa) in a strategy designed to stimulate the flavonoid pathway and alter the composition of flavonoids produced in forage. Lc constructs included a full-length gene and a gene with a shortened 5′-untranslated region. Lc RNA was strongly expressed in Lc transgenic alfalfa foliage, but accumulation of red-purple anthocyanin was observed only under conditions of high light intensity or low temperature. These stress conditions induced chalcone synthase and flavanone 3-hydroxylase expression in Lc transgenic alfalfa foliage compared with non-transformed plants. Genotypes containing the Lc transgene construct with a full-length 5′-untranslated region responded more quickly to stress conditions and with a more extreme phenotype. High-performance liquid chromatography analysis of field-grown tissue indicated that flavone content was reduced in forage of the Lc transgenic plants. Leucocyanidin reductase, the enzyme that controls entry of metabolites into the proanthocyanidin pathway, was activated both in foliage and in developing seeds of the Lc transgenic alfalfa genotypes. Proanthocyanidin polymer was accumulated in the forage, but (+)-catechin monomers were not detected. B-Peru transgenic and C1 transgenic populations displayed no visible phenotypic changes, although these transgenes were expressed at detectable levels. These results support the emerging picture of Lc transgene-specific patterns of expression in different recipient species. These results demonstrate that proanthocyanidin biosynthesis can be stimulated in alfalfa forage using an myc-like transgene, and they pave the way for the development of high quality, bloat-safe cultivars with ruminal protein bypass. PMID:12857826

The Biology of Linguistic Expression Impacts Neural Correlates for Spatial Language

PubMed Central

Emmorey, Karen; McCullough, Stephen; Mehta, Sonya; Ponto, Laura L. B.; Grabowski, Thomas J.

2013-01-01

Biological differences between signed and spoken languages may be most evident in the expression of spatial information. PET was used to investigate the neural substrates supporting the production of spatial language in American Sign Language as expressed by classifier constructions, in which handshape indicates object type and the location/motion of the hand iconically depicts the location/motion of a referent object. Deaf native signers performed a picture description task in which they overtly named objects or produced classifier constructions that varied in location, motion, or object type. In contrast to the expression of location and motion, the production of both lexical signs and object type classifier morphemes engaged left inferior frontal cortex and left inferior temporal cortex, supporting the hypothesis that unlike the location and motion components of a classifier construction, classifier handshapes are categorical morphemes that are retrieved via left hemisphere language regions. In addition, lexical signs engaged the anterior temporal lobes to a greater extent than classifier constructions, which we suggest reflects increased semantic processing required to name individual objects compared with simply indicating the type of object. Both location and motion classifier constructions engaged bilateral superior parietal cortex, with some evidence that the expression of static locations differentially engaged the left intraparietal sulcus. We argue that bilateral parietal activation reflects the biological underpinnings of sign language. To express spatial information, signers must transform visual–spatial representations into a body-centered reference frame and reach toward target locations within signing space. PMID:23249348

24 CFR 941.203 - Design and construction standards.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 24 Housing and Urban Development 4 2011-04-01 2011-04-01 false Design and construction standards... URBAN DEVELOPMENT PUBLIC HOUSING DEVELOPMENT PHA Eligibility and Program Requirements § 941.203 Design and construction standards. (a) Physical structures shall be designed, constructed and equipped so as...

High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

PubMed Central

Minaba, Masaomi

2014-01-01

Synthetic biologists construct complex biological circuits by combinations of various genetic parts. Many genetic parts that are orthogonal to one another and are independent of existing cellular processes would be ideal for use in synthetic biology. However, our toolbox is still limited with respect to the bacterium Escherichia coli, which is important for both research and industrial use. The site-specific incorporation of unnatural amino acids is a technique that incorporates unnatural amino acids into proteins using a modified exogenous aminoacyl-tRNA synthetase/tRNA pair that is orthogonal to any native pairs in a host and is independent from other cellular functions. Focusing on the orthogonality and independency that are suitable for the genetic parts, we designed novel AND gate and translational switches using the unnatural amino acid 3-iodo-l-tyrosine incorporation system in E. coli. A translational switch was turned on after addition of 3-iodo-l-tyrosine in the culture medium within minutes and allowed tuning of switchability and translational efficiency. As an application, we also constructed a gene expression system that produced large amounts of proteins under induction conditions and exhibited zero-leakage expression under repression conditions. Similar translational switches are expected to be applicable also for eukaryotes such as yeasts, nematodes, insects, mammalian cells, and plants. PMID:24375139

Biosynthesis of adipic acid via microaerobic hydrogenation of cis,cis-muconic acid by oxygen-sensitive enoate reductase.

PubMed

Sun, Jing; Raza, Muslim; Sun, Xinxiao; Yuan, Qipeng

2018-06-06

Adipic acid (AA) is an important dicarboxylic acid used for the manufacture of nylon and polyurethane plastics. In this study, a novel adipic acid biosynthetic pathway was designed by extending the cis,cis-muconic acid (MA) biosynthesis through biohydrogenation. Enoate reductase from Clostridium acetobutylicum (CaER), an oxygen-sensitive reductase, was demonstrated to have in vivo enzyme activity of converting cis,cis-muconic acid to adipic acid under microaerobic condition. Engineered Escherichia coli strains were constructed to express the whole pathway and accumulated 5.8 ± 0.9 mg/L adipic acid from simple carbon sources. Considering the different oxygen demands between cis,cis-muconic acid biosynthesis and its degradation, a co-culture system was constructed. To improve production, T7 promoter instead of lac promoter was used for higher level expression of the key enzyme CaER and the titer of adipic acid increased to 18.3 ± 0.6 mg/L. To decrease the oxygen supply to downstream strains expressing CaER, Vitreoscilla hemoglobin (VHb) was introduced to upstream strains for its ability on oxygen obtaining. This attempt further improved the production of this novel pathway and 27.6 ± 1.3 mg/L adipic acid was accumulated under microaerobic condition. Copyright © 2018. Published by Elsevier B.V.

Generation and analysis of the improved human HAL9/10 antibody phage display libraries.

PubMed

Kügler, Jonas; Wilke, Sonja; Meier, Doris; Tomszak, Florian; Frenzel, André; Schirrmann, Thomas; Dübel, Stefan; Garritsen, Henk; Hock, Björn; Toleikis, Lars; Schütte, Mark; Hust, Michael

2015-02-19

Antibody phage display is a proven key technology that allows the generation of human antibodies for diagnostics and therapy. From naive antibody gene libraries - in theory - antibodies against any target can be selected. Here we describe the design, construction and characterization of an optimized antibody phage display library. The naive antibody gene libraries HAL9 and HAL10, with a combined theoretical diversity of 1.5×10(10) independent clones, were constructed from 98 healthy donors using improved phage display vectors. In detail, most common phagemids employed for antibody phage display are using a combined His/Myc tag for detection and purification. We show that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display. For several published antibody libraries, the selected number of kappa scFvs were lower compared to lambda scFvs, probably due to a lower kappa scFv or Fab expression rate. Deletion of a phenylalanine at the end of the CL linker sequence in our new phagemid design increased scFv production rate and frequency of selected kappa antibodies significantly. The HAL libraries and 834 antibodies selected against 121 targets were analyzed regarding the used germline V-genes, used V-gene combinations and CDR-H3/-L3 length and composition. The amino acid diversity and distribution in the CDR-H3 of the initial library was retrieved in the CDR-H3 of selected antibodies showing that all CDR-H3 amino acids occurring in the human antibody repertoire can be functionally used and is not biased by E. coli expression or phage selection. Further, the data underline the importance of CDR length variations. The highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv phagemid vector design. Analysis of selected antibodies revealed that the complete amino acid diversity in the CDR-H3 was also found in selected scFvs showing the functionality of the naive CDR-H3 diversity.

[Construction of the superantigen SEA transfected laryngocarcinoma cells].

PubMed

Ji, Xiaobin; Jingli, J V; Liu, Qicai; Xie, Jinghua

2013-04-01

To construct an eukaryotic expression vectors containing superantigen staphylococcal enterotoxin A (SEA) gene, and to identify its expression in laryngeal squamous carcinoma cells. SEA full-length gene fragment was obtained from ATCC13565 genome of the staphylococcus, referencing standard strains producing SEA. Coding sequence of SEA was artificially synthetized. Than, SEA gene fragments was subcloned into eukaryotic expression vector pIRES-EGFP. The recombinant plasmid pSEA-IRES-EGFP was constructed and was transfected to laryngocarcinoma Hep-2 cells. Resistant clones were screened by G418. The expression of SEA in laryngocarcinoma cells was identified with ELISA and RT-PCR method. The subclone of artificially synthetized SEA gene was subclone to eukaryotic expression vector pires-EGFP. Flanking sequence confirmed that SEA sequence was fully identical to the coding sequence of standard staphylococcus strains ATCC13565 in Genbank. After recombinant plasmid transfected to laryngocarcinoma cells, the resistant clones was obtained after screening for two weeks. The clones were selected. The specific gene fragment was obtained by RT-PCR amplification. ELISA assay confirmed that the content of SEA protein in supernatant fluid of cell culture had reached about Pg level. The recombinant eukaryotic expression vector containing superantigen SEA gene is successfully constructed, and is capable of effective expression and continued secretion of SEA protein in laryngochrcinoma Hep-2 cells after recombinant plasmid transfected to laryngocarcinoma cells.

Design and construction of a first-generation high-throughput integrated robotic molecular biology platform for bioenergy applications.

PubMed

Hughes, Stephen R; Butt, Tauseef R; Bartolett, Scott; Riedmuller, Steven B; Farrelly, Philip

2011-08-01

The molecular biological techniques for plasmid-based assembly and cloning of gene open reading frames are essential for elucidating the function of the proteins encoded by the genes. High-throughput integrated robotic molecular biology platforms that have the capacity to rapidly clone and express heterologous gene open reading frames in bacteria and yeast and to screen large numbers of expressed proteins for optimized function are an important technology for improving microbial strains for biofuel production. The process involves the production of full-length complementary DNA libraries as a source of plasmid-based clones to express the desired proteins in active form for determination of their functions. Proteins that were identified by high-throughput screening as having desired characteristics are overexpressed in microbes to enable them to perform functions that will allow more cost-effective and sustainable production of biofuels. Because the plasmid libraries are composed of several thousand unique genes, automation of the process is essential. This review describes the design and implementation of an automated integrated programmable robotic workcell capable of producing complementary DNA libraries, colony picking, isolating plasmid DNA, transforming yeast and bacteria, expressing protein, and performing appropriate functional assays. These operations will allow tailoring microbial strains to use renewable feedstocks for production of biofuels, bioderived chemicals, fertilizers, and other coproducts for profitable and sustainable biorefineries. Published by Elsevier Inc.

Improving the Expression and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers by Targeted Sequence Changes

PubMed Central

Ringe, Rajesh P.; Ozorowski, Gabriel; Yasmeen, Anila; Cupo, Albert; Cruz Portillo, Victor M.; Pugach, Pavel; Golabek, Michael; Rantalainen, Kimmo; Holden, Lauren G.; Cottrell, Christopher A.; Wilson, Ian A.; Sanders, Rogier W.; Ward, Andrew B.; Klasse, P. J.

2017-01-01

ABSTRACT Soluble, recombinant native-like envelope glycoprotein (Env) trimers of various human immunodeficiency virus type 1 (HIV-1) genotypes are being developed for structural studies and as vaccine candidates aimed at the induction of broadly neutralizing antibodies (bNAbs). The prototypic design is designated SOSIP.664, but many HIV-1 env genes do not yield fully native-like trimers efficiently. One such env gene is CZA97.012 from a neutralization-resistant (tier 2) clade C virus. As appropriately purified, native-like CZA97.012 SOSIP.664 trimers induce autologous neutralizing antibodies (NAbs) efficiently in immunized rabbits, we sought to improve the efficiency with which they can be produced and to better understand the limitations to the original design. By using structure- and antigenicity-guided mutagenesis strategies focused on the V2 and V3 regions and the gp120-gp41 interface, we developed the CZA97 SOSIP.v4.2-M6.IT construct. Fully native-like, stable trimers that display multiple bNAb epitopes could be expressed from this construct in a stable CHO cell line and purified at an acceptable yield using either a PGT145 or a 2G12 bNAb affinity column. We also show that similar mutagenesis strategies can be used to improve the yields and properties of SOSIP.664 trimers of the DU422, 426c, and 92UG037 genotypes. IMPORTANCE Recombinant trimeric proteins based on HIV-1 env genes are being developed for future vaccine trials in humans. A feature of these proteins is their mimicry of the envelope glycoprotein (Env) structure on virus particles that is targeted by neutralizing antibodies, i.e., antibodies that prevent cells from becoming infected. The vaccine concept under exploration is that recombinant trimers may be able to elicit virus-neutralizing antibodies when delivered as immunogens. Because HIV-1 is extremely variable, a practical vaccine may need to incorporate Env trimers derived from multiple different virus sequences. Accordingly, we need to understand how to make recombinant trimers from many different env genes. Here, we show how to produce trimers from a clade C virus, CZA97.012, by using an array of protein engineering techniques to improve a prototypic construct. We also show that the methods may have wider utility for other env genes, thereby further guiding immunogen design. PMID:28381572

An iterative synthetic approach to engineer a high-performing PhoB-specific reporter.

PubMed

Stoudenmire, Julie L; Essock-Burns, Tara; Weathers, Erena N; Solaimanpour, Sina; Mrázek, Jan; Stabb, Eric V

2018-05-11

Transcriptional reporters are common tools for analyzing either the transcription of a gene of interest or the activity of a specific transcriptional regulator. Unfortunately, the latter application has the shortcoming that native promoters did not evolve as optimal readouts for the activity of a particular regulator. We sought to synthesize an optimized transcriptional reporter for assessing PhoB activity, aiming for maximal "on" expression when PhoB is active, minimal background in the "off" state, and no control elements for other regulators. We designed specific sequences for promoter elements with appropriately spaced PhoB-binding sites, and at nineteen additional intervening nucleotide positions for which we did not predict sequence-specific effects the bases were randomized. Eighty-three such constructs were screened in Vibrio fischeri , enabling us to identify bases at particular randomized positions that significantly correlated with high "on" or low "off" expression. A second round of promoter design rationally constrained thirteen additional positions, leading to a reporter with high PhoB-dependent expression, essentially no background, and no other known regulatory elements. As expressed reporters, we used both stable and destabilized GFP, the latter with a half-life of eighty-one minutes in V. fischeri In culture, PhoB induced the reporter when phosphate was depleted below 10 μM. During symbiotic colonization of its host squid Euprymna scolopes , the reporter indicated heterogeneous phosphate availability in different light-organ microenvironments. Finally, testing this construct in other Proteobacteria demonstrated its broader utility. The results illustrate how a limited ability to predict synthetic promoter-reporter performance can be overcome through iterative screening and re-engineering. IMPORTANCE Transcriptional reporters can be powerful tools for assessing when a particular regulator is active; however, native promoters may not be ideal for this purpose. Optimal reporters should be specific to the regulator being examined and should maximize the difference between "on" and "off" states; however, these properties are distinct from the selective pressures driving the evolution of natural promoters. Synthetic promoters offer a promising alternative, but our understanding often does not enable fully predictive promoter design, and the large number of alternative sequence possibilities can be intractable. In a synthetic promoter region with over thirty-four billion sequence variants, we identified bases correlated with favorable performance by screening only eighty-three candidates, allowing us to rationally constrain our design. We thereby generated an optimized reporter that is induced by PhoB and used it to explore the low-phosphate response of V. fischeri This promoter-design strategy will facilitate the engineering of other regulator-specific reporters. Copyright © 2018 American Society for Microbiology.

Design and testing of regulatory cassettes for optimal activity in skeletal and cardiac muscles.

PubMed

Himeda, Charis L; Chen, Xiaolan; Hauschka, Stephen D

2011-01-01

Gene therapy for muscular dystrophies requires efficient gene delivery to the striated musculature and specific, high-level expression of the therapeutic gene in a physiologically diverse array of muscles. This can be achieved by the use of recombinant adeno-associated virus vectors in conjunction with muscle-specific regulatory cassettes. We have constructed several generations of regulatory cassettes based on the enhancer and promoter of the muscle creatine kinase gene, some of which include heterologous enhancers and individual elements from other muscle genes. Since the relative importance of many control elements varies among different anatomical muscles, we are aiming to tailor these cassettes for high-level expression in cardiac muscle, and in fast and slow skeletal muscles. With the achievement of efficient intravascular gene delivery to isolated limbs, selected muscle groups, and heart in large animal models, the design of cassettes optimized for activity in different muscle types is now a practical goal. In this protocol, we outline the key steps involved in the design of regulatory cassettes for optimal activity in skeletal and cardiac muscle, and testing in mature muscle fiber cultures. The basic principles described here can also be applied to engineering tissue-specific regulatory cassettes for other cell types.

76 FR 58079 - Notice of Passenger Facility Charge (PFC) Approvals and Disapprovals

Federal Register 2010, 2011, 2012, 2013, 2014

2011-09-19

...--construction. Rehabilitate taxiways B, A, and B-1--design. Pavement condition update. Acquire aircraft rescue..., phase II. Construct taxiway to runway 10/28. Design/construct air cargo/general aviation apron. Design... center controls for new control tower. Design and construct new south general aviation apron. Remove...

29 CFR 1926.1433 - Design, construction and testing.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 29 Labor 8 2013-07-01 2013-07-01 false Design, construction and testing. 1926.1433 Section 1926... Construction § 1926.1433 Design, construction and testing. The following requirements apply to equipment that... locomotive cranes manufactured prior to November 8, 2010 must meet the applicable requirements for design...

29 CFR 1926.1433 - Design, construction and testing.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 29 Labor 8 2014-07-01 2014-07-01 false Design, construction and testing. 1926.1433 Section 1926... Construction § 1926.1433 Design, construction and testing. The following requirements apply to equipment that... locomotive cranes manufactured prior to November 8, 2010 must meet the applicable requirements for design...

29 CFR 1926.1433 - Design, construction and testing.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 29 Labor 8 2011-07-01 2011-07-01 false Design, construction and testing. 1926.1433 Section 1926... Construction § 1926.1433 Design, construction and testing. The following requirements apply to equipment that... locomotive cranes manufactured prior to November 8, 2010 must meet the applicable requirements for design...

29 CFR 1926.1433 - Design, construction and testing.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 29 Labor 8 2012-07-01 2012-07-01 false Design, construction and testing. 1926.1433 Section 1926... Construction § 1926.1433 Design, construction and testing. The following requirements apply to equipment that... locomotive cranes manufactured prior to November 8, 2010 must meet the applicable requirements for design...

Gender roles, sex and the expression of driving anger.

PubMed

Sullman, M J M; Paxion, J; Stephens, A N

2017-09-01

The present study investigated the validity of the 25-item Driving Anger Expression Inventory (DAX) as well as the role of sex and gender-roles in relation to the expression of driving anger in a sample of 378 French drivers (males=38%, M=32.9years old). Confirmatory Factor Analysis supported the four-factor structure of the 25-item DAX (Adaptive/Constructive Expression; Use of the Vehicle to Express Anger; Verbal Aggressive Expression and Personal Physical Aggressive Expression) and two of the three aggressive factors were found to have significant positive relationships with driving anger, while adaptive/constructive expression was negatively related to driving anger. Use of the vehicle to express anger was not significantly related to crash involvement, but was significantly related to all other crash-related conditions (traffic tickets, loss of concentration, loss of control of the vehicle, near crash). The presence of feminine traits, but not sex, was predictive of adaptive/constructive behaviours, while masculine traits predicted more frequent verbal aggressive expression, use of the vehicle to express anger, personal physical aggressive expression and total aggressive expression. This finding may account for the inconsistent relationship found between driving anger and sex in previous research. This research also found that the 25-item DAX is a valid tool to measure the expression of driving anger and that the endorsement of masculine traits are related to more aggressive forms of driving anger expression. Copyright © 2017 Elsevier Ltd. All rights reserved.

Synthetic gene circuits for metabolic control: design trade-offs and constraints

PubMed Central

Oyarzún, Diego A.; Stan, Guy-Bart V.

2013-01-01

A grand challenge in synthetic biology is to push the design of biomolecular circuits from purely genetic constructs towards systems that interface different levels of the cellular machinery, including signalling networks and metabolic pathways. In this paper, we focus on a genetic circuit for feedback regulation of unbranched metabolic pathways. The objective of this feedback system is to dampen the effect of flux perturbations caused by changes in cellular demands or by engineered pathways consuming metabolic intermediates. We consider a mathematical model for a control circuit with an operon architecture, whereby the expression of all pathway enzymes is transcriptionally repressed by the metabolic product. We address the existence and stability of the steady state, the dynamic response of the network under perturbations, and their dependence on common tuneable knobs such as the promoter characteristic and ribosome binding site (RBS) strengths. Our analysis reveals trade-offs between the steady state of the enzymes and the intermediates, together with a separation principle between promoter and RBS design. We show that enzymatic saturation imposes limits on the parameter design space, which must be satisfied to prevent metabolite accumulation and guarantee the stability of the network. The use of promoters with a broad dynamic range and a small leaky expression enlarges the design space. Simulation results with realistic parameter values also suggest that the control circuit can effectively upregulate enzyme production to compensate flux perturbations. PMID:23054953

A Java-based tool for the design of classification microarrays.

PubMed

Meng, Da; Broschat, Shira L; Call, Douglas R

2008-08-04

Classification microarrays are used for purposes such as identifying strains of bacteria and determining genetic relationships to understand the epidemiology of an infectious disease. For these cases, mixed microarrays, which are composed of DNA from more than one organism, are more effective than conventional microarrays composed of DNA from a single organism. Selection of probes is a key factor in designing successful mixed microarrays because redundant sequences are inefficient and limited representation of diversity can restrict application of the microarray. We have developed a Java-based software tool, called PLASMID, for use in selecting the minimum set of probe sequences needed to classify different groups of plasmids or bacteria. The software program was successfully applied to several different sets of data. The utility of PLASMID was illustrated using existing mixed-plasmid microarray data as well as data from a virtual mixed-genome microarray constructed from different strains of Streptococcus. Moreover, use of data from expression microarray experiments demonstrated the generality of PLASMID. In this paper we describe a new software tool for selecting a set of probes for a classification microarray. While the tool was developed for the design of mixed microarrays-and mixed-plasmid microarrays in particular-it can also be used to design expression arrays. The user can choose from several clustering methods (including hierarchical, non-hierarchical, and a model-based genetic algorithm), several probe ranking methods, and several different display methods. A novel approach is used for probe redundancy reduction, and probe selection is accomplished via stepwise discriminant analysis. Data can be entered in different formats (including Excel and comma-delimited text), and dendrogram, heat map, and scatter plot images can be saved in several different formats (including jpeg and tiff). Weights generated using stepwise discriminant analysis can be stored for analysis of subsequent experimental data. Additionally, PLASMID can be used to construct virtual microarrays with genomes from public databases, which can then be used to identify an optimal set of probes.

Construction of pTM series plasmids for gene expression in Brucella species.

PubMed

Tian, Mingxing; Qu, Jing; Bao, Yanqing; Gao, Jianpeng; Liu, Jiameng; Wang, Shaohui; Sun, Yingjie; Ding, Chan; Yu, Shengqing

2016-04-01

Brucellosis, the most common widespread zoonotic disease, is caused by Brucella spp., which are facultative, intracellular, Gram-negative bacteria. With the development of molecular biology techniques, more and more virulence-associated factors have been identified in Brucella spp. A suitable plasmid system is an important tool to study virulence genes in Brucella. In this study, we constructed three constitutive replication plasmids (pTM1-Cm, pTM2-Amp, and pTM3-Km) using the replication origin (rep) region derived from the pBBR1-MCS vector. Also, a DNA fragment containing multiple cloning sites (MCSs) and a terminator sequence derived from the pCold vector were produced for complementation of the deleted genes. Besides pGH-6×His, a plasmid containing the groE promoter of Brucella spp. was constructed to express exogenous proteins in Brucella with high efficiency. Furthermore, we constructed the inducible expression plasmid pZT-6×His, containing the tetracycline-inducible promoter pzt1, which can induce expression by the addition of tetracycline in the Brucella culture medium. The constructed pTM series plasmids will play an important role in the functional investigation of Brucella spp. Copyright © 2016 Elsevier B.V. All rights reserved.

[Construction and eukaryotic expression of PVAX1-hPV58mE6E7fcGB composite gene vaccine].

PubMed

Wang, He; Yu, Jiyun; Li, Li

2013-10-01

To construct and express a composite gene vaccine for human papillomavirus 58(HPV58)-associated cervical cancer, we inserted HPV58mE6E7 fusion gene into pCI-Fc-GPI eukaryotic expression vector, constructing a recombinant plasmid named pCI-sig-HPV58mE6E7-Fc-GPI. Then we further inserted fragment of sig-HPV58mE6E7Fc-GPI into the novel vaccine vector PVAX1-IRES-GM/B7, constructing PVAX1-HPV58mE6E7FcGB composite gene vaccine. PVAX1-HPV58mE6E7FcGB vaccine was successfully constructed and identified by restriction endonuclease and sequencing analysis. Eukaryotic expression of fusion antigen sig-HPV58mE6E7-Fc-GPI and molecular ad-juvant GM-CSF and B7. 1 were proved to be realized at the same time by flow cytometry and immunofluorescence. So PVAX1-HPV58mE6E7FcGB can be taken as a candidate of therapeutic vaccine for HPV58-associated tumors and their precancerous transformations.

10 CFR Appendix N to Part 52 - Standardization of Nuclear Power Plant Designs: Combined Licenses To Construct and Operate...

Code of Federal Regulations, 2011 CFR

2011-01-01

... Licenses To Construct and Operate Nuclear Power Reactors of Identical Design at Multiple Sites N Appendix N... Designs: Combined Licenses To Construct and Operate Nuclear Power Reactors of Identical Design at Multiple... construct and operate nuclear power reactors of identical design (“common design”) to be located at multiple...

Highly specific gene silencing in a monocot species by artificial microRNAs derived from chimeric miRNA precursors

DOE PAGES

Carbonell, Alberto; Fahlgren, Noah; Mitchell, Skyler; ...

2015-05-20

Artificial microRNAs (amiRNAs) are used for selective gene silencing in plants. However, current methods to produce amiRNA constructs for silencing transcripts in monocot species are not suitable for simple, cost-effective and large-scale synthesis. Here, a series of expression vectors based on Oryza sativa MIR390 (OsMIR390) precursor was developed for high-throughput cloning and high expression of amiRNAs in monocots. Four different amiRNA sequences designed to target specifically endogenous genes and expressed from OsMIR390-based vectors were validated in transgenic Brachypodium distachyon plants. Surprisingly, amiRNAs accumulated to higher levels and were processed more accurately when expressed from chimeric OsMIR390-based precursors that include distalmore » stem-loop sequences from Arabidopsis thaliana MIR390a (AtMIR390a). In all cases, transgenic plants displayed the predicted phenotypes induced by target gene repression, and accumulated high levels of amiRNAs and low levels of the corresponding target transcripts. Genome-wide transcriptome profiling combined with 5-RLM-RACE analysis in transgenic plants confirmed that amiRNAs were highly specific. Finally, significance Statement A series of amiRNA vectors based on Oryza sativa MIR390 (OsMIR390) precursor were developed for simple, cost-effective and large-scale synthesis of amiRNA constructs to silence genes in monocots. Unexpectedly, amiRNAs produced from chimeric OsMIR390-based precursors including Arabidopsis thaliana MIR390a distal stem-loop sequences accumulated elevated levels of highly effective and specific amiRNAs in transgenic Brachypodium distachyon plants.« less

Stable expression and purification of a functional processed Fab' fragment from a single nascent polypeptide in CHO cells expressing the mCAT-1 retroviral receptor.

PubMed

Camper, Nicolas; Byrne, Teresa; Burden, Roberta E; Lowry, Jenny; Gray, Breena; Johnston, James A; Migaud, Marie E; Olwill, Shane A; Buick, Richard J; Scott, Christopher J

2011-09-30

Monoclonal antibodies and derivative formats such as Fab' fragments are used in a broad range of therapeutic, diagnostic and research applications. New systems and methodologies that can improve the production of these proteins are consequently of much interest. Here we present a novel approach for the rapid production of processed Fab' fragments in a CHO cell line that has been engineered to express the mouse cationic amino acid transporter receptor 1 (mCAT-1). This facilitated the introduction of the target antibody gene through retroviral transfection, rapidly producing stable expression. Using this system, we designed a single retroviral vector construct for the expression of a target Fab' fragment as a single polypeptide with a furin cleavage site and a FMDV 2A self-cleaving peptide introduced to bridge the light and truncated heavy chain regions. The introduction of these cleavage motifs ensured equimolar expression and processing of the heavy and light domains as exemplified by the production of an active chimeric Fab' fragment against the Fas receptor, routinely expressed in 1-2mg/L yield in spinner-flask cell cultures. These results demonstrate that this method could have application in the facile production of bioactive Fab' fragments. Copyright © 2011 Elsevier B.V. All rights reserved.

Macroarray expression analysis of barley susceptibility and nonhost resistance to Blumeria graminis.

PubMed

Eichmann, Ruth; Biemelt, Sophia; Schäfer, Patrick; Scholz, Uwe; Jansen, Carin; Felk, Angelika; Schäfer, Wilhelm; Langen, Gregor; Sonnewald, Uwe; Kogel, Karl-Heinz; Hückelhoven, Ralph

2006-04-01

Different formae speciales of the grass powdery mildew fungus Blumeria graminis undergo basic-compatible or basic-incompatible (nonhost) interactions with barley. Background resistance in compatible interactions and nonhost resistance require common genetic and mechanistic elements of plant defense. To build resources for differential screening for genes that potentially distinguish a compatible from an incompatible interaction on the level of differential gene expression of the plant, we constructed eight dedicated cDNA libraries, established 13.000 expressed sequence tag (EST) sequences and designed DNA macroarrays. Using macroarrays based on cDNAs derived from epidermal peels of plants pretreated with the chemical resistance activating compound acibenzolar-S-methyl, we compared the expression of barley gene transcripts in the early host interaction with B. graminis f.sp. hordei or the nonhost pathogen B. graminis f.sp. tritici, respectively. We identified 102 spots corresponding to 94 genes on the macroarray that gave significant B. graminis-responsive signals at 12 and/or 24 h after inoculation. In independent expression analyses, we confirmed the macroarray results for 11 selected genes. Although the majority of genes showed a similar expression profile in compatible versus incompatible interactions, about 30 of the 94 genes were expressed on slightly different levels in compatible versus incompatible interactions.

Parental Expressivity and Parenting Styles in Chinese Families: Prospective and Unique Relations to Children's Psychological Adjustment.

PubMed

Chen, Stephen H; Zhou, Qing; Eisenberg, Nancy; Valiente, Carlos; Wang, Yun

2011-01-01

OBJECTIVES: Parents from different cultures differ in how frequently they express emotions. However, the generalizability of the relations between parental expressivity and child adjustment in non-Western cultures has not been extensively studied. The goal of the present study was to investigate prospective relations between parental expressivity within the family (positive, negative dominant, and negative submissive expressivity) and Chinese children's psychological adjustment, above and beyond parenting styles. DESIGN: The study used two waves (3.8 years apart) of longitudinal data from a sample (n= 425) of children in Beijing (mean ages = 7.7 years at T1 and 11.6 years at T2). Parental expressivity and parenting styles were self-reported. To reduce the potential measurement overlap, items that tap parental expression of emotions toward the child were removed from the parenting style measure. Children's adjustment was measured with parents', teachers', and peers' or children's reports. RESULTS: Consistent with findings with European American samples, parental negative dominant expressivity uniquely and positively predicted Chinese children's externalizing problems controlling for prior externalizing problems, parenting styles, and family SES. Neither parental expressivity nor parenting styles uniquely predicted social competence. CONCLUSIONS: Despite previously reported cultural differences in the mean levels of parental expressivity, some of the socialization functions of parental expressivity found in Western countries can be generalized to Chinese families. Although parental expressivity and parenting styles are related constructs, their unique relations to child's adjustment suggest that they should be examined as distinct processes.

Mass of materials: the impact of designers on construction ergonomics.

PubMed

Smallwood, John

2012-01-01

Many construction injuries are musculoskeletal related in the form of sprains and strains arising from the handling of materials, which are specified by designers. The paper presents the results of a study conducted among delegates attending two 'designing for H&S' (DfH&S) seminars using a questionnaire. The salient findings include: the level of knowledge relative to the mass and density of materials is limited; designers generally do not consider the mass and density of materials when designing structures and elements and specifying materials; to a degree designers appreciate that the mass and density of materials impact on construction ergonomics; designers rate their knowledge of the mass and density of materials as limited, and designers appreciate the potential of the consideration of the mass and density of materials to contribute to an improvement in construction ergonomics. Conclusions include: designers lack the requisite knowledge relative to the mass and density of materials; designers are thus precluded from conducting optimum design hazard identification and risk assessments, and tertiary built environment designer education does not enlighten designers relative to construction ergonomics. Recommendations include: tertiary built environment designer education should construction ergonomics; professional associations should raise the level of awareness relative to construction ergonomics, and design practices should include a category 'mass and density of materials' in their practice libraries.

Lack of awareness for spatial and verbal constructive apraxia.

PubMed

Rinaldi, Maria Cristina; Piras, Federica; Pizzamiglio, Luigi

2010-05-01

It is still a matter of debate whether constructive apraxia (CA) should be considered a form of apraxia or, rather, the motor expression of a more pervasive impairment in visuo-spatial processing. Constructive disorders were linked to visuo-spatial disorders and to deficits in appreciating spatial relations among component sub-parts or problems in reproducing three-dimensionality. We screened a large population of brain-damaged patients for CA. Only patients with constructive disorders and no signs of neglect and/or aphasia were selected. Five apractic subjects were tested with both visuo-spatial and verbal tasks requiring constructive abilities. The former ones were tests such as design copying, while the latter were experimental tasks built to transpose into the linguistic domain the constructive process as phrasing by arranging paper scraps into a sentence. A first result showed a constructive impairment in both the visuo-spatial and the linguistic domain; this finding challenges the idea that CA is confined to the visuo-spatial domain. A second result showed a systematic association between CA and unawareness for constructive disorders. Third, lack of awareness was always associated with a lesion in the right dorsolateral prefrontal cortex, a region deemed as involved in managing a conflict between intentions and sensory feed-back. Anosognosia for constructive disorders and the potential role of the right prefrontal cortex in generating the impairment, are discussed in the light of current models of action control. The core of CA could be the inability to detect any inconsistency between intended and executed action rather than a deficit in reproducing spatial relationship. 2010 Elsevier Ltd. All rights reserved.

Construction and characterization of recombinant adenovirus carrying a mouse TIGIT-GFP gene.

PubMed

Zheng, J M; Cui, J L; He, W T; Yu, D W; Gao, Y; Wang, L; Chen, Z K; Zhou, H M

2015-12-29

Recombinant adenovirus vector systems have been used extensively in protein research and gene therapy. However, the construction and characterization of recombinant adenovirus is a tedious and time-consuming process. TIGIT is a recently discovered immunosuppressive molecule that plays an important role in maintaining immunological balance. The construction of recombinant adenovirus mediating TIGIT expression must be simplified to facilitate its use in the study of TIGIT. In this study, the TIGIT gene was combined with green fluorescent protein (GFP); the TIGIT-GFP gene was inserted into a gateway plasmid to construct a TIGIT-GFP adenovirus. HEK 293A cells were infected with the adenovirus, which was then purified and subjected to virus titering. TIGIT-GFP adenovirus was characterized by flow cytometry and immunofluorescence, and its expression in mouse liver was detected by infection through caudal vein injection. The results showed the successful construction of the TIGIT-GFP adenovirus (5 x 10(10) PFU/mL). Co-expression of TIGIT and GFP was identified in 293A and liver cells; synthesis and positioning of TIGIT-GFP was viewed under a fluorescence microscope. TIGIT-GFP was highly expressed on liver cells 1 day (25.53%) after infection and faded 3 days (11.36%) after injection. In conclusion, the fusion of TIGIT with GFP allows easy, rapid, and uncomplicated detection of TIGIT translation. The construction of a TIGIT-GFP adenovirus, mediating TIGIT expression in vitro and in vivo, lays the foundation for further research into TIGIT function and gene therapy. Moreover, the TIGIT-GFP adenovirus is a helpful tool for studying other proteins (which could replace the TIGIT gene).

Different applications of virus-like particles in biology and medicine: Vaccination and delivery systems.

PubMed

Shirbaghaee, Zeinab; Bolhassani, Azam

2016-03-01

Virus-like particles (VLPs) mimic the whole construct of virus particles devoid of viral genome as used in subunit vaccine design. VLPs can elicit efficient protective immunity as direct immunogens compared to soluble antigens co-administered with adjuvants in several booster injections. Up to now, several prokaryotic and eukaryotic systems such as insect, yeast, plant, and E. coli were used to express recombinant proteins, especially for VLP production. Recent studies are also generating VLPs in plants using different transient expression vectors for edible vaccines. VLPs and viral particles have been applied for different functions such as gene therapy, vaccination, nanotechnology, and diagnostics. Herein, we describe VLP production in different systems as well as its applications in biology and medicine. © 2015 Wiley Periodicals, Inc.

The Imperative in Chinese.

ERIC Educational Resources Information Center

Hashimoto, Anne Yue

A preliminary study of the syntactic characteristics of the imperative construction in modern Chinese is presented. The term "imperative" is used to refer to the type of syntactic construction which is marked by an implicit or explicit second person subject, and which expresses a direct command. Indirect or implied commands expressed by a…

Dual reporter transgene driven by 2.3Col1a1 promoter is active in differentiated osteoblasts

NASA Technical Reports Server (NTRS)

Marijanovic, Inga; Jiang, Xi; Kronenberg, Mark S.; Stover, Mary Louise; Erceg, Ivana; Lichtler, Alexander C.; Rowe, David W.

2003-01-01

AIM: As quantitative and spatial analyses of promoter reporter constructs are not easily performed in intact bone, we designed a reporter gene specific to bone, which could be analyzed both visually and quantitatively by using chloramphenicol acetyltransferase (CAT) and a cyan version of green fluorescent protein (GFPcyan), driven by a 2.3-kb fragment of the rat collagen promoter (Col2.3). METHODS: The construct Col2.3CATiresGFPcyan was used for generating transgenic mice. Quantitative measurement of promoter activity was performed by CAT analysis of different tissues derived from transgenic animals; localization was performed by visualized GFP in frozen bone sections. To assess transgene expression during in vitro differentiation, marrow stromal cell and neonatal calvarial osteoblast cultures were analyzed for CAT and GFP activity. RESULTS: In mice, CAT activity was detected in the calvaria, long bone, teeth, and tendon, whereas histology showed that GFP expression was limited to osteoblasts and osteocytes. In cell culture, increased activity of CAT correlated with increased differentiation, and GFP activity was restricted to mineralized nodules. CONCLUSION: The concept of a dual reporter allows a simultaneous visual and quantitative analysis of transgene activity in bone.

Cell of Origin: Exploring an Alternative Contributor to Ovarian Cancer

DTIC Science & Technology

2014-09-01

previously shown that DDX4 is expressed in ovarian carcinomas and its expression is associated with age and the serous histophenotype. Thus, we analyzed...oncogenic alleles of human TP53, AKT1, KRAS, and PIK3CA were constructed and initially validated in both a human endometrial cancer cell line and mouse...AKT1, KRAS, or PIK3CA were successfully constructed. 9. The viral constructs were initially validated in a human endometrial cancer cell line and

APPLICATION OF THE 3D MODEL OF RAILWAY VIADUCTS TO COST ESTIMATION AND CONSTRUCTION

NASA Astrophysics Data System (ADS)

Fujisawa, Yasuo; Yabuki, Nobuyoshi; Igarashi, Zenichi; Yoshino, Hiroyuki

Three dimensional models of civil engineering structures are only partially used in either design or construction but not both. Research on integration of design, cost estimation and construction by 3Dmodels has not been heard in civil engineering domain yet. Using continuously a 3D product model of a structure from design to construction through estimation should improve the efficiency and decrease the occurrence of mistakes, hence enhancing the quality. In this research, we investigated the current practices of flow from design to construction, particularly focusing on cost estimation. Then, we identified advantages and issues on utilization of 3D design models to estimation and construction by applying 3D models to an actual railway construction project.

Fuel Crime Conceptualization through Specialization of Ontology for Investigation Management System

NASA Astrophysics Data System (ADS)

Cybulka, Jolanta

We undertook the task of building the conceptual model of a particular economic offense, called "a fuel crime". This model is thought of as a part of a larger conceptualization, which comprises consensual semantics underlying the knowledge base of a system, aimed at supporting the teamwork of investigators of economic crimes. Because such a knowledge-based system represents a perspective on economic crimes, it should be carefully modeled. This can be done with the help of an expressive enough ontology. To achieve our goal we use the constructive descriptions and situations (c.DnS) design pattern, which enables us to construct an extensible, layered ontology in a top-down manner: c.DnS top layer is specialized by the reference ontology for investigation management system, that in turn, is specialized by the ontology of the fuel crime.

41 CFR 102-76.30 - What seismic safety standards must Federal agencies follow in the design and construction of...

Code of Federal Regulations, 2013 CFR

2013-07-01

... (Continued) FEDERAL MANAGEMENT REGULATION REAL PROPERTY 76-DESIGN AND CONSTRUCTION Design and Construction... 41 Public Contracts and Property Management 3 2013-07-01 2013-07-01 false What seismic safety standards must Federal agencies follow in the design and construction of Federal facilities? 102-76.30...

41 CFR 102-76.30 - What seismic safety standards must Federal agencies follow in the design and construction of...

Code of Federal Regulations, 2011 CFR

2011-01-01

... (Continued) FEDERAL MANAGEMENT REGULATION REAL PROPERTY 76-DESIGN AND CONSTRUCTION Design and Construction... 41 Public Contracts and Property Management 3 2011-01-01 2011-01-01 false What seismic safety standards must Federal agencies follow in the design and construction of Federal facilities? 102-76.30...

41 CFR 102-76.30 - What seismic safety standards must Federal agencies follow in the design and construction of...

Code of Federal Regulations, 2014 CFR

2014-01-01

... (Continued) FEDERAL MANAGEMENT REGULATION REAL PROPERTY 76-DESIGN AND CONSTRUCTION Design and Construction... 41 Public Contracts and Property Management 3 2014-01-01 2014-01-01 false What seismic safety standards must Federal agencies follow in the design and construction of Federal facilities? 102-76.30...

41 CFR 102-76.30 - What seismic safety standards must Federal agencies follow in the design and construction of...

Code of Federal Regulations, 2012 CFR

2012-01-01

... (Continued) FEDERAL MANAGEMENT REGULATION REAL PROPERTY 76-DESIGN AND CONSTRUCTION Design and Construction... 41 Public Contracts and Property Management 3 2012-01-01 2012-01-01 false What seismic safety standards must Federal agencies follow in the design and construction of Federal facilities? 102-76.30...

41 CFR 102-76.30 - What seismic safety standards must Federal agencies follow in the design and construction of...

Code of Federal Regulations, 2010 CFR

2010-07-01

... (Continued) FEDERAL MANAGEMENT REGULATION REAL PROPERTY 76-DESIGN AND CONSTRUCTION Design and Construction... 41 Public Contracts and Property Management 3 2010-07-01 2010-07-01 false What seismic safety standards must Federal agencies follow in the design and construction of Federal facilities? 102-76.30...

Genetic circuit design automation.

PubMed

Nielsen, Alec A K; Der, Bryan S; Shin, Jonghyeon; Vaidyanathan, Prashant; Paralanov, Vanya; Strychalski, Elizabeth A; Ross, David; Densmore, Douglas; Voigt, Christopher A

2016-04-01

Computation can be performed in living cells by DNA-encoded circuits that process sensory information and control biological functions. Their construction is time-intensive, requiring manual part assembly and balancing of regulator expression. We describe a design environment, Cello, in which a user writes Verilog code that is automatically transformed into a DNA sequence. Algorithms build a circuit diagram, assign and connect gates, and simulate performance. Reliable circuit design requires the insulation of gates from genetic context, so that they function identically when used in different circuits. We used Cello to design 60 circuits forEscherichia coli(880,000 base pairs of DNA), for which each DNA sequence was built as predicted by the software with no additional tuning. Of these, 45 circuits performed correctly in every output state (up to 10 regulators and 55 parts), and across all circuits 92% of the output states functioned as predicted. Design automation simplifies the incorporation of genetic circuits into biotechnology projects that require decision-making, control, sensing, or spatial organization. Copyright © 2016, American Association for the Advancement of Science.

Hydrostatic pressure modulates mRNA expressions for matrix proteins in human meniscal cells.

PubMed

Suzuki, Toru; Toyoda, Takashi; Suzuki, Hiroshi; Hisamori, Noriyuki; Matsumoto, Hideo; Toyama, Yoshiaki

2006-01-01

There have been few reports describing the effects of mechanical loading on the metabolism of meniscal cells. The aim of this study was to investigate the effects of hydrostatic pressure on meniscal cell metabolism. Human meniscal cells were cultured in alginate beads for 3 days. They were then subjected to 4 MPa hydrostatic pressure for 4 hours in either a static or cyclic (1 Hz) mode using a specially designed and constructed system. Immediately after the pressure application, the messenger RNA levels for aggrecan, type I collagen, matrix metalloproteinases (MMP) -1, -3, -9, -13 and tissue inhibitors of metalloproteinases (TIMP) -1 and -2 were measured. It was found that the application of static hydrostatic pressure caused a significant decrease in mRNA expression for MMP-1 and -13 (p<0.05). In contrast, the application of cyclic hydrostatic pressure was associated with a significant increase in type I collagen (p<0.01), TIMP-1 and -2 mRNA expression (p<0.01). These results would suggest that hydrostatic pressure in isolation can modulate mRNA expressions for matrix proteins in meniscal cells.

Ugene, a newly identified protein that is commonly over-expressed in cancer, and that binds uracil DNA-glycosylase

PubMed Central

Guo, Chunguang; Zhang, Xiaodong; Fink, Stephen P; Platzer, Petra; Wilson, Keith; Willson, James K. V.; Wang, Zhenghe; Markowitz, Sanford D

2008-01-01

Expression microarrays identified a novel transcript, designated as Ugene, whose expression is absent in normal colon and colon adenomas, but that is commonly induced in malignant colon cancers. These findings were validated by real-time PCR and Northern blot analysis in an independent panel of colon cancer cases. In addition, Ugene expression was found to be elevated in many other common cancer types, including, breast, lung, uterus, and ovary. Immunofluorescence of V5-tagged Ugene revealed it to have a nuclear localization. In a pull-down assay, uracil DNA-glycosylase 2 (UNG2), an important enzyme in the base excision repair pathway, was identified as a partner protein that binds to Ugene. Co-immunoprecipitation and Western blot analysis confirmed the binding between the endogenous Ugene and UNG2 proteins. Using deletion constructs, we find that Ugene binds to the first 25 amino acids of the UNG2 NH2-terminus. We suggest Ugene induction in cancer may contribute to the cancer phenotype by interacting with the base excision repair pathway. PMID:18676834

Construction of minitransposons for constitutive and inducible expression of pertussis toxin in bvg-negative Bordetella bronchiseptica.

PubMed Central

Walker, M J; Rohde, M; Wehland, J; Timmis, K N

1991-01-01

Appropriately detoxified pertussis toxin (PT) of Bordetella pertussis is considered to be an essential component of new-generation whooping cough vaccines, but the development of a procedure to obtain high levels of purified toxin has been and continues to be a major difficulty. To produce a system enabling the biological separation of PT from other virulence determinants of B. pertussis and the attainment of high yields of the toxin, minitransposons containing the PT operon were constructed and stably integrated into the chromosome of Bordetella virulence regulatory gene (bvg)-negative Bordetella bronchiseptica ATCC 10580. Since the minitransposons introduced into Bordetella spp. lack the cognate transposase function, they are unable to undergo further transposition events or mediate gene deletions and rearrangements that lead to strain instability. The TnPtacPT minitransposon contains the PT operon under the control of the tac promoter and directs IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible expression of PT in B. bronchiseptica ATCC 10580. The level of IPTG-induced PT expression was, however, lower than that found for the wild-type B. pertussis Tohama I strain. The TnfusPT minitransposon contains a promoterless PT operon which is only expressed after insertion of the transposon downstream of an appropriately oriented indigenous promoter. After "promoter probing" of B. bronchiseptica with the transposon, clones were screened for PT production by immunoblotting with specific monoclonal antibodies. One clone, designated B. bronchiseptica 10580:: TnfusPT1, expresses significantly higher levels of PT than does B. pertussis Tohama I. The recombinant toxin produced was biologically active in the Chinese hamster ovary cell-clustering assay. High-level expression of PT from a B. bronchiseptica host promoter should provide better yields of the toxin from bacteria not producing other bvg-regulated pathogenesis factors that may play a role in the undesired side effects of current pertussis vaccine preparations. Images PMID:1682257

Positive deviance study to inform a Chagas disease control program in southern Ecuador.

PubMed

Nieto-Sanchez, Claudia; Baus, Esteban G; Guerrero, Darwin; Grijalva, Mario J

2015-05-01

Chagas disease is caused by Trypanosoma cruzi, which is mainly transmitted by the faeces of triatomine insects that find favourable environments in poorly constructed houses. Previous studies have documented persistent triatomine infestation in houses in the province of Loja in southern Ecuador despite repeated insecticide and educational interventions. We aim to develop a sustainable strategy for the interruption of Chagas disease transmission by promoting living environments that are designed to prevent colonisation of rural houses by triatomines. This study used positive deviance to inform the design of an anti-triatomine prototype house by identifying knowledge, attitudes and practices used by families that have remained triatomine-free (2010-2012). Positive deviants reported practices that included maintenance of structural elements of the house, fumigation of dwellings and animal shelters, sweeping with "insect repellent" plants and relocation of domestic animals away from the house, among others. Participants favoured construction materials that do not drastically differ from those currently used (adobe walls and tile roofs). They also expressed their belief in a clear connection between a clean house and health. The family's economic dynamics affect space use and must be considered in the prototype's design. Overall, the results indicate a positive climate for the introduction of housing improvements as a protective measure against Chagas disease in this region.

Positive deviance study to inform a Chagas disease control program in southern Ecuador

PubMed Central

Nieto-Sanchez, Claudia; Baus, Esteban G; Guerrero, Darwin; Grijalva, Mario J

2015-01-01

Chagas disease is caused by Trypanosoma cruzi, which is mainly transmitted by the faeces of triatomine insects that find favourable environments in poorly constructed houses. Previous studies have documented persistent triatomine infestation in houses in the province of Loja in southern Ecuador despite repeated insecticide and educational interventions. We aim to develop a sustainable strategy for the interruption of Chagas disease transmission by promoting living environments that are designed to prevent colonisation of rural houses by triatomines. This study used positive deviance to inform the design of an anti-triatomine prototype house by identifying knowledge, attitudes and practices used by families that have remained triatomine-free (2010-2012). Positive deviants reported practices that included maintenance of structural elements of the house, fumigation of dwellings and animal shelters, sweeping with "insect repellent" plants and relocation of domestic animals away from the house, among others. Participants favoured construction materials that do not drastically differ from those currently used (adobe walls and tile roofs). They also expressed their belief in a clear connection between a clean house and health. The family's economic dynamics affect space use and must be considered in the prototype's design. Overall, the results indicate a positive climate for the introduction of housing improvements as a protective measure against Chagas disease in this region. PMID:25807468

A Novel Cassette Method for Probe Evaluation in the Designed Biochips

PubMed Central

Zinkevich, Vitaly; Sapojnikova, Nelly; Mitchell, Julian; Kartvelishvili, Tamar; Asatiani, Nino; Alkhalil, Samia; Bogdarina, Irina; Al-Humam, Abdulmohsen A.

2014-01-01

A critical step in biochip design is the selection of probes with identical hybridisation characteristics. In this article we describe a novel method for evaluating DNA hybridisation probes, allowing the fine-tuning of biochips, that uses cassettes with multiple probes. Each cassette contains probes in equimolar proportions so that their hybridisation performance can be assessed in a single reaction. The model used to demonstrate this method was a series of probes developed to detect TORCH pathogens. DNA probes were designed for Toxoplasma gondii, Chlamidia trachomatis, Rubella, Cytomegalovirus, and Herpes virus and these were used to construct the DNA cassettes. Five cassettes were constructed to detect TORCH pathogens using a variety of genes coding for membrane proteins, viral matrix protein, an early expressed viral protein, viral DNA polymerase and the repetitive gene B1 of Toxoplasma gondii. All of these probes, except that for the B1 gene, exhibited similar profiles under the same hybridisation conditions. The failure of the B1 gene probe to hybridise was not due to a position effect, and this indicated that the probe was unsuitable for inclusion in the biochip. The redesigned probe for the B1 gene exhibited identical hybridisation properties to the other probes, suitable for inclusion in a biochip. PMID:24897111

Upcoming Purchasing and Subcontracting Opportunities | NREL

Science.gov Websites

Services Fall 2022 Construction@nrel.gov Design-Build and Construction Spring 2018 Construction@nrel.gov Design-Build for Minor Construction Spring 2020 Construction@nrel.gov Testing of Fuel and Oil Samples

Bacterial cell-free expression technology to in vitro systems engineering and optimization.

PubMed

Caschera, Filippo

2017-06-01

Cell-free expression system is a technology for the synthesis of proteins in vitro . The system is a platform for several bioengineering projects, e.g. cell-free metabolic engineering, evolutionary design of experiments, and synthetic minimal cell construction. Bacterial cell-free protein synthesis system (CFPS) is a robust tool for synthetic biology. The bacteria lysate, the DNA, and the energy module, which are the three optimized sub-systems for in vitro protein synthesis, compose the integrated system. Currently, an optimized E. coli cell-free expression system can produce up to ∼2.3 mg/mL of a fluorescent reporter protein. Herein, I will describe the features of ATP-regeneration systems for in vitro protein synthesis, and I will present a machine-learning experiment for optimizing the protein yield of E. coli cell-free protein synthesis systems. Moreover, I will introduce experiments on the synthesis of a minimal cell using liposomes as dynamic containers, and E. coli cell-free expression system as biochemical platform for metabolism and gene expression. CFPS can be further integrated with other technologies for novel applications in environmental, medical and material science.

Deep learning of the regulatory grammar of yeast 5′ untranslated regions from 500,000 random sequences

PubMed Central

Groves, Benjamin; Kuchina, Anna; Rosenberg, Alexander B.; Jojic, Nebojsa; Fields, Stanley; Seelig, Georg

2017-01-01

Our ability to predict protein expression from DNA sequence alone remains poor, reflecting our limited understanding of cis-regulatory grammar and hampering the design of engineered genes for synthetic biology applications. Here, we generate a model that predicts the protein expression of the 5′ untranslated region (UTR) of mRNAs in the yeast Saccharomyces cerevisiae. We constructed a library of half a million 50-nucleotide-long random 5′ UTRs and assayed their activity in a massively parallel growth selection experiment. The resulting data allow us to quantify the impact on protein expression of Kozak sequence composition, upstream open reading frames (uORFs), and secondary structure. We trained a convolutional neural network (CNN) on the random library and showed that it performs well at predicting the protein expression of both a held-out set of the random 5′ UTRs as well as native S. cerevisiae 5′ UTRs. The model additionally was used to computationally evolve highly active 5′ UTRs. We confirmed experimentally that the great majority of the evolved sequences led to higher protein expression rates than the starting sequences, demonstrating the predictive power of this model. PMID:29097404

Anatomically shaped tissue-engineered cartilage with tunable and inducible anticytokine delivery for biological joint resurfacing

PubMed Central

Moutos, Franklin T.; Glass, Katherine A.; Compton, Sarah A.; Ross, Alison K.; Gersbach, Charles A.; Estes, Bradley T.

2016-01-01

Biological resurfacing of entire articular surfaces represents an important but challenging strategy for treatment of cartilage degeneration that occurs in osteoarthritis. Not only does this approach require anatomically sized and functional engineered cartilage, but the inflammatory environment within an arthritic joint may also inhibit chondrogenesis and induce degradation of native and engineered cartilage. The goal of this study was to use adult stem cells to engineer anatomically shaped, functional cartilage constructs capable of tunable and inducible expression of antiinflammatory molecules, specifically IL-1 receptor antagonist (IL-1Ra). Large (22-mm-diameter) hemispherical scaffolds were fabricated from 3D woven poly(ε-caprolactone) (PCL) fibers into two different configurations and seeded with human adipose-derived stem cells (ASCs). Doxycycline (dox)-inducible lentiviral vectors containing eGFP or IL-1Ra transgenes were immobilized to the PCL to transduce ASCs upon seeding, and constructs were cultured in chondrogenic conditions for 28 d. Constructs showed biomimetic cartilage properties and uniform tissue growth while maintaining their anatomic shape throughout culture. IL-1Ra–expressing constructs produced nearly 1 µg/mL of IL-1Ra upon controlled induction with dox. Treatment with IL-1 significantly increased matrix metalloprotease activity in the conditioned media of eGFP-expressing constructs but not in IL-1Ra–expressing constructs. Our findings show that advanced textile manufacturing combined with scaffold-mediated gene delivery can be used to tissue engineer large anatomically shaped cartilage constructs that possess controlled delivery of anticytokine therapy. Importantly, these cartilage constructs have the potential to provide mechanical functionality immediately upon implantation, as they will need to replace a majority, if not the entire joint surface to restore function. PMID:27432980

Symbolic computation of the Birkhoff normal form in the problem of stability of the triangular libration points

NASA Astrophysics Data System (ADS)

Shevchenko, I. I.

2008-05-01

The problem of stability of the triangular libration points in the planar circular restricted three-body problem is considered. A software package, intended for normalization of autonomous Hamiltonian systems by means of computer algebra, is designed so that normalization problems of high analytical complexity could be solved. It is used to obtain the Birkhoff normal form of the Hamiltonian in the given problem. The normalization is carried out up to the 6th order of expansion of the Hamiltonian in the coordinates and momenta. Analytical expressions for the coefficients of the normal form of the 6th order are derived. Though intermediary expressions occupy gigabytes of the computer memory, the obtained coefficients of the normal form are compact enough for presentation in typographic format. The analogue of the Deprit formula for the stability criterion is derived in the 6th order of normalization. The obtained floating-point numerical values for the normal form coefficients and the stability criterion confirm the results by Markeev (1969) and Coppola and Rand (1989), while the obtained analytical and exact numeric expressions confirm the results by Meyer and Schmidt (1986) and Schmidt (1989). The given computational problem is solved without constructing a specialized algebraic processor, i.e., the designed computer algebra package has a broad field of applicability.

Chemical stress sensitive luminescent human cells: Molecular biology approach using inducible Drosophila melanogaster hsp22 promoter

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

Mandon, C.A.; Diaz, C.; Arrigo, A.-P.

2005-09-23

A whole-cell bioassay has been developed for the total toxicity testing of liquid samples. The method is based on the induction of the bioluminescent activity of genetically manipulated mammalian cells. For that purpose, transfection was used to introduce, in HeLa cells, a DNA sensing element that responds to chemical stress agents (heavy metals, genotoxic agents, and endocrine-disrupting chemicals). Such element was designed to direct the expression of a reporting gene (firefly luciferase) through the activation of Drosophila melanogaster hsp22 promoter. A molecular approach was conducted to optimize hsp22 promoter element in order to decrease the background expression level of themore » reporting gene and to increase the sensitivity of the bioassay for testing endocrine disruptors. As a result, in the presence of 20-100 {mu}M cadmium chloride, a 6-fold increase in luciferase expression was obtained using a specially designed truncated hsp22 promoter construction. The following chemicals known to be found in the polluted samples were tested: CdCl{sub 2}, Cd(NO{sub 3}){sub 2}, NaAsO{sub 2}, alachlore, fentine acetate, thiram, and maneb. The stressing effect of each of them was sensitively detected by the present bioassay in the 0.05-50 {mu}M concentration range.« less

Development of a GFP expression vector for Cucurbit chlorotic yellows virus.

PubMed

Wei, Ying; Han, Xiaoyu; Wang, Zhenyue; Gu, Qinsheng; Li, Honglian; Chen, Linlin; Sun, Bingjian; Shi, Yan

2018-05-24

Cucurbit chlorotic yellows virus (CCYV), a bipartite crinivirus, causes chlorotic leaf spots and yellowing symptoms on cucurbit leaves. We previously developed an infectious clone of CCYV. Limited work has been conducted on the construction of a crinivirus green fluorescence protein (GFP) expression vector to date. We constructed a CCYV GFP expression vector using the "add a gene" strategy based on CCYV RNA2 cDNA constrcut. Three resultant clones, pCCYVGFP SGC , pCCYVGFP CGC , and pCCYVGFP CGS, were constructed with different promoters used to initiate GFP and CP expression. At 25 dpi GFP fluorescence was detectable not only in leaf veins but also in the surrounding cells. pCCYVGFP CGC -infected cucumber leaves exhibited cell spread at 25 dpi, whereas pCCYVGFP SGC and pCCYVGFP CGS were mainly found in single cells. Further observation of pCCYVGFP CGC GFP expression at 30 dpi, 40 dpi, and 50 dpi showed phloem-limited localization in the systemic leaves. We developed of a CCYV GFP expression vector that will be useful for further study of CCYV movement in cucurbits.

Engineering starch accumulation by manipulation of phosphate metabolism of starch.

PubMed

Weise, Sean E; Aung, Kimberly; Jarou, Zach J; Mehrshahi, Payam; Li, Ziru; Hardy, Anna C; Carr, David J; Sharkey, Thomas D

2012-06-01

A new understanding of leaf starch degradation has emerged in the last 10 years. It has been shown that starch phosphorylation and dephosphorylation are critical components of this process. Glucan, water dikinase (GWD) (and phosphoglucan, water dikinase) adds phosphate to starch, and phosphoglucan phosphatase (SEX4) removes these phosphates. To explore the use of this metabolism to manipulate starch accumulation, Arabidopsis (Arabidopsis thaliana) plants were engineered by introducing RNAi constructs designed to reduce expression of AtGWD and AtSEX4. The timing of starch build-up was altered with ethanol-inducible and senescence-induced gene promoters. Ethanol induction of RNAi lines reduced transcript for AtGWD and AtSEX4 by 50%. The transgenic lines had seven times more starch than wild type at the end of the dark period but similar growth rates and total biomass. Elevated leaf starch content in maize leaves was engineered by making an RNAi construct against a gene in maize that appeared to be homologous to AtGWD. The RNAi construct was expressed using the constitutive ubiquitin promoter. Leaf starch content at the end of a night period in engineered maize plants was 20-fold higher than in untransformed plants with no impact on total plant biomass. We conclude that plants can be engineered to accumulate starch in the leaves with little impact on vegetative biomass. © 2012 The Authors. Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

[Promoting effect of cyclin D1 overexpression on proliferation and epithelial mesenchymal transition of cervical squamous cell carcinoma SiHa cells].

PubMed

Wang, P; Liu, S; Cheng, B; Wu, X Z; Ding, S S; Xu, L; Liu, Y; Duan, L; Sun, S Z

2017-03-08

Objective: To study effects of cyclin D1 overexpression on the proliferation and differentiation of cervical squamous cell carcinoma SiHa cells and to investigate related signaling molecules. Methods: Primers were designed to amplify the full length of cyclin D1 gene and cyclin D1 gene was amplified by PCR for constructing pcDNA3.1 plasmid vector. The construct was then transfected into SiHa cells, and the cells with stable overexpression of cyclin D1 were established, cyclin D1 gene and protein expression were detected by RT-PCR and Western blot, respectively. Cell growth curve was documented by MTT assay. CK7, E-cadherin, vimentin, Snail gene and protein expression in transfected cells were detected by RT-PCR and Western blot. RT-PCR was used to detect the mRNA expression of proliferation and differentiation-related genes like CDK4, CDK2, p21, p27, cyclin E, Rb, E2F, E6/E7 and Ki-67. After synchronization of cells, RT-PCR was used to detect of cyclin D1 and p21 mRNA expression at different time points of the cell cycle. Results: The G-3 cells with cyclin D1 overexpression were successfully established. The growth curve and Ki-67 mRNA expression accelerated in G-3 cells.Vimentin and Snail expression significantly increased at both gene and protein levels, while E-cadherin, CK7 gene and protein expression significantly decreased, indicating epithelial mesenchymal transitionoccurred in G-3 cells.Meanwhile, mRNA expression of cyclin D1, CDK4, CDK2, p21, p27, cyclin E, E2F and Rb increased, while E6/E7 and p16 showed no significant change. The expression trends of p21 and cyclin D1 were almost identical with fluctuation at different time points in the cell cycle. Conclusions: Overexpression of cyclin D1 induced by gene transfection promotes proliferation and epithelial mesenchymal transition in SiHa cells.The process is accompanied by up-regulation of CDK4, CDK2, p21, p27 and cyclin E genes.p21 expression increases synchronously with cyclin D1, suggesting a regulatory role in epithelial mesenchymal transition by affecting expression of vimentin in G-3 cells.

Grammatical Constructions as Relational Categories.

PubMed

Goldwater, Micah B

2017-07-01

This paper argues that grammatical constructions, specifically argument structure constructions that determine the "who did what to whom" part of sentence meaning and how this meaning is expressed syntactically, can be considered a kind of relational category. That is, grammatical constructions are represented as the abstraction of the syntactic and semantic relations of the exemplar utterances that are expressed in that construction, and it enables the generation of novel exemplars. To support this argument, I review evidence that there are parallel behavioral patterns between how children learn relational categories generally and how they learn grammatical constructions specifically. Then, I discuss computational simulations of how grammatical constructions are abstracted from exemplar sentences using a domain-general relational cognitive architecture. Last, I review evidence from adult language processing that shows parallel behavioral patterns with expert behavior from other cognitive domains. After reviewing the evidence, I consider how to integrate this account with other theories of language development. Copyright © 2017 Cognitive Science Society, Inc.

[The expression of interferon-lambda1 in CHO cell].

PubMed

Yuan, Wu-Mei; Ma, Fen-Lian; Zhang, Qian; Zheng, Wen-Zhi; Zheng, Li-Shu

2013-06-01

To construct the eukaryotic expression vector PCI-dhfr-lambda1 and PCI-dhfr-SP163-lambda1 which linked the enhancer SP163 with interferon lambda1. Then express the interferon lambda1 in CHO (dhfr-) cells. Using PCR method to introduce the restriction enzyme sites and through the fusion PCR binding the enhancer with the interferon Lambda1. After sequenced, lambda1 and SP163-lambda1 was inserted into PCI-dhfr forming the expression vector PCI-dhfr-lambda1 and PCI-dhfr-SP163-lambda1 which was constructed successfully confirming by sequencing. Then the expressing vectors were transfected into CHO (dhfr-) cells using liposome transfection method and interferon lambda1 protein was assayed with indirect immunofluorescence and Western Blot. Using cytopathic effect inhibition evaluated the antiviral activity of interferon lambda1. Successfully constructing the eukaryotic expression vectors of interferon lambda and the vectors could express interferon lambda1. The result of immunofluorescence showed the enhancer developed the expression of interferon lambda1. Detecting the interferon lambda1 in CHO (dhfr-) cells after transfecting 48 hour using Western Blot. The cytopathic effect inhibition showed the expressed interferon lambda1 has the antiviral activity. Successfully expressed the interferon lambda1 in CHO (dhfr-) cells and the protein possesses antiviral activity, which may supply a valuable basis for building the stable cell line of interferon lambda1.

[Construction of the eukaryotic recombinant vector and expression of the outer membrane protein LipL32 gene from Leptospira serovar Lai].

PubMed

Huang, Bi; Bao, Lang; Zhong, Qi; Shang, Zheng-ling; Zhang, Hui-dong; Zhang, Ying

2008-02-01

To construct the eukaryotic experssion vector of LipL32 gene from Leptospira serovar Lai and express the recombinant plasmid in COS-7 cell. The LipL32 gene was amplified from Leptospira strain 017 genomic DNA by PCR and cloned into pcDNA3.1, through restriction nuclease enzyme digestion. Then the recombinant plasmid was transformed into E.coli DH5alpha. After identified by nuclease digestion, PCR and sequencing analysis, the recombinant vector was transfected into COS-7 cell with lipsome. The expression of the target gene was detected by RT-PCR and Western blot. The eukaryotic experssion vector pcDNA3.1-LipL32 was successfully constructed and stably expressed in COS-7 cell. The eukaryotic recombinant vector of outer membrane protein LipL32 gene from Leptospira serovar Lai can be expressed in mammalian cell, which provides an experimental basis for the application of the Leptospira DNA vaccine.

Expressed Sense of Self by People With Alzheimer's Disease in a Support Group Interpreted in Terms of Agency and Communion.

PubMed

Hedman, Ragnhild; Hansebo, Görel; Ternestedt, Britt-Marie; Hellström, Ingrid; Norberg, Astrid

2016-04-01

The self is constructed in cooperation with other people and social context influences how people perceive and express it. People with Alzheimer's disease (AD) often receive insufficient support in constructing their preferred selves, but little is known about how they express themselves together with other people with AD. In accordance with Harré's social constructionist theory of self, this study aimed to describe how five people with mild and moderate AD express their Self 2 (i.e., their personal attributes and life histories) in a support group with a facilitator experienced in communicating with people with AD. The participants' expressions of their Self 2 were analyzed with qualitative abductive content analysis and interpreted in terms of agency and communion and a lack of agency and communion. The findings highlight the importance of supporting a sense of agency and communion when assisting people with AD in constructing their self. © The Author(s) 2014.

The trajectories of Prevention through Design in construction.

PubMed

Toole, T Michael; Gambatese, John

2008-01-01

Construction Hazards Prevention through Design (CHPtD) is a process in which engineers and architects explicitly consider the safety of construction workers during the design process. Although articles on CHPtD have appeared in top construction journals, the literature has not addressed technical principles underlying CHPtD to help designers better perform CHPtD, to facilitate the development of additional CHPtD tools, and to predict the future path of CHPtD. This theoretical paper uses the existing literature on CHPtD and current action research associated with several CHPtD workgroups to analyze how CHPtD will likely evolve over the coming decades. There are four trajectories along which CHPtD will progress. (a) Designs will increasingly facilitate prefabricated construction; (b) designers will increasingly choose materials and systems that are inherently safer than alternatives; (c) designers will increasingly perform construction engineering; and (d) designers will increasingly apply spatial considerations to reduce worker hazards. By understanding how CHPtD may be manifested in the engineering-procurement-construction (EPC) industry, practitioners can better prepare for adopting CHPtD within their organizations and construction and engineering educators can better prepare their graduates to perform CHPtD.

The Efficiency of Split Panel Designs in an Analysis of Variance Model

PubMed Central

Wang, Wei-Guo; Liu, Hai-Jun

2016-01-01

We consider split panel design efficiency in analysis of variance models, that is, the determination of the cross-sections series optimal proportion in all samples, to minimize parametric best linear unbiased estimators of linear combination variances. An orthogonal matrix is constructed to obtain manageable expression of variances. On this basis, we derive a theorem for analyzing split panel design efficiency irrespective of interest and budget parameters. Additionally, relative estimator efficiency based on the split panel to an estimator based on a pure panel or a pure cross-section is present. The analysis shows that the gains from split panel can be quite substantial. We further consider the efficiency of split panel design, given a budget, and transform it to a constrained nonlinear integer programming. Specifically, an efficient algorithm is designed to solve the constrained nonlinear integer programming. Moreover, we combine one at time designs and factorial designs to illustrate the algorithm’s efficiency with an empirical example concerning monthly consumer expenditure on food in 1985, in the Netherlands, and the efficient ranges of the algorithm parameters are given to ensure a good solution. PMID:27163447

Domain selection combined with improved cloning strategy for high throughput expression of higher eukaryotic proteins

PubMed Central

Chen, Yunjia; Qiu, Shihong; Luan, Chi-Hao; Luo, Ming

2007-01-01

Background Expression of higher eukaryotic genes as soluble, stable recombinant proteins is still a bottleneck step in biochemical and structural studies of novel proteins today. Correct identification of stable domains/fragments within the open reading frame (ORF), combined with proper cloning strategies, can greatly enhance the success rate when higher eukaryotic proteins are expressed as these domains/fragments. Furthermore, a HTP cloning pipeline incorporated with bioinformatics domain/fragment selection methods will be beneficial to studies of structure and function genomics/proteomics. Results With bioinformatics tools, we developed a domain/domain boundary prediction (DDBP) method, which was trained by available experimental data. Combined with an improved cloning strategy, DDBP had been applied to 57 proteins from C. elegans. Expression and purification results showed there was a 10-fold increase in terms of obtaining purified proteins. Based on the DDBP method, the improved GATEWAY cloning strategy and a robotic platform, we constructed a high throughput (HTP) cloning pipeline, including PCR primer design, PCR, BP reaction, transformation, plating, colony picking and entry clones extraction, which have been successfully applied to 90 C. elegans genes, 88 Brucella genes, and 188 human genes. More than 97% of the targeted genes were obtained as entry clones. This pipeline has a modular design and can adopt different operations for a variety of cloning/expression strategies. Conclusion The DDBP method and improved cloning strategy were satisfactory. The cloning pipeline, combined with our recombinant protein HTP expression pipeline and the crystal screening robots, constitutes a complete platform for structure genomics/proteomics. This platform will increase the success rate of purification and crystallization dramatically and promote the further advancement of structure genomics/proteomics. PMID:17663785

Affective loop experiences: designing for interactional embodiment.

PubMed

Höök, Kristina

2009-12-12

Involving our corporeal bodies in interaction can create strong affective experiences. Systems that both can be influenced by and influence users corporeally exhibit a use quality we name an affective loop experience. In an affective loop experience, (i) emotions are seen as processes, constructed in the interaction, starting from everyday bodily, cognitive or social experiences; (ii) the system responds in ways that pull the user into the interaction, touching upon end users' physical experiences; and (iii) throughout the interaction the user is an active, meaning-making individual choosing how to express themselves-the interpretation responsibility does not lie with the system. We have built several systems that attempt to create affective loop experiences with more or less successful results. For example, eMoto lets users send text messages between mobile phones, but in addition to text, the messages also have colourful and animated shapes in the background chosen through emotion-gestures with a sensor-enabled stylus pen. Affective Diary is a digital diary with which users can scribble their notes, but it also allows for bodily memorabilia to be recorded from body sensors mapping to users' movement and arousal and placed along a timeline. Users can see patterns in their bodily reactions and relate them to various events going on in their lives. The experiences of building and deploying these systems gave us insights into design requirements for addressing affective loop experiences, such as how to design for turn-taking between user and system, how to create for 'open' surfaces in the design that can carry users' own meaning-making processes, how to combine modalities to create for a 'unity' of expression, and the importance of mirroring user experience in familiar ways that touch upon their everyday social and corporeal experiences. But a more important lesson gained from deploying the systems is how emotion processes are co-constructed and experienced inseparable from all other aspects of everyday life. Emotion processes are part of our social ways of being in the world; they dye our dreams, hopes and bodily experiences of the world. If we aim to design for affective interaction experiences, we need to place them into this larger picture.

Affective loop experiences: designing for interactional embodiment

PubMed Central

Höök, Kristina

2009-01-01

Involving our corporeal bodies in interaction can create strong affective experiences. Systems that both can be influenced by and influence users corporeally exhibit a use quality we name an affective loop experience. In an affective loop experience, (i) emotions are seen as processes, constructed in the interaction, starting from everyday bodily, cognitive or social experiences; (ii) the system responds in ways that pull the user into the interaction, touching upon end users' physical experiences; and (iii) throughout the interaction the user is an active, meaning-making individual choosing how to express themselves—the interpretation responsibility does not lie with the system. We have built several systems that attempt to create affective loop experiences with more or less successful results. For example, eMoto lets users send text messages between mobile phones, but in addition to text, the messages also have colourful and animated shapes in the background chosen through emotion-gestures with a sensor-enabled stylus pen. Affective Diary is a digital diary with which users can scribble their notes, but it also allows for bodily memorabilia to be recorded from body sensors mapping to users' movement and arousal and placed along a timeline. Users can see patterns in their bodily reactions and relate them to various events going on in their lives. The experiences of building and deploying these systems gave us insights into design requirements for addressing affective loop experiences, such as how to design for turn-taking between user and system, how to create for ‘open’ surfaces in the design that can carry users' own meaning-making processes, how to combine modalities to create for a ‘unity’ of expression, and the importance of mirroring user experience in familiar ways that touch upon their everyday social and corporeal experiences. But a more important lesson gained from deploying the systems is how emotion processes are co-constructed and experienced inseparable from all other aspects of everyday life. Emotion processes are part of our social ways of being in the world; they dye our dreams, hopes and bodily experiences of the world. If we aim to design for affective interaction experiences, we need to place them into this larger picture. PMID:19884153

Semirational Approach for Ultrahigh Poly(3-hydroxybutyrate) Accumulation in Escherichia coli by Combining One-Step Library Construction and High-Throughput Screening.

PubMed

Li, Teng; Ye, Jianwen; Shen, Rui; Zong, Yeqing; Zhao, Xuejin; Lou, Chunbo; Chen, Guo-Qiang

2016-11-18

As a product of a multistep enzymatic reaction, accumulation of poly(3-hydroxybutyrate) (PHB) in Escherichia coli (E. coli) can be achieved by overexpression of the PHB synthesis pathway from a native producer involving three genes phbC, phbA, and phbB. Pathway optimization by adjusting expression levels of the three genes can influence properties of the final product. Here, we reported a semirational approach for highly efficient PHB pathway optimization in E. coli based on a phbCAB operon cloned from the native producer Ralstonia entropha (R. entropha). Rationally designed ribosomal binding site (RBS) libraries with defined strengths for each of the three genes were constructed based on high or low copy number plasmids in a one-pot reaction by an oligo-linker mediated assembly (OLMA) method. Strains with desired properties were evaluated and selected by three different methodologies, including visual selection, high-throughput screening, and detailed in-depth analysis. Applying this approach, strains accumulating 0%-92% PHB contents in cell dry weight (CDW) were achieved. PHB with various weight-average molecular weights (M w ) of 2.7-6.8 × 10 6 were also efficiently produced in relatively high contents. These results suggest that the semirational approach combining library design, construction, and proper screening is an efficient way to optimize PHB and other multienzyme pathways.

Biomimetic perfusion and electrical stimulation applied in concert improved the assembly of engineered cardiac tissue

PubMed Central

Lee, Eun Jung; Luo, Jianwen; Duan, Yi; Yeager, Keith; Konofagou, Elisa; Vunjak-Novakovic, Gordana

2012-01-01

Maintenance of normal myocardial function depends intimately on synchronous tissue contraction driven by electrical activation and on adequate nutrient perfusion in support thereof. Bioreactors have been used to mimic aspects of these factors in vitro to engineer cardiac tissue, but due to design limitations, previous bioreactor systems have yet to simultaneously support nutrient perfusion, electrical stimulation, and unconstrained (i.e., not isometric) tissue contraction. To the best of our knowledge, the bioreactor system described herein is the first to integrate in concert these three key factors. We present the design of our bioreactor and characterize its capability in integrated experimental and mathematical modeling studies. We then culture cardiac cells obtained from neonatal rats in porous, channeled elastomer scaffolds with the simultaneous application of perfusion and electrical stimulation, with controls excluding either one or both of these two conditions. After eight days of culture, constructs grown with the simultaneous perfusion and electrical stimulation exhibited substantially improved functional properties, as evidenced by a significant increase in contraction amplitude (0.23±0.10% vs. 0.14±0.05, 0.13±0.08, or 0.09±0.02% in control constructs grown without stimulation, without perfusion, or either stimulation or perfusion, respectively). Consistently, these constructs had significantly improved DNA contents, cell distribution throughout the scaffold thickness, cardiac protein expression, cell morphology and overall tissue organization than either control group. Thus, the simultaneous application of medium perfusion and electrical conditioning enabled by the use of the novel bioreactor system may accelerate the generation of fully functional, clinically sized cardiac tissue constructs. PMID:22170772

Scaling relations for a needle-like electron beam plasma from the self-similar behavior in beam propagation

NASA Astrophysics Data System (ADS)

Bai, Xiaoyan; Chen, Chen; Li, Hong; Liu, Wandong; Chen, Wei

2017-10-01

Scaling relations of the main parameters of a needle-like electron beam plasma (EBP) to the initial beam energy, beam current, and discharge pressures are presented. The relations characterize the main features of the plasma in three parameter space and can provide great convenience in plasma design with electron beams. First, starting from the self-similar behavior of electron beam propagation, energy and charge depositions in beam propagation were expressed analytically as functions of the three parameters. Second, according to the complete coupled theoretical model of an EBP and appropriate assumptions, independent equations controlling the density and space charges were derived. Analytical expressions for the density and charges versus functions of energy and charge depositions were obtained. Finally, with the combination of the expressions derived in the above two steps, scaling relations of the density and potential to the three parameters were constructed. Meanwhile, numerical simulations were used to test part of the scaling relations.

Genomic localization of the Z/EG transgene in the mouse genome.

PubMed

Colombo, Sophie; Kumasaka, Mayuko; Lobe, Corrinne; Larue, Lionel

2010-02-01

The Z/EG transgenic mouse line, produced by Novak et al., displays tissue-specific EGFP expression after Cre-mediated recombination. The autofluorescence of EGFP allows the visualization of cells of interest displaying Cre recombination. The initial construct was designed such that cells without Cre recombination express the beta-galactosidase marker, facilitating counterselection. We used inverse PCR to identify the site of integration of the Z/EG transgene, to improve the efficiency of homozygous Z/EG mouse production. Recombined cells produced large amounts of EGFP protein, resulting in higher levels of fluorescence and therefore greater contrast with nonrecombined cells. We mapped the transgene to the G1 region of chromosome 5. This random insertion was found to have occurred 230-bp upstream from the start codon of the Rasa4 gene. The insertion of the Z/EG transgene in the C57BL/6 genetic background had no effect on Rasa4 expression. Homozygous Z/EG mice therefore had no obvious phenotype. (c) 2009 Wiley-Liss, Inc.

The character of gene expression of human periosteum used to form new tissue in allograft bone.

PubMed

Kemppainen, Jessica; Yu, Qing; Alexander, John; Jacquet, Robin; Scharschmidt, Thomas; Landis, William

2014-08-01

Of more than 2 million segmental bone defects repaired annually with bone autografts and allografts, 15-40% fail. Improving healing rates may be approached with tissue engineering and use of periosteum overlying an allograft. The present study documents gene expression in human periosteum-allograft constructs compared to allografts alone. Strips of human cadaveric periosteum (26 years, f, distal femur) were sutured about sterilized human femoral cortical strut bone allograft (54 years, m) segments. After construct incubation (M199 supplemented medium) for 8 d, constructs and allografts alone were implanted in nude mice. At 10 and 20 weeks, constructs (N = 4, each group) and allografts (N = 2, each group) were retrieved and placed in RNAlater for quantitative PCR to determine expression of human- and murine-specific genes relevant to remodeling. Specimens were frozen-ground to powders and RNA was extracted, purified, reverse-transcribed, and amplified. Ribosomal protein (P0) was used to normalize sample quantities. Fold change plots were generated following statistical analyses comparing 20- to 10-week gene expression data. Allografts alone yielded no human-specific gene expression. Notable fold changes of human-specific alkaline phosphatase, bone sialoprotein, type I collagen, decorin, RANKL, RANK, cathepsin K, and osteocalcin in 20-week compared to 10-week specimens were found. Murine-specific expression of genes indicative of host mouse vascularization (RANK, type I collagen) was detected in both allograft alone and periosteum-allograft samples. Gene data confirm viable periosteum in constructs after 20 weeks. Relatively higher fold-change values of RANK, RANKL and cathepsin K indicate activities of osteoclast precursors, osteoclasts and osteoblasts involved in allograft remodeling during implantation. All additional genes of interest indicate osteoblast activity in new bone matrix formation. Gene data are directly correlated with previous and present histology work. The results of this study suggest that further investigations could help to establish whether autologous periosteum-allograft constructs could be used for the repair of bone defects.

46 CFR 58.05-1 - Material, design and construction.

Code of Federal Regulations, 2014 CFR

2014-10-01

... AUXILIARY MACHINERY AND RELATED SYSTEMS Main Propulsion Machinery § 58.05-1 Material, design and construction. (a) The material, design, construction, workmanship, and arrangement of main propulsion machinery...

46 CFR 58.05-1 - Material, design and construction.

Code of Federal Regulations, 2012 CFR

2012-10-01

... AUXILIARY MACHINERY AND RELATED SYSTEMS Main Propulsion Machinery § 58.05-1 Material, design and construction. (a) The material, design, construction, workmanship, and arrangement of main propulsion machinery...

46 CFR 58.05-1 - Material, design and construction.

Code of Federal Regulations, 2011 CFR

2011-10-01

... AUXILIARY MACHINERY AND RELATED SYSTEMS Main Propulsion Machinery § 58.05-1 Material, design and construction. (a) The material, design, construction, workmanship, and arrangement of main propulsion machinery...

46 CFR 58.05-1 - Material, design and construction.

Code of Federal Regulations, 2010 CFR

2010-10-01

... AUXILIARY MACHINERY AND RELATED SYSTEMS Main Propulsion Machinery § 58.05-1 Material, design and construction. (a) The material, design, construction, workmanship, and arrangement of main propulsion machinery...

46 CFR 58.05-1 - Material, design and construction.

Code of Federal Regulations, 2013 CFR

2013-10-01

... AUXILIARY MACHINERY AND RELATED SYSTEMS Main Propulsion Machinery § 58.05-1 Material, design and construction. (a) The material, design, construction, workmanship, and arrangement of main propulsion machinery...

A native promoter and inclusion of an intron is necessary for efficient expression of GFP or mRFP in Armillaria mellea

PubMed Central

Ford, Kathryn L.; Baumgartner, Kendra; Henricot, Béatrice; Bailey, Andy M.; Foster, Gary D.

2016-01-01

Armillaria mellea is a significant pathogen that causes Armillaria root disease on numerous hosts in forests, gardens and agricultural environments worldwide. Using a yeast-adapted pCAMBIA0380 Agrobacterium vector, we have constructed a series of vectors for transformation of A. mellea, assembled using yeast-based recombination methods. These have been designed to allow easy exchange of promoters and inclusion of introns. The vectors were first tested by transformation into basidiomycete Clitopilus passeckerianus to ascertain vector functionality then used to transform A. mellea. We show that heterologous promoters from the basidiomycetes Agaricus bisporus and Phanerochaete chrysosporium that were used successfully to control the hygromycin resistance cassette were not able to support expression of mRFP or GFP in A. mellea. The endogenous A. mellea gpd promoter delivered efficient expression, and we show that inclusion of an intron was also required for transgene expression. GFP and mRFP expression was stable in mycelia and fluorescence was visible in transgenic fruiting bodies and GFP was detectable in planta. Use of these vectors has been successful in giving expression of the fluorescent proteins GFP and mRFP in A. mellea, providing an additional molecular tool for this pathogen. PMID:27384974

[Eukaryotic expression of Leptospira interrogans lipL32/1-ompL1/1 fusion gene encoding genus-specific protein antigens and the immunoreactivity of expression products].

PubMed

Yan, Jie; Zhao, Shou-feng; Mao, Ya-fei; Ruan, Ping; Luo, Yi-hui; Li, Shu-ping; Li, Li-wei

2005-01-01

To construct the eukaryotic expression system of L.interrogans lipL32/1-ompL1/1 fusion gene and to identify the immunoreactivity of expression products. PCR with linking primer was used to construct the fusion gene lipL32/1-ompL1/1. The P.pastoris eukaryotic expression system of the fusion gene, pPIC9K-lipL32/1-ompL1/1-P. pastorisGS115, was constructed after the fusion gene was cloned and sequenced. Colony with phenotype His(+)Mut(+) was isolated by using MD and MM plates and His(+) Mut(+) transformant with high resistance to G418 was screened out by using YPD plate. Using lysate of His(+) Mut(+) colony with high copies of the target gene digested with yeast lyase as the template and 5'AOX1 and 3'AOX1 as the primers, the target fusion gene in chromosome DNA of the constructed P. pastoris engineering strain was detected by PCR. Methanol in BMMY medium was used to induce the target recombinant protein rLipL32/1-rOmpL1/1 expression. rLipL32/1-rOmpL1/1 in the medium supernatant was extracted by using ammonium sulfate precipitation and Ni-NTA affinity chromatography. Output and immunoreactivity of rLipL32/1-rOmpL1/1 were measured by SDS-PAGE and Western blot methods, respectively. Amplification fragments of the obtained fusion gene lipL32/1-ompL1/1 was 1794 bp in size. The homogeneity of nucleotide and putative amino acid sequences of the fusion gene were as high as 99.94 % and 100 %, respectively, compared with the sequences of original lipL32/1 and ompL1/1 genotypes. The constructed eukaryotic expression system was able to secrete rLipL32/1-rOmpL1/1 with an output of 10 % of the total proteins in the supernatant, which located the expected position after SDS-PAGE. The rabbit anti-rLipL32/1 and anti-rOmpL1/1 sera could combine the expressed rLipL32/1-rOmpL1/1. An eukaryotic expression system with high efficiency in P.pastoris of L.interrogans lipL32/1-ompL1/1 fusion gene was successfully constructed in this study. The expressed fusion protein shows specific immunoreactivity, which can be used as a potential antigen for developing a novel vaccine of L.interrogans.

40 CFR 125.99 - What are approved design and construction technologies?

Code of Federal Regulations, 2011 CFR

2011-07-01

... construction technologies? 125.99 Section 125.99 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY...) of the Act § 125.99 What are approved design and construction technologies? (a) The following technologies constitute approved design and construction technologies for purposes of § 125.94(a)(4): (1...

40 CFR 125.99 - What are approved design and construction technologies?

Code of Federal Regulations, 2013 CFR

2013-07-01

... construction technologies? 125.99 Section 125.99 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY...) of the Act § 125.99 What are approved design and construction technologies? (a) The following technologies constitute approved design and construction technologies for purposes of § 125.94(a)(4): (1...

40 CFR 125.99 - What are approved design and construction technologies?

Code of Federal Regulations, 2014 CFR

2014-07-01

... construction technologies? 125.99 Section 125.99 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY...) of the Act § 125.99 What are approved design and construction technologies? (a) The following technologies constitute approved design and construction technologies for purposes of § 125.94(a)(4): (1...

40 CFR 125.99 - What are approved design and construction technologies?

Code of Federal Regulations, 2012 CFR

2012-07-01

... construction technologies? 125.99 Section 125.99 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY...) of the Act § 125.99 What are approved design and construction technologies? (a) The following technologies constitute approved design and construction technologies for purposes of § 125.94(a)(4): (1...

36 CFR 223.38 - Standards for road design and construction.

Code of Federal Regulations, 2010 CFR

2010-07-01

... Provisions § 223.38 Standards for road design and construction. Road construction authorized under timber... 36 Parks, Forests, and Public Property 2 2010-07-01 2010-07-01 false Standards for road design and construction. 223.38 Section 223.38 Parks, Forests, and Public Property FOREST SERVICE, DEPARTMENT OF...

40 CFR 125.99 - What are approved design and construction technologies?

Code of Federal Regulations, 2010 CFR

2010-07-01

... construction technologies? 125.99 Section 125.99 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY...) of the Act § 125.99 What are approved design and construction technologies? (a) The following technologies constitute approved design and construction technologies for purposes of § 125.94(a)(4): (1...

SCHOOL CONSTRUCTION SYSTEMS DEVELOPMENT PROJECT.

ERIC Educational Resources Information Center

BOICE, JOHN,; AND OTHERS

ONE-HUNDRED MANUFACTURERS EXPRESSED INTEREST IN BIDDING FOR A SYSTEM ON SCHOOL CONSTRUCTION CALLED SCSD OR SCHOOL CONSTRUCTION SYSTEMS DEVELOPMENT TO THE FIRST CALIFORNIA COMMISSION ON SCHOOL CONSTRUCTION SYSTEMS. TWENTY-TWO BUILDINGS COMPRISED THE PROJECT. THE OBJECTIVE WAS TO DEVELOP AN INTEGRATED SYSTEM OF STANDARD SCHOOL BUILDING COMPONENTS…

45 CFR 1232.15 - New construction.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 45 Public Welfare 4 2014-10-01 2014-10-01 false New construction. 1232.15 Section 1232.15 Public... Accessibility § 1232.15 New construction. (a) Design, construction, and alteration. New facilities shall be designed and constructed to be readily accessible to and usable by handicapped persons. construction shall...

45 CFR 1232.15 - New construction.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 45 Public Welfare 4 2011-10-01 2011-10-01 false New construction. 1232.15 Section 1232.15 Public... Accessibility § 1232.15 New construction. (a) Design, construction, and alteration. New facilities shall be designed and constructed to be readily accessible to and usable by handicapped persons. construction shall...

45 CFR 1232.15 - New construction.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 45 Public Welfare 4 2013-10-01 2013-10-01 false New construction. 1232.15 Section 1232.15 Public... Accessibility § 1232.15 New construction. (a) Design, construction, and alteration. New facilities shall be designed and constructed to be readily accessible to and usable by handicapped persons. construction shall...

49 CFR 41.110 - New DOT owned buildings and additions to buildings.

Code of Federal Regulations, 2011 CFR

2011-10-01

... architect's authenticated verifications of seismic design codes, standards, and practices used in the design... for the design and construction of new DOT Federally owned buildings will ensure that each building is designed and constructed in accord with the seismic design and construction standards set out in § 41.120...

LEAP: constructing gene co-expression networks for single-cell RNA-sequencing data using pseudotime ordering.

PubMed

Specht, Alicia T; Li, Jun

2017-03-01

To construct gene co-expression networks based on single-cell RNA-Sequencing data, we present an algorithm called LEAP, which utilizes the estimated pseudotime of the cells to find gene co-expression that involves time delay. R package LEAP available on CRAN. jun.li@nd.edu. Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

78 FR 57211 - Notice of Passenger Facility Charge (PFC) Approvals and Disapprovals

Federal Register 2010, 2011, 2012, 2013, 2014

2013-09-17

... rehabilitation design. Taxiway/apron/fuel road pavement replacement. Taxiway/apron/fuel road pavement replacement... Description of Projects Approved for Collection and Use: Taxiway F improvements (design and construction). Drainage ditch improvements (design and construction). Apron C expansion (design and construction...

Disclosing the Parameters Leading to High Productivity of Retroviral Producer Cells Lines: Evaluating Random Versus Targeted Integration.

PubMed

Bandeira, Vanessa S; Tomás, Hélio A; Alici, Evren; Carrondo, Manuel J T; Coroadinha, Ana S

2017-04-01

Gammaretrovirus and lentivirus are the preferred viral vectors to genetically modify T and natural killer cells to be used in immune cell therapies. The transduction efficiency of hematopoietic and T cells is more efficient using gibbon ape leukemia virus (GaLV) pseudotyping. In this context gammaretroviral vector producer cells offer competitive higher titers than transient lentiviral vectors productions. The main aim of this work was to identify the key parameters governing GaLV-pseudotyped gammaretroviral vector productivity in stable producer cells, using a retroviral vector expression cassette enabling positive (facilitating cell enrichment) and negative cell selection (allowing cell elimination). The retroviral vector contains a thymidine kinase suicide gene fused with a ouabain-resistant Na + ,K + -ATPase gene, a potential safer and faster marker. The establishment of retroviral vector producer cells is traditionally performed by randomly integrating the retroviral vector expression cassette codifying the transgene. More recently, recombinase-mediated cassette exchange methodologies have been introduced to achieve targeted integration. Herein we compared random and targeted integration of the retroviral vector transgene construct. Two retroviral producer cell lines, 293 OuaS and 293 FlexOuaS, were generated by random and targeted integration, respectively, producing high titers (on the order of 10 7 infectious particles·ml -1 ). Results showed that the retroviral vector transgene cassette is the key retroviral vector component determining the viral titers notwithstanding, single-copy integration is sufficient to provide high titers. The expression levels of the three retroviral constructs (gag-pol, GaLV env, and retroviral vector transgene) were analyzed. Although gag-pol and GaLV env gene expression levels should surpass a minimal threshold, we found that relatively modest expression levels of these two expression cassettes are required. Their levels of expression should not be maximized. We concluded, to establish a high producer retroviral vector cell line only the expression level of the genomic retroviral RNA, that is, the retroviral vector transgene cassette, should be maximized, both through (1) the optimization of its design (i.e., genetic elements composition) and (2) the selection of high expressing chromosomal locus for its integration. The use of methodologies identifying and promoting integration into high-expression loci, as targeted integration or high-throughput screening are in this perspective highly valuable.

Statistical plant set estimation using Schroeder-phased multisinusoidal input design

NASA Technical Reports Server (NTRS)

Bayard, D. S.

1992-01-01

A frequency domain method is developed for plant set estimation. The estimation of a plant 'set' rather than a point estimate is required to support many methods of modern robust control design. The approach here is based on using a Schroeder-phased multisinusoid input design which has the special property of placing input energy only at the discrete frequency points used in the computation. A detailed analysis of the statistical properties of the frequency domain estimator is given, leading to exact expressions for the probability distribution of the estimation error, and many important properties. It is shown that, for any nominal parametric plant estimate, one can use these results to construct an overbound on the additive uncertainty to any prescribed statistical confidence. The 'soft' bound thus obtained can be used to replace 'hard' bounds presently used in many robust control analysis and synthesis methods.

Transfer matrix method for dynamics modeling and independent modal space vibration control design of linear hybrid multibody system

NASA Astrophysics Data System (ADS)

Rong, Bao; Rui, Xiaoting; Lu, Kun; Tao, Ling; Wang, Guoping; Ni, Xiaojun

2018-05-01

In this paper, an efficient method of dynamics modeling and vibration control design of a linear hybrid multibody system (MS) is studied based on the transfer matrix method. The natural vibration characteristics of a linear hybrid MS are solved by using low-order transfer equations. Then, by constructing the brand-new body dynamics equation, augmented operator and augmented eigenvector, the orthogonality of augmented eigenvector of a linear hybrid MS is satisfied, and its state space model expressed in each independent model space is obtained easily. According to this dynamics model, a robust independent modal space-fuzzy controller is designed for vibration control of a general MS, and the genetic optimization of some critical control parameters of fuzzy tuners is also presented. Two illustrative examples are performed, which results show that this method is computationally efficient and with perfect control performance.

Exploring of the molecular mechanism of rhinitis via bioinformatics methods

PubMed Central

Song, Yufen; Yan, Zhaohui

2018-01-01

The aim of this study was to analyze gene expression profiles for exploring the function and regulatory network of differentially expressed genes (DEGs) in pathogenesis of rhinitis by a bioinformatics method. The gene expression profile of GSE43523 was downloaded from the Gene Expression Omnibus database. The dataset contained 7 seasonal allergic rhinitis samples and 5 non-allergic normal samples. DEGs between rhinitis samples and normal samples were identified via the limma package of R. The webGestal database was used to identify enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the DEGs. The differentially co-expressed pairs of the DEGs were identified via the DCGL package in R, and the differential co-expression network was constructed based on these pairs. A protein-protein interaction (PPI) network of the DEGs was constructed based on the Search Tool for the Retrieval of Interacting Genes database. A total of 263 DEGs were identified in rhinitis samples compared with normal samples, including 125 downregulated ones and 138 upregulated ones. The DEGs were enriched in 7 KEGG pathways. 308 differential co-expression gene pairs were obtained. A differential co-expression network was constructed, containing 212 nodes. In total, 148 PPI pairs of the DEGs were identified, and a PPI network was constructed based on these pairs. Bioinformatics methods could help us identify significant genes and pathways related to the pathogenesis of rhinitis. Steroid biosynthesis pathway and metabolic pathways might play important roles in the development of allergic rhinitis (AR). Genes such as CDC42 effector protein 5, solute carrier family 39 member A11 and PR/SET domain 10 might be also associated with the pathogenesis of AR, which provided references for the molecular mechanisms of AR. PMID:29257233

[Expression and identification of eukaryotic expression vectors of Brucella melitensis lipoprotein OMP19].

PubMed

He, Zuoping; Luo, Peifang; Hu, Feihuan; Weng, Yunceng; Wang, Wenjing; Li, Chengyao

2016-04-01

To construct eukaryotic expression vectors carrying Brucella melitensis outer membrane protein 19 (OMP19), express them in transfected Huh7.5.1 and JEG-3 cells, and analyze their role in cell apoptosis. Brucella melitensis lipidated OMP19 (L-OMP19) gene and unlipidated OMP19 (U-OMP19) gene were amplified by PCR and inserted into the vector pZeroBack/blunt. The correct L-OMP19 and U-OMP19 genes verified by XbaI and BamHI double digestion and sequencing were cloned into the lentivirus expression vector pHAGE-CMV-MCS-IZsGreen to construct vectors pHAGE-L-OMP19 and pHAGE-U-OMP19, which were separately transfected into 293FT cells, Huh7.5.1 and JEG-3 cells. L-OMP19 and U-OMP19 in the cells were detected by Western blotting and immunofluorescence technique. Flow cytometry combined with annexin V-PE/7-AAD staining was used to detect the cell apoptosis. The lentiviral vectors pHAGE-L-OMP19 and pHAGE-U-OMP19 were constructed correctly and the recombinant lipoproteins L-OMP19 and U-OMP19 expressed in the above cells were well recognized by the specific antibodies against L-OMP19 in Western blotting and immunofluorescence technique. L-OMP19 and U-OMP19 induced JEG-3 cell death, but did not induce the apoptosis of Huh7.5.1 cells. The eukaryotic expression vectors of L-OMP19 and U-OMP19 have been constructed successfully. Recombinant lipoproteins L-OMP19 and U-OMP19 expressed in cells have a good antigenicity, which could be used as experimental materials for the research on the relationship between host cells and lipoproteins in Brucella infection.

A Recombinant Raccoon Poxvirus Vaccine Expressing both Yersinia pestis F1 and Truncated V Antigens Protects Animals against Lethal Plague

PubMed Central

Rocke, Tonie E.; Kingstad-Bakke, Brock; Berlier, Willy; Osorio, Jorge E.

2014-01-01

In previous studies, we demonstrated in mice and prairie dogs that simultaneous administration of two recombinant raccoon poxviruses (rRCN) expressing Yersinia pestis antigens (F1 and V307—a truncated version of the V protein) provided superior protection against plague challenge compared to individual single antigen constructs. To reduce costs of vaccine production and facilitate implementation of a sylvatic plague vaccine (SPV) control program for prairie dogs, a dual antigen construct is more desirable. Here we report the construction and characterization of a novel RCN-vectored vaccine that simultaneously expresses both F1 and V307 antigens. This dual antigen vaccine provided similar levels of protection against plague in both mice and prairie dogs as compared to simultaneous administration of the two single antigen constructs and was also shown to protect mice against an F1 negative strain of Y. pestis. The equivalent safety, immunogenicity and efficacy profile of the dual RCN-F1/V307 construct warrants further evaluation in field efficacy studies in sylvatic plague endemic areas. PMID:26344891

A recombinant raccoon poxvirus vaccine expressing both Yersinia pestis F1 and truncated V antigens protects animals against lethal plague.

USGS Publications Warehouse

Rocke, Tonie E.; Kingstad-Bakke, B; Berlier, W; Osorio, J.E.

2014-01-01

In previous studies, we demonstrated in mice and prairie dogs that simultaneous administration of two recombinant raccoon poxviruses (rRCN) expressing Yersinia pestis antigens (F1 and V307-a truncated version of the V protein) provided superior protection against plague challenge compared to individual single antigen constructs. To reduce costs of vaccine production and facilitate implementation of a sylvatic plague vaccine (SPV) control program for prairie dogs, a dual antigen construct is more desirable. Here we report the construction and characterization of a novel RCN-vectored vaccine that simultaneously expresses both F1 and V307 antigens. This dual antigen vaccine provided similar levels of protection against plague in both mice and prairie dogs as compared to simultaneous administration of the two single antigen constructs and was also shown to protect mice against an F1 negative strain of Y. pestis.. The equivalent safety, immunogenicity and efficacy profile of the dual RCN-F1/V307 construct warrants further evaluation in field efficacy studies in sylvatic plague endemic areas.

Parental Expressivity and Parenting Styles in Chinese Families: Prospective and Unique Relations to Children’s Psychological Adjustment

PubMed Central

Chen, Stephen H.; Zhou, Qing; Eisenberg, Nancy; Valiente, Carlos; Wang, Yun

2012-01-01

SYNOPSIS Objectives Parents from different cultures differ in how frequently they express emotions. However, the generalizability of the relations between parental expressivity and child adjustment in non-Western cultures has not been extensively studied. The goal of the present study was to investigate prospective relations between parental expressivity within the family (positive, negative dominant, and negative submissive expressivity) and Chinese children’s psychological adjustment, above and beyond parenting styles. Design The study used two waves (3.8 years apart) of longitudinal data from a sample (n= 425) of children in Beijing (mean ages = 7.7 years at T1 and 11.6 years at T2). Parental expressivity and parenting styles were self-reported. To reduce the potential measurement overlap, items that tap parental expression of emotions toward the child were removed from the parenting style measure. Children’s adjustment was measured with parents’, teachers’, and peers’ or children’s reports. Results Consistent with findings with European American samples, parental negative dominant expressivity uniquely and positively predicted Chinese children’s externalizing problems controlling for prior externalizing problems, parenting styles, and family SES. Neither parental expressivity nor parenting styles uniquely predicted social competence. Conclusions Despite previously reported cultural differences in the mean levels of parental expressivity, some of the socialization functions of parental expressivity found in Western countries can be generalized to Chinese families. Although parental expressivity and parenting styles are related constructs, their unique relations to child’s adjustment suggest that they should be examined as distinct processes. PMID:23226715

Analytic solution of field distribution and demagnetization function of ideal hollow cylindrical field source

NASA Astrophysics Data System (ADS)

Xu, Xiaonong; Lu, Dingwei; Xu, Xibin; Yu, Yang; Gu, Min

2017-09-01

The Halbach type hollow cylindrical permanent magnet array (HCPMA) is a volume compact and energy conserved field source, which have attracted intense interests in many practical applications. Here, using the complex variable integration method based on the Biot-Savart Law (including current distributions inside the body and on the surfaces of magnet), we derive analytical field solutions to an ideal multipole HCPMA in entire space including the interior of magnet. The analytic field expression inside the array material is used to construct an analytic demagnetization function, with which we can explain the origin of demagnetization phenomena in HCPMA by taking into account an ideal magnetic hysteresis loop with finite coercivity. These analytical field expressions and demagnetization functions provide deeper insight into the nature of such permanent magnet array systems and offer guidance in designing optimized array system.

Reliable transformation system for Microbotryum lychnidis-dioicae informed by genome and transcriptome project.

PubMed

Toh, Su San; Treves, David S; Barati, Michelle T; Perlin, Michael H

2016-10-01

Microbotryum lychnidis-dioicae is a member of a species complex infecting host plants in the Caryophyllaceae. It is used as a model system in many areas of research, but attempts to make this organism tractable for reverse genetic approaches have not been fruitful. Here, we exploited the recently obtained genome sequence and transcriptome analysis to inform our design of constructs for use in Agrobacterium-mediated transformation techniques currently available for other fungi. Reproducible transformation was demonstrated at the genomic, transcriptional and functional levels. Moreover, these initial proof-of-principle experiments provide evidence that supports the findings from initial global transcriptome analysis regarding expression from the respective promoters under different growth conditions of the fungus. The technique thus provides for the first time the ability to stably introduce transgenes and over-express target M. lychnidis-dioicae genes.

Knockdown of Rice microRNA166 by Short Tandem Target Mimic (STTM).

PubMed

Teotia, Sachin; Zhang, Dabing; Tang, Guiliang

2017-01-01

Small RNAs, including microRNAs (miRNAs), are abundant in plants and play key roles in controlling plant development and physiology. miRNAs regulate the expression of the target genes involved in key plant processes. Due to functional redundancy among miRNA family members in plants, an ideal approach to silence the expression of all members simultaneously, for their functional characterization, is desirable. Target mimic (TM) was the first approach to achieve this goal. Short tandem target mimic (STTM) is a potent approach complementing TM for silencing miRNAs in plants. STTMs have been successfully used in dicots to block miRNA functions. Here, we describe in detail the protocol for designing STTM construct to block miRNA functions in rice. Such approach can be applied to silence miRNAs in other monocots as well.

46 CFR 161.010-2 - Design, construction, and test requirements.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 6 2013-10-01 2013-10-01 false Design, construction, and test requirements. 161.010-2 Section 161.010-2 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) EQUIPMENT, CONSTRUCTION, AND MATERIALS: SPECIFICATIONS AND APPROVAL ELECTRICAL EQUIPMENT Floating Electric Waterlight § 161.010-2 Design, construction, and test...

46 CFR 160.132-7 - Design, construction, and performance of davits.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 6 2013-10-01 2013-10-01 false Design, construction, and performance of davits. 160.132-7 Section 160.132-7 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) EQUIPMENT, CONSTRUCTION, AND MATERIALS: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Launching Appliances-Davits § 160.132-7 Design, construction, and...

46 CFR 161.010-2 - Design, construction, and test requirements.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 6 2010-10-01 2010-10-01 false Design, construction, and test requirements. 161.010-2 Section 161.010-2 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) EQUIPMENT, CONSTRUCTION, AND MATERIALS: SPECIFICATIONS AND APPROVAL ELECTRICAL EQUIPMENT Floating Electric Waterlight § 161.010-2 Design, construction, and test...

46 CFR 161.010-2 - Design, construction, and test requirements.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 6 2014-10-01 2014-10-01 false Design, construction, and test requirements. 161.010-2 Section 161.010-2 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) EQUIPMENT, CONSTRUCTION, AND MATERIALS: SPECIFICATIONS AND APPROVAL ELECTRICAL EQUIPMENT Floating Electric Waterlight § 161.010-2 Design, construction, and test...

46 CFR 160.132-7 - Design, construction, and performance of davits.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 6 2014-10-01 2014-10-01 false Design, construction, and performance of davits. 160.132-7 Section 160.132-7 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) EQUIPMENT, CONSTRUCTION, AND MATERIALS: SPECIFICATIONS AND APPROVAL LIFESAVING EQUIPMENT Launching Appliances-Davits § 160.132-7 Design, construction, and...

46 CFR 161.010-2 - Design, construction, and test requirements.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 6 2011-10-01 2011-10-01 false Design, construction, and test requirements. 161.010-2 Section 161.010-2 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) EQUIPMENT, CONSTRUCTION, AND MATERIALS: SPECIFICATIONS AND APPROVAL ELECTRICAL EQUIPMENT Floating Electric Waterlight § 161.010-2 Design, construction, and test...

Co-expression of five genes in E coli for L-phenylalanine in Brevibacterium flavum

PubMed Central

Wu, Yong-Qing; Jiang, Pei-Hong; Fan, Chang-Sheng; Wang, Jian-Gang; Shang, Liang; Huang, Wei-Da

2003-01-01

AIM: To study the effect of co-expression of ppsA, pckA, aroG, pheA and tyrB genes on the production of L-phenylalanine, and to construct a genetic engineering strain for L-phenylalanine. METHODS: ppsA and pckA genes were amplified from genomic DNA of E. coli by polymerase chain reaction, and then introduced into shuttle vectors between E coli and Brevibacterium flavum to generate constructs pJN2 and pJN5. pJN2 was generated by inserting ppsA and pckA genes into vector pCZ; whereas pJN5 was obtained by introducing ppsA and pckA genes into pCZ-GAB, which was originally constructed for co-expression of aroG, pheA and tyrB genes. The recombinant plasmids were then introduced into B. flavum by electroporation and the transformants were used for L-phenylalanine fermentation. RESULTS: Compared with the original B. flavum cells, all the transformants were showed to have increased five enzyme activities specifically, and have enhanced L-phenylalanine biosynthesis ability variably. pJN5 transformant was observed to have the highest elevation of L-phenylalanine production by a 3.4-fold. Co-expression of ppsA and pckA increased activity of DAHP synthetase significantly. CONCLUSION: Co-expression of ppsA and pckA genes in B. flavum could remarkably increase the expression of DAHP synthetase; Co-expression of ppsA, pckA, aroG, pheA and tyrB of E. coli in B. flavum was a feasible approach to construct a strain for phenylalanine production. PMID:12532463

Space construction system analysis. Part 2: Platform definition

NASA Technical Reports Server (NTRS)

Hart, R. J.; Myers, H. L.; Abramson, R. D.; Dejong, P. N.; Donavan, R. D.; Greenberg, H. S.; Indrikis, J.; Jandrasi, J. S.; Manoff, M.; Mcbaine, C. K.

1980-01-01

The top level system requirements are summarized and the accompanying conceptual design for an engineering and technology verification platform (ETVP) system is presented. An encompassing statement of the system objectives which drive the system requirements is presented and the major mission and subsystem requirements are described with emphasis on the advanced communications technology mission payload. The platform design is defined and used as a reference configuration for an end to space construction analyses. The preferred construction methods and processes, the important interactions between the platform design and the construction system design and operation, and the technology development efforts required to support the design and space construction of the ETVP are outlined.

24 CFR 941.402 - Project design and construction.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 24 Housing and Urban Development 4 2011-04-01 2011-04-01 false Project design and construction... URBAN DEVELOPMENT PUBLIC HOUSING DEVELOPMENT Project Development § 941.402 Project design and.... A PHA may certify that its proposed design and construction plans for the development are in...

7 CFR 3575.42 - Design and construction requirements.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 15 2011-01-01 2011-01-01 false Design and construction requirements. 3575.42 Section..., DEPARTMENT OF AGRICULTURE GENERAL Community Programs Guaranteed Loans § 3575.42 Design and construction... all funds were utilized for authorized purposes. The borrower and the lender will authorize designs...

Probabilistic pathway construction.

PubMed

Yousofshahi, Mona; Lee, Kyongbum; Hassoun, Soha

2011-07-01

Expression of novel synthesis pathways in host organisms amenable to genetic manipulations has emerged as an attractive metabolic engineering strategy to overproduce natural products, biofuels, biopolymers and other commercially useful metabolites. We present a pathway construction algorithm for identifying viable synthesis pathways compatible with balanced cell growth. Rather than exhaustive exploration, we investigate probabilistic selection of reactions to construct the pathways. Three different selection schemes are investigated for the selection of reactions: high metabolite connectivity, low connectivity and uniformly random. For all case studies, which involved a diverse set of target metabolites, the uniformly random selection scheme resulted in the highest average maximum yield. When compared to an exhaustive search enumerating all possible reaction routes, our probabilistic algorithm returned nearly identical distributions of yields, while requiring far less computing time (minutes vs. years). The pathways identified by our algorithm have previously been confirmed in the literature as viable, high-yield synthesis routes. Prospectively, our algorithm could facilitate the design of novel, non-native synthesis routes by efficiently exploring the diversity of biochemical transformations in nature. Copyright © 2011 Elsevier Inc. All rights reserved.

Carbon footprint hotspots of prefabricated sandwich panels for hostel construction in Perlis

NASA Astrophysics Data System (ADS)

Razali, Norashikin; Ayob, Afizah; Chandra, Muhammad Erwan Shah; Zaki, Mohd Faiz Mohammad; Ahmad, Abdul Ghapar

2017-10-01

Sustainable design and construction have gained increasing research interest, and reduction of carbon from building construction has become the main focus of environmental strategies in Malaysia. This study uses life cycle assessment and life cycle inventory analysis frameworks to estimate the amount of carbon footprint expressed in carbon dioxide equivalent tons (CO2e) produced by manufacturing prefabricated Industrialized Building System sandwich panels and its installation for a five-story hostel in Perlis, Malaysia. Results show that the carbon footprint hotspots were centered on boiler machine operation and cement with 4.52 and 369.04 tons CO2e, respectively. This finding is due to the extensive energy used for steam heating and high engine rating for the boiler. However, for cement, the carbon footprint hotspots are caused by the large quantity of cement applied in shotcrete mixture and its high extraction and production CO2 emission values. The overall onsite materials generated 96.36% of the total carbon footprint. These carbon footprint hotspot results constitute a necessary base for the Malaysian government in accomplishing an adequate dimensioning of carbon emissions in the building sector.

Expression of sunflower cytoplasmic male sterility-associated open reading frame, orfH522 induces male sterility in transgenic tobacco plants.

PubMed

Nizampatnam, Narasimha Rao; Doodhi, Harinath; Kalinati Narasimhan, Yamini; Mulpuri, Sujatha; Viswanathaswamy, Dinesh Kumar

2009-03-01

Sterility in the universally exploited PET1-CMS system of sunflower is associated with the expression of orfH522, a novel mitochondrial gene. Definitive evidence that ORFH522 is directly responsible for male sterility is lacking. To test the hypothesis that ORFH522 is sufficient to induce male sterility, a set of chimeric constructs were developed. The cDNA of orfH522 was cloned in-frame with yeast coxIV pre-sequence, and was expressed under tapetum-specific promoter TA29 (construct designated as TCON). For developing control vectors, orfH522 was cloned without the transit peptide under TA29 promoter (TON) or orfH522 was cloned with or without transit peptide under the constitutive CaMV35S promoter (SCOP and SOP). Among several independent transformants obtained with each of the gene cassettes, one third of the transgenics (6/17) with TCON were completely male sterile while more than 10 independent transformants obtained with each of the control vectors were fertile. The male sterile plants were morphologically similar to fertile plants, but had anthers that remained below the stigmatic surface at anthesis. RT-PCR analysis of the sterile plants confirmed the anther-specific expression of orfH522 and bright-field microscopy demonstrated ablation of the tapetal cell layer. Premature DNA fragmentation and programmed cell death was observed at meiosis stage in the anthers of sterile plants. Stable transmission of induced male sterility trait was confirmed in test cross progeny. This constitutes the first report at demonstrating the induction of male sterility by introducing orfH522 gene that could be useful for genetic engineering of male sterility.

Recombinant raccoon pox vaccine protects mice against lethal plague

USGS Publications Warehouse

Osorio, J.E.; Powell, T.D.; Frank, R.S.; Moss, K.; Haanes, E.J.; Smith, S.R.; Rocke, T.E.; Stinchcomb, D.T.

2003-01-01

Using a raccoon poxvirus (RCN) expression system, we have developed new recombinant vaccines that can protect mice against lethal plague infection. We tested the effects of a translation enhancer (EMCV-IRES) in combination with a secretory (tPA) signal or secretory (tPA) and membrane anchoring (CHV-gG) signals on in vitro antigen expression of F1 antigen in tissue culture and the induction of antibody responses and protection against Yersinia pestis challenge in mice. The RCN vector successfully expressed the F1 protein of Y. pestis in vitro. In addition, the level of expression was increased by the insertion of the EMCV-IRES and combinations of this and the secretory signal or secretory and anchoring signals. These recombinant viruses generated protective immune responses that resulted in survival of 80% of vaccinated mice upon challenge with Y. pestis. Of the RCN-based vaccines we tested, the RCN-IRES-tPA-YpF1 recombinant construct was the most efficacious. Mice vaccinated with this construct withstood challenge with as many as 1.5 million colony forming units of Y. pestis (7.7×104 LD50). Interestingly, vaccination with F1 fused to the anchoring signal (RCN-IRES-tPA-YpF1-gG) elicited significant anti-F1 antibody titers, but failed to protect mice from plague challenge. Our studies demonstrate, in vitro and in vivo, the potential importance of the EMCV-IRES and secretory signals in vaccine design. These molecular tools provide a new approach for improving the efficacy of vaccines. In addition, these novel recombinant vaccines could have human, veterinary, and wildlife applications in the prevention of plague.

Principles of Good School Building Design. School Buildings Planning, Design, and Construction Series No. 3.

ERIC Educational Resources Information Center

Odell, John H.

A school construction guide offers key personnel in school development projects information on the complex task of master planning and construction of schools in Australia. This chapter of the guide provides advice on school building design issues, such as the fundamentals of good design and designs that accommodate change, issues affecting…

Multiscale Embedded Gene Co-expression Network Analysis

PubMed Central

Song, Won-Min; Zhang, Bin

2015-01-01

Gene co-expression network analysis has been shown effective in identifying functional co-expressed gene modules associated with complex human diseases. However, existing techniques to construct co-expression networks require some critical prior information such as predefined number of clusters, numerical thresholds for defining co-expression/interaction, or do not naturally reproduce the hallmarks of complex systems such as the scale-free degree distribution of small-worldness. Previously, a graph filtering technique called Planar Maximally Filtered Graph (PMFG) has been applied to many real-world data sets such as financial stock prices and gene expression to extract meaningful and relevant interactions. However, PMFG is not suitable for large-scale genomic data due to several drawbacks, such as the high computation complexity O(|V|3), the presence of false-positives due to the maximal planarity constraint, and the inadequacy of the clustering framework. Here, we developed a new co-expression network analysis framework called Multiscale Embedded Gene Co-expression Network Analysis (MEGENA) by: i) introducing quality control of co-expression similarities, ii) parallelizing embedded network construction, and iii) developing a novel clustering technique to identify multi-scale clustering structures in Planar Filtered Networks (PFNs). We applied MEGENA to a series of simulated data and the gene expression data in breast carcinoma and lung adenocarcinoma from The Cancer Genome Atlas (TCGA). MEGENA showed improved performance over well-established clustering methods and co-expression network construction approaches. MEGENA revealed not only meaningful multi-scale organizations of co-expressed gene clusters but also novel targets in breast carcinoma and lung adenocarcinoma. PMID:26618778

Multiscale Embedded Gene Co-expression Network Analysis.

PubMed

Song, Won-Min; Zhang, Bin

2015-11-01

Gene co-expression network analysis has been shown effective in identifying functional co-expressed gene modules associated with complex human diseases. However, existing techniques to construct co-expression networks require some critical prior information such as predefined number of clusters, numerical thresholds for defining co-expression/interaction, or do not naturally reproduce the hallmarks of complex systems such as the scale-free degree distribution of small-worldness. Previously, a graph filtering technique called Planar Maximally Filtered Graph (PMFG) has been applied to many real-world data sets such as financial stock prices and gene expression to extract meaningful and relevant interactions. However, PMFG is not suitable for large-scale genomic data due to several drawbacks, such as the high computation complexity O(|V|3), the presence of false-positives due to the maximal planarity constraint, and the inadequacy of the clustering framework. Here, we developed a new co-expression network analysis framework called Multiscale Embedded Gene Co-expression Network Analysis (MEGENA) by: i) introducing quality control of co-expression similarities, ii) parallelizing embedded network construction, and iii) developing a novel clustering technique to identify multi-scale clustering structures in Planar Filtered Networks (PFNs). We applied MEGENA to a series of simulated data and the gene expression data in breast carcinoma and lung adenocarcinoma from The Cancer Genome Atlas (TCGA). MEGENA showed improved performance over well-established clustering methods and co-expression network construction approaches. MEGENA revealed not only meaningful multi-scale organizations of co-expressed gene clusters but also novel targets in breast carcinoma and lung adenocarcinoma.

Impact of age and vector construct on striatal and nigral transgene expression

PubMed Central

Polinski, Nicole K; Manfredsson, Fredric P; Benskey, Matthew J; Fischer, D Luke; Kemp, Christopher J; Steece-Collier, Kathy; Sandoval, Ivette M; Paumier, Katrina L; Sortwell, Caryl E

2016-01-01

Therapeutic protein delivery using viral vectors has shown promise in preclinical models of Parkinson’s disease (PD) but clinical trial success remains elusive. This may partially be due to a failure to include advanced age as a covariate despite aging being the primary risk factor for PD. We investigated transgene expression following intracerebral injections of recombinant adeno-associated virus pseudotypes 2/2 (rAAV2/2), 2/5 (rAAV2/5), 2/9 (rAAV2/9), and lentivirus (LV) expressing green fluorescent protein (GFP) in aged versus young adult rats. Both rAAV2/2 and rAAV2/5 yielded lower GFP expression following injection to either the aged substantia nigra or striatum. rAAV2/9-mediated GFP expression was deficient in the aged striatonigral system but displayed identical transgene expression between ages in the nigrostriatal system. Young and aged rats displayed equivalent GFP levels following LV injection to the striatonigral system but LV-delivered GFP was deficient in delivering GFP to the aged nigrostriatal system. Notably, age-related transgene expression deficiencies revealed by protein quantitation were poorly predicted by GFP-immunoreactive cell counts. Further, in situ hybridization for the viral CβA promoter revealed surprisingly limited tropism for astrocytes compared to neurons. Our results demonstrate that aging is a critical covariate to consider when designing gene therapy approaches for PD. PMID:27933309

PAX6 MiniPromoters drive restricted expression from rAAV in the adult mouse retina

PubMed Central

Hickmott, Jack W; Chen, Chih-yu; Arenillas, David J; Korecki, Andrea J; Lam, Siu Ling; Molday, Laurie L; Bonaguro, Russell J; Zhou, Michelle; Chou, Alice Y; Mathelier, Anthony; Boye, Sanford L; Hauswirth, William W; Molday, Robert S; Wasserman, Wyeth W; Simpson, Elizabeth M

2016-01-01

Current gene therapies predominantly use small, strong, and readily available ubiquitous promoters. However, as the field matures, the availability of small, cell-specific promoters would be greatly beneficial. Here we design seven small promoters from the human paired box 6 (PAX6) gene and test them in the adult mouse retina using recombinant adeno-associated virus. We chose the retina due to previous successes in gene therapy for blindness, and the PAX6 gene since it is: well studied; known to be driven by discrete regulatory regions; expressed in therapeutically interesting retinal cell types; and mutated in the vision-loss disorder aniridia, which is in need of improved therapy. At the PAX6 locus, 31 regulatory regions were bioinformatically predicted, and nine regulatory regions were constructed into seven MiniPromoters. Driving Emerald GFP, these MiniPromoters were packaged into recombinant adeno-associated virus, and injected intravitreally into postnatal day 14 mice. Four MiniPromoters drove consistent retinal expression in the adult mouse, driving expression in combinations of cell-types that endogenously express Pax6: ganglion, amacrine, horizontal, and Müller glia. Two PAX6-MiniPromoters drive expression in three of the four cell types that express PAX6 in the adult mouse retina. Combined, they capture all four cell types, making them potential tools for research, and PAX6-gene therapy for aniridia. PMID:27556059

PAX6 MiniPromoters drive restricted expression from rAAV in the adult mouse retina.

PubMed

Hickmott, Jack W; Chen, Chih-Yu; Arenillas, David J; Korecki, Andrea J; Lam, Siu Ling; Molday, Laurie L; Bonaguro, Russell J; Zhou, Michelle; Chou, Alice Y; Mathelier, Anthony; Boye, Sanford L; Hauswirth, William W; Molday, Robert S; Wasserman, Wyeth W; Simpson, Elizabeth M

2016-01-01

Current gene therapies predominantly use small, strong, and readily available ubiquitous promoters. However, as the field matures, the availability of small, cell-specific promoters would be greatly beneficial. Here we design seven small promoters from the human paired box 6 (PAX6) gene and test them in the adult mouse retina using recombinant adeno-associated virus. We chose the retina due to previous successes in gene therapy for blindness, and the PAX6 gene since it is: well studied; known to be driven by discrete regulatory regions; expressed in therapeutically interesting retinal cell types; and mutated in the vision-loss disorder aniridia, which is in need of improved therapy. At the PAX6 locus, 31 regulatory regions were bioinformatically predicted, and nine regulatory regions were constructed into seven MiniPromoters. Driving Emerald GFP, these MiniPromoters were packaged into recombinant adeno-associated virus, and injected intravitreally into postnatal day 14 mice. Four MiniPromoters drove consistent retinal expression in the adult mouse, driving expression in combinations of cell-types that endogenously express Pax6: ganglion, amacrine, horizontal, and Müller glia. Two PAX6-MiniPromoters drive expression in three of the four cell types that express PAX6 in the adult mouse retina. Combined, they capture all four cell types, making them potential tools for research, and PAX6-gene therapy for aniridia.

Patient's Communication Perceived Self-efficacy Scale (PCSS): construction and validation of a new measure in a socio-cognitive perspective.

PubMed

Capone, Vincenza; Petrillo, Giovanna

2014-06-01

In two studies we constructed and validated the Patient's Communication Perceived Self-efficacy Scale (PCSS) designed to assess patients' beliefs about their capability to successfully manage problematic situations related to communication with doctor. The 20-item scale was administered to 179 outpatients (study 1). An Exploratory Factor Analysis revealed a three-factor solution. In study 2, the 16-item scale was administered to 890 outpatients. Exploratory and Confirmatory Factor Analyses supported the 3-factor solution (Provide and Collect information, Express concerns and doubts, Verify information) that showed good psychometric properties and was invariant for gender. PCSS is an easily administered, reliable, and valid test of patients' communication self-efficacy beliefs. It can be applied optimally in the empirical study of factors influencing doctor-patient communication and used in training aimed at strengthening patients' communication skills. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

7 CFR 3560.559 - Design and construction requirements.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 15 2011-01-01 2011-01-01 false Design and construction requirements. 3560.559... § 3560.559 Design and construction requirements. (a) General. The requirements established in § 3560.60... encouraged that the design of off-farm labor housing incorporate outdoor shower, boot washing station, and/or...

49 CFR 192.476 - Internal corrosion control: Design and construction of transmission line.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 3 2010-10-01 2010-10-01 false Internal corrosion control: Design and... STANDARDS Requirements for Corrosion Control § 192.476 Internal corrosion control: Design and construction of transmission line. (a) Design and construction. Except as provided in paragraph (b) of this...

49 CFR 192.476 - Internal corrosion control: Design and construction of transmission line.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 49 Transportation 3 2014-10-01 2014-10-01 false Internal corrosion control: Design and... STANDARDS Requirements for Corrosion Control § 192.476 Internal corrosion control: Design and construction of transmission line. (a) Design and construction. Except as provided in paragraph (b) of this...

49 CFR 192.476 - Internal corrosion control: Design and construction of transmission line.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 49 Transportation 3 2012-10-01 2012-10-01 false Internal corrosion control: Design and... STANDARDS Requirements for Corrosion Control § 192.476 Internal corrosion control: Design and construction of transmission line. (a) Design and construction. Except as provided in paragraph (b) of this...

49 CFR 192.476 - Internal corrosion control: Design and construction of transmission line.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 49 Transportation 3 2013-10-01 2013-10-01 false Internal corrosion control: Design and... STANDARDS Requirements for Corrosion Control § 192.476 Internal corrosion control: Design and construction of transmission line. (a) Design and construction. Except as provided in paragraph (b) of this...

49 CFR 192.476 - Internal corrosion control: Design and construction of transmission line.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 49 Transportation 3 2011-10-01 2011-10-01 false Internal corrosion control: Design and... STANDARDS Requirements for Corrosion Control § 192.476 Internal corrosion control: Design and construction of transmission line. (a) Design and construction. Except as provided in paragraph (b) of this...

49 CFR 41.115 - New buildings to be leased for DOT occupancy.

Code of Federal Regulations, 2011 CFR

2011-10-01

... compliance may include the engineer's and architect's authenticated verifications of seismic design codes... design and construction of new buildings to be leased for DOT occupancy or use will ensure that each building is designed and constructed in accord with the seismic design and construction standards set out...

49 CFR 41.115 - New buildings to be leased for DOT occupancy.

Code of Federal Regulations, 2010 CFR

2010-10-01

... compliance may include the engineer's and architect's authenticated verifications of seismic design codes... design and construction of new buildings to be leased for DOT occupancy or use will ensure that each building is designed and constructed in accord with the seismic design and construction standards set out...

18 CFR 2.13 - Design and construction.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 18 Conservation of Power and Water Resources 1 2011-04-01 2011-04-01 false Design and construction... Interpretations Under the Federal Power Act § 2.13 Design and construction. (a) The Commission recognizes the... for the Protection of Natural, Historic, Scenic, and Recreational Values in the Design and Location of...

Efficient production of artificially designed gelatins with a Bacillus brevis system.

PubMed

Kajino, T; Takahashi, H; Hirai, M; Yamada, Y

2000-01-01

Artificially designed gelatins comprising tandemly repeated 30-amino-acid peptide units derived from human alphaI collagen were successfully produced with a Bacillus brevis system. The DNA encoding the peptide unit was synthesized by taking into consideration the codon usage of the host cells, but no clones having a tandemly repeated gene were obtained through the above-mentioned strategy. Minirepeat genes could be selected in vivo from a mixture of every possible sequence encoding an artificial gelatin by randomly ligating the mixed sequence unit and transforming it into Escherichia coli. Larger repeat genes constructed by connecting minirepeat genes obtained by in vivo selection were also stable in the expression host cells. Gelatins derived from the eight-unit and six-unit repeat genes were extracellularly produced at the level of 0.5 g/liter and easily purified by ammonium sulfate fractionation and anion-exchange chromatography. The purified artificial gelatins had the predicted N-terminal sequences and amino acid compositions and a solgel property similar to that of the native gelatin. These results suggest that the selection of a repeat unit sequence stable in an expression host is a shortcut for the efficient production of repetitive proteins and that it can conveniently be achieved by the in vivo selection method. This study revealed the possible industrial application of artificially designed repetitive proteins.

Isolation and Expression of the Lysis Genes of Actinomyces naeslundii Phage Av-1

PubMed Central

Delisle, Allan L.; Barcak, Gerard J.; Guo, Ming

2006-01-01

Like most gram-positive oral bacteria, Actinomyces naeslundii is resistant to salivary lysozyme and to most other lytic enzymes. We are interested in studying the lysins of phages of this important oral bacterium as potential diagnostic and therapeutic agents. To identify the Actinomyces phage genes encoding these species-specific enzymes in Escherichia coli, we constructed a new cloning vector, pAD330, that can be used to enrich for and isolate phage holin genes, which are located adjacent to the lysin genes in most phage genomes. Cloned holin insert sequences were used to design sequencing primers to identify nearby lysin genes by using whole phage DNA as the template. From partial digestions of A. naeslundii phage Av-1 genomic DNA we were able to clone, in independent experiments, inserts that complemented the defective λ holin in pAD330, as evidenced by extensive lysis after thermal induction. The DNA sequence of the inserts in these plasmids revealed that both contained the complete lysis region of Av-1, which is comprised of two holin-like genes, designated holA and holB, and an endolysin gene, designated lysA. We were able to subclone and express these genes and determine some of the functional properties of their gene products. PMID:16461656

Molecular Simulation of Receptor Occupancy and Tumor Penetration of an Antibody and Smaller Scaffolds: Application to Molecular Imaging.

PubMed

Orcutt, Kelly D; Adams, Gregory P; Wu, Anna M; Silva, Matthew D; Harwell, Catey; Hoppin, Jack; Matsumura, Manabu; Kotsuma, Masakatsu; Greenberg, Jonathan; Scott, Andrew M; Beckman, Robert A

2017-10-01

Competitive radiolabeled antibody imaging can determine the unlabeled intact antibody dose that fully blocks target binding but may be confounded by heterogeneous tumor penetration. We evaluated the hypothesis that smaller radiolabeled constructs can be used to more accurately evaluate tumor expressed receptors. The Krogh cylinder distributed model, including bivalent binding and variable intervessel distances, simulated distribution of smaller constructs in the presence of increasing doses of labeled antibody forms. Smaller constructs <25 kDa accessed binding sites more uniformly at large distances from blood vessels compared with larger constructs and intact antibody. These observations were consistent for different affinity and internalization characteristics of constructs. As predicted, a higher dose of unlabeled intact antibody was required to block binding to these distant receptor sites. Small radiolabeled constructs provide more accurate information on total receptor expression in tumors and reveal the need for higher antibody doses for target receptor blockade.

Adaptive Façade: Variant-Finding using Shape Grammar

NASA Astrophysics Data System (ADS)

Tomasowa, Riva; Utama Sjarifudin, Firza

2017-12-01

Modular façade construction has never been better since the birth of computer-aided manufacturing which bridges the modeling phase into the manufacturing phase for escalating the mass production. This comes to a result that the identity of a product or a building façade will commonly generate in the same way that the initial design was intended to. Rectifying the early model will then greatly impact the process later. The aim of this paper is to propose a way to solve these two challenges, without risking the manufacturing process, but more to explore the potential designs. Shape grammar is used to conceive more designs in the early stage, derived from the initial product - the modular adaptive façade system. The derivations are then tested through simulation to state the efficacy of the models. We find that the workflow somehow contributes to the better design and engineering process as well as the solution allows diversification in the façade expressions.

Measures against mechanical noise from large wind turbines: A design guide

NASA Astrophysics Data System (ADS)

Ljunggren, Sten; Johansson, Melker

1991-06-01

The noise generated by the machinery of the two Swedish prototypes contains pure tones which are very important with respect to the environmental impact. A discussion of the results of noise measurements carried out at these turbines, that are meant to be used as a guide as to how to predict and control the noise around a large wind turbine during the design stage, is presented. The design targets are discussed, stressing the importance of the audibility of pure tones and not only the annoyance; a simple criterion is cited. The main noise source is the gearbox and a simple empirical expression for the sound power level is shown to give good agreement with the measurement results. The influence of the noise of the gearbox design is discussed in some detail. Formulas for the prediction of the airborne sound transmission to the ground outside the nacelle are presented, together with a number of empirical data on the sound reduction indices for single and double constructions. The structure-borne noise transmission is discussed.

Treatment of CHP in LEED® for Building Design and Construction: New Construction and Major Renovations

EPA Pesticide Factsheets

This factsheet introduces CHP and its benefits to architects and engineers, and summarizes how CHP is treated under the LEED® for Building Design and Construction: New Construction and Major Renovations (LEED BD+C: New Construction) rating system.

10 CFR 4.128 - New construction.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 10 Energy 1 2012-01-01 2012-01-01 false New construction. 4.128 Section 4.128 Energy NUCLEAR... 1973, as Amended Discriminatory Practices § 4.128 New construction. (a) Design, construction, and... Federal Accessibility Standards. (1) Effective as of January 18, 1991, design, construction, or alteration...

45 CFR 1170.33 - New construction.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 45 Public Welfare 3 2014-10-01 2014-10-01 false New construction. 1170.33 Section 1170.33 Public... ASSISTED PROGRAMS OR ACTIVITIES Accessibility § 1170.33 New construction. (a) Design, construction, and... Federal Accessibility Standards. (1) Effective as of January 18, 1991, design, construction, or alteration...

10 CFR 4.128 - New construction.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 10 Energy 1 2010-01-01 2010-01-01 false New construction. 4.128 Section 4.128 Energy NUCLEAR... 1973, as Amended Discriminatory Practices § 4.128 New construction. (a) Design, construction, and... Federal Accessibility Standards. (1) Effective as of January 18, 1991, design, construction, or alteration...

45 CFR 1170.33 - New construction.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 45 Public Welfare 3 2013-10-01 2013-10-01 false New construction. 1170.33 Section 1170.33 Public... ASSISTED PROGRAMS OR ACTIVITIES Accessibility § 1170.33 New construction. (a) Design, construction, and... Federal Accessibility Standards. (1) Effective as of January 18, 1991, design, construction, or alteration...

45 CFR 1170.33 - New construction.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 45 Public Welfare 3 2012-10-01 2012-10-01 false New construction. 1170.33 Section 1170.33 Public... ASSISTED PROGRAMS OR ACTIVITIES Accessibility § 1170.33 New construction. (a) Design, construction, and... Federal Accessibility Standards. (1) Effective as of January 18, 1991, design, construction, or alteration...

A Dual-Mode Bioreactor System for Tissue Engineered Vascular Models.

PubMed

Bono, N; Meghezi, S; Soncini, M; Piola, M; Mantovani, D; Fiore, Gianfranco Beniamino

2017-06-01

In the past decades, vascular tissue engineering has made great strides towards bringing engineered vascular tissues to the clinics and, in parallel, obtaining in-lab tools for basic research. Herein, we propose the design of a novel dual-mode bioreactor, useful for the fabrication (construct mode) and in vitro stimulation (culture mode) of collagen-based tubular constructs. Collagen-based gels laden with smooth muscle cells (SMCs) were molded directly within the bioreactor culture chamber. Based on a systematic characterization of the bioreactor culture mode, constructs were subjected to 10% cyclic strain at 0.5 Hz for 5 days. The effects of cyclic stimulation on matrix re-arrangement and biomechanical/viscoelastic properties were examined and compared vs. statically cultured constructs. A thorough comparison of cell response in terms of cell localization and expression of contractile phenotypic markers was carried out as well. We found that cyclic stimulation promoted cell-driven collagen matrix bi-axial compaction, enhancing the mechanical strength of strained samples with respect to static controls. Moreover, cyclic strain positively affected SMC behavior: cells maintained their contractile phenotype and spread uniformly throughout the whole wall thickness. Conversely, static culture induced a noticeable polarization of cell distribution to the outer rim of the constructs and a sharp reduction in total cell density. Overall, coupling the use of a novel dual-mode bioreactor with engineered collagen-gel-based tubular constructs demonstrated to be an interesting technology to investigate the modulation of cell and tissue behavior under controlled mechanically conditioned in vitro maturation.

Expression-invariant representations of faces.

PubMed

Bronstein, Alexander M; Bronstein, Michael M; Kimmel, Ron

2007-01-01

Addressed here is the problem of constructing and analyzing expression-invariant representations of human faces. We demonstrate and justify experimentally a simple geometric model that allows to describe facial expressions as isometric deformations of the facial surface. The main step in the construction of expression-invariant representation of a face involves embedding of the facial intrinsic geometric structure into some low-dimensional space. We study the influence of the embedding space geometry and dimensionality choice on the representation accuracy and argue that compared to its Euclidean counterpart, spherical embedding leads to notably smaller metric distortions. We experimentally support our claim showing that a smaller embedding error leads to better recognition.

Food-grade host/vector expression system for Lactobacillus casei based on complementation of plasmid-associated phospho-beta-galactosidase gene lacG.

PubMed

Takala, T M; Saris, P E J; Tynkkynen, S S H

2003-01-01

A new food-grade host/vector system for Lactobacillus casei based on lactose selection was constructed. The wild-type non-starter host Lb. casei strain E utilizes lactose via a plasmid-encoded phosphotransferase system. For food-grade cloning, a stable lactose-deficient mutant was constructed by deleting a 141-bp fragment from the phospho-beta-galactosidase gene lacG via gene replacement. The deletion resulted in an inactive phospho-beta-galactosidase enzyme with an internal in-frame deletion of 47 amino acids. A complementation plasmid was constructed containing a replicon from Lactococcus lactis, the lacG gene from Lb. casei, and the constitutive promoter of pepR for lacG expression from Lb. rhamnosus. The expression of the lacG gene from the resulting food-grade plasmid pLEB600 restored the ability of the lactose-negative mutant strain to grow on lactose to the wild-type level. The vector pLEB600 was used for expression of the proline iminopeptidase gene pepI from Lb. helveticus in Lb. casei. The results show that the food-grade expression system reported in this paper can be used for expression of foreign genes in Lb. casei.

A Modular Lentiviral and Retroviral Construction System to Rapidly Generate Vectors for Gene Expression and Gene Knockdown In Vitro and In Vivo

PubMed Central

Geiling, Benjamin; Vandal, Guillaume; Posner, Ada R.; de Bruyns, Angeline; Dutchak, Kendall L.; Garnett, Samantha; Dankort, David

2013-01-01

The ability to express exogenous cDNAs while suppressing endogenous genes via RNAi represents an extremely powerful research tool with the most efficient non-transient approach being accomplished through stable viral vector integration. Unfortunately, since traditional restriction enzyme based methods for constructing such vectors are sequence dependent, their construction is often difficult and not amenable to mass production. Here we describe a non-sequence dependent Gateway recombination cloning system for the rapid production of novel lentiviral (pLEG) and retroviral (pREG) vectors. Using this system to recombine 3 or 4 modular plasmid components it is possible to generate viral vectors expressing cDNAs with or without inhibitory RNAs (shRNAmirs). In addition, we demonstrate a method to rapidly produce and triage novel shRNAmirs for use with this system. Once strong candidate shRNAmirs have been identified they may be linked together in tandem to knockdown expression of multiple targets simultaneously or to improve the knockdown of a single target. Here we demonstrate that these recombinant vectors are able to express cDNA and effectively knockdown protein expression using both cell culture and animal model systems. PMID:24146852

[Establishment of RAW264.7 cell strain stably expressing RFP-GFP-LC3].

PubMed

Wang, Wan; Zhang, Qing; Zhao, Runpeng; Xu, Xuewei; Xing, Yingru; Zhang, Rongbo; Wu, Jing; Hu, Dong

2015-09-01

To establish murine macrophage RAW264.7 cell strain with stable expression of red fluorescent protein-green fluorescent protein-microtubule associated protein light chain 3 (RFP-GFP-LC3). A lentiviral vector containing RFP-GFP-LC3 gene was constructed and then packaged in HEK293T cells with the packaging plasmids. The viral supernatant was collected to infect RAW264.7 cells. The RAW264.7 cell strain with stable expression of RFP-GFP-LC3 was screened with puromycin and analyzed with flow cytometry and fluorescent microscopy for infection efficiency. The number of RFP-GFP-LC3 puncta was observed using florescence microscopy following starvation treatment. The recombinant lentivirus pLV-CMV-RFP-GFP-LC3 was successfully constructed. The RAW264.7 cells with stable expression of RFP-GFP-LC3 were obtained by viral infection and puromycin screening. Fluorescent microscopy and flow cytometry demonstrated the expression rates of RFP and GFP reached to 100%. The number of autophagic puncta significantly increased after starvation treatment. The RAW264.7 cell strain with stable expression of RFP-GFP-LC3 has been successfully constructed, which provides a reliable cellular platform for autophagy research.

Characterization of Novel Plant Symbiosis Mutants Using a New Multiple Gene-Expression Reporter Sinorhizobium meliloti Strain

PubMed Central

Lang, Claus; Smith, Lucinda S.; Haney, Cara H.; Long, Sharon R.

2018-01-01

The formation of nitrogen fixing root nodules by Medicago truncatula and Sinorhizobium meliloti requires communication between both organisms and coordinated differentiation of plant and bacterial cells. After an initial signal exchange, the bacteria invade the tissue of the growing nodule via plant-derived tubular structures, called infection threads. The bacteria are released from the infection threads into invasion-competent plant cells, where they differentiate into nitrogen-fixing bacteroids. Both organisms undergo dramatic transcriptional, metabolic and morphological changes during nodule development. To identify plant processes that are essential for the formation of nitrogen fixing nodules after nodule development has been initiated, large scale mutageneses have been conducted to discover underlying plant symbiosis genes. Such screens yield numerous uncharacterized plant lines with nitrogen fixation deficient nodules. In this study, we report construction of a S. meliloti strain carrying four distinct reporter constructs to reveal stages of root nodule development. The strain contains a constitutively expressed lacZ reporter construct; a PexoY-mTFP fusion that is expressed in infection threads but not in differentiated bacteroids; a PbacA-mcherry construct that is expressed in infection threads and during bacteroid differentiation; and a PnifH-uidA construct that is expressed during nitrogen fixation. We used this strain together with fluorescence microscopy to study nodule development over time in wild type nodules and to characterize eight plant mutants from a fast neutron bombardment screen. Based on the signal intensity and the localization patterns of the reporter genes, we grouped mutants with similar phenotypes and placed them in a developmental context. PMID:29467773

Characterization of Novel Plant Symbiosis Mutants Using a New Multiple Gene-Expression Reporter Sinorhizobium meliloti Strain.

PubMed

Lang, Claus; Smith, Lucinda S; Long, Sharon R

2018-01-01

The formation of nitrogen fixing root nodules by Medicago truncatula and Sinorhizobium meliloti requires communication between both organisms and coordinated differentiation of plant and bacterial cells. After an initial signal exchange, the bacteria invade the tissue of the growing nodule via plant-derived tubular structures, called infection threads. The bacteria are released from the infection threads into invasion-competent plant cells, where they differentiate into nitrogen-fixing bacteroids. Both organisms undergo dramatic transcriptional, metabolic and morphological changes during nodule development. To identify plant processes that are essential for the formation of nitrogen fixing nodules after nodule development has been initiated, large scale mutageneses have been conducted to discover underlying plant symbiosis genes. Such screens yield numerous uncharacterized plant lines with nitrogen fixation deficient nodules. In this study, we report construction of a S. meliloti strain carrying four distinct reporter constructs to reveal stages of root nodule development. The strain contains a constitutively expressed lacZ reporter construct; a P exoY -mTFP fusion that is expressed in infection threads but not in differentiated bacteroids; a P bacA -mcherry construct that is expressed in infection threads and during bacteroid differentiation; and a P nifH -uidA construct that is expressed during nitrogen fixation. We used this strain together with fluorescence microscopy to study nodule development over time in wild type nodules and to characterize eight plant mutants from a fast neutron bombardment screen. Based on the signal intensity and the localization patterns of the reporter genes, we grouped mutants with similar phenotypes and placed them in a developmental context.

tCRISPRi: tunable and reversible, one-step control of gene expression

NASA Astrophysics Data System (ADS)

Li, Xin-Tian; Jun, Yonggun; Erickstad, Michael J.; Brown, Steven D.; Parks, Adam; Court, Donald L.; Jun, Suckjoon

2016-12-01

The ability to control the level of gene expression is a major quest in biology. A widely used approach employs deletion of a nonessential gene of interest (knockout), or multi-step recombineering to move a gene of interest under a repressible promoter (knockdown). However, these genetic methods are laborious, and limited for quantitative study. Here, we report a tunable CRISPR-cas system, “tCRISPRi”, for precise and continuous titration of gene expression by more than 30-fold. Our tCRISPRi system employs various previous advancements into a single strain: (1) We constructed a new strain containing a tunable arabinose operon promoter PBAD to quantitatively control the expression of CRISPR-(d)Cas protein over two orders of magnitude in a plasmid-free system. (2) tCRISPRi is reversible, and gene expression is repressed under knockdown conditions. (3) tCRISPRi shows significantly less than 10% leaky expression. (4) Most important from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step of oligo recombineering. Our results show that tCRISPRi, in combination with recombineering, provides a simple and easy-to-implement tool for gene expression control, and is ideally suited for construction of both individual strains and high-throughput tunable knockdown libraries.

The murine SP-C promoter directs type II cell-specific expression in transgenic mice.

PubMed

Glasser, Stephan W; Eszterhas, Susan K; Detmer, Emily A; Maxfield, Melissa D; Korfhagen, Thomas R

2005-04-01

Genomic DNA from the mouse pulmonary surfactant protein C (SP-C) gene was analyzed in transgenic mice to identify DNA essential for alveolar type II cell-specific expression. SP-C promoter constructs extending either 13 or 4.8 kb upstream of the transcription start site directed lung-specific expression of the bacterial chloramphenicol acetyl transferase (CAT) reporter gene. In situ hybridization analysis demonstrated alveolar cell-specific expression in the lungs of adult transgenic mice, and the pattern of 4.8 SP-C-CAT expression during development paralleled that of the endogenous SP-C gene. With the use of deletion constructs, lung-specific, low-level CAT activity was detected in tissue assays of SP-C-CAT transgenic mice retaining 318 bp of the promoter. In transient and stable cell transfection experiments, the 4.8-kb SP-C promoter was 90-fold more active as a stably integrated gene. These findings indicate that 1) the 4.8-kb SP-C promoter is sufficient to direct cell-specific and developmental expression, 2) an enhancer essential for lung-specific expression maps to the proximal 318-bp promoter, and 3) the activity of the 4.8-kb SP-C promoter construct is highly dependent on its chromatin environment.

Construction of two vectors for gene expression in Trichoderma reesei.

PubMed

Lv, Dandan; Wang, Wei; Wei, Dongzhi

2012-01-01

We report the construction of two filamentous fungi Trichoderma reesei expression vectors, pWEF31 and pWEF32. Both vectors possess the hygromycin phosphotransferase B gene expression cassette and the strong promoter and terminator of the cellobiohydrolase 1 gene (cbh1) from T. reesei. The two newly constructed vectors can be efficiently transformed into T. reesei with Agrobacterium-mediated transformation. The difference between pWEF31 and pWEF32 is that pWEF32 has two longer homologous arms. As a result, pWEF32 easily undergoes homologous recombination. On the other hand, pWEF31 undergoes random recombination. The applicability of both vectors was tested by first generating the expression vectors pWEF31-red and pWEF32-red and then detecting the expression of the DsRed2 gene in T. reesei Rut C30. Additionally, we measured the exo-1,4-β-glucanase activity of the recombinant cells. Our work provides an effective transformation system for homologous and heterologous gene expression and gene knockout in T. reesei. It also provides a method for recombination at a specific chromosomal location. Finally, both vectors will be useful for the large-scale gene expression industry. Copyright © 2011 Elsevier Inc. All rights reserved.

Rapid evolution of regulatory element libraries for tunable transcriptional and translational control of gene expression.

PubMed

Jin, Erqing; Wong, Lynn; Jiao, Yun; Engel, Jake; Holdridge, Benjamin; Xu, Peng

2017-12-01

Engineering cell factories for producing biofuels and pharmaceuticals has spurred great interests to develop rapid and efficient synthetic biology tools customized for modular pathway engineering. Along the way, combinatorial gene expression control through modification of regulatory element offered tremendous opportunity for fine-tuning gene expression and generating digital-like genetic circuits. In this report, we present an efficient evolutionary approach to build a range of regulatory control elements. The reported method allows for rapid construction of promoter, 5'UTR, terminator and trans -activating RNA libraries. Synthetic overlapping oligos with high portion of degenerate nucleotides flanking the regulatory element could be efficiently assembled to a vector expressing fluorescence reporter. This approach combines high mutation rate of the synthetic DNA with the high assembly efficiency of Gibson Mix. Our constructed library demonstrates broad range of transcriptional or translational gene expression dynamics. Specifically, both the promoter library and 5'UTR library exhibits gene expression dynamics spanning across three order of magnitude. The terminator library and trans -activating RNA library displays relatively narrowed gene expression pattern. The reported study provides a versatile toolbox for rapidly constructing a large family of prokaryotic regulatory elements. These libraries also facilitate the implementation of combinatorial pathway engineering principles and the engineering of more efficient microbial cell factory for various biomanufacturing applications.

An independent confirmatory factor analysis of the Wechsler Intelligence Scale for Children-fourth Edition (WISC-IV) integrated: what do the process approach subtests measure?

PubMed

Benson, Nicholas; Hulac, David M; Bernstein, Joshua D

2013-09-01

The Wechsler intelligence scale for children--fourth edition (WISC-IV) Integrated contains the WISC-IV core and supplemental subtests along with process approach subtests designed to facilitate a process-oriented approach to score interpretation. The purpose of this study was to examine the extent to which WISC-IV Integrated subtests measure the constructs they are purported to measure. In addition to examining the measurement and scoring model provided in the manual, this study also tested hypotheses regarding Cattell-Horn-Carroll abilities that might be measured along with other substantive questions regarding the factor structure of the WISC-IV Integrated and the nature of abilities measured by process approach subtests. Results provide insight regarding the constructs measured by these subtests. Many subtests appear to be good to excellent measures of psychometric g (i.e., the general factor presumed to cause the positive correlation of mental tasks). Other abilities measured by subtests are described. For some subtests, the majority of variance is not accounted for by theoretical constructs included in the scoring model. Modifications made to remove demands such as memory recall and verbal expression were found to reduce construct-irrelevant variance. The WISC-IV Integrated subtests appear to measure similar constructs across ages 6-16, although strict factorial invariance was not supported.

Transmit Designs for the MIMO Broadcast Channel With Statistical CSI

NASA Astrophysics Data System (ADS)

Wu, Yongpeng; Jin, Shi; Gao, Xiqi; McKay, Matthew R.; Xiao, Chengshan

2014-09-01

We investigate the multiple-input multiple-output broadcast channel with statistical channel state information available at the transmitter. The so-called linear assignment operation is employed, and necessary conditions are derived for the optimal transmit design under general fading conditions. Based on this, we introduce an iterative algorithm to maximize the linear assignment weighted sum-rate by applying a gradient descent method. To reduce complexity, we derive an upper bound of the linear assignment achievable rate of each receiver, from which a simplified closed-form expression for a near-optimal linear assignment matrix is derived. This reveals an interesting construction analogous to that of dirty-paper coding. In light of this, a low complexity transmission scheme is provided. Numerical examples illustrate the significant performance of the proposed low complexity scheme.

Type Specialization in Aldor

NASA Astrophysics Data System (ADS)

Dragan, Laurentiu; Watt, Stephen M.

Computer algebra in scientific computation squarely faces the dilemma of natural mathematical expression versus efficiency. While higher-order programming constructs and parametric polymorphism provide a natural and expressive language for mathematical abstractions, they can come at a considerable cost. We investigate how deeply nested type constructions may be optimized to achieve performance similar to that of hand-tuned code written in lower-level languages.

Lateral support systems and underpinning, volume I : design and construction.

DOT National Transportation Integrated Search

1976-04-01

"This volume is a convenient reference on the design and construction of lateral support systems and underpinning which are often required in conjunction with cut-and-cover or soft ground tunneling. The design recommendations and construction methods...

Identification of Tools and Techniques to Enhance Interdisciplinary Collaboration During Design and Construction Projects.

PubMed

Keys, Yolanda; Silverman, Susan R; Evans, Jennie

2017-10-01

The purpose of this study was to collect the perceptions of design professionals and clinicians regarding design process success strategies and elements of interprofessional engagement and communication during healthcare design and construction projects. Additional objectives were to gather best practices to maximize clinician engagement and provide tools and techniques to improve interdisciplinary collaboration for future projects. Strategies are needed to enhance the design and construction process and create interactions that benefit not only the project but the individuals working to see its completion. Meaningful interprofessional collaboration is essential to any healthcare design project and making sure the various players communicate is a critical element. This was a qualitative study conducted via an online survey. Respondents included architects, construction managers, interior designers, and healthcare personnel who had recently been involved in a building renovation or new construction project for a healthcare facility. Responses to open-ended questions were analyzed for themes, and descriptive statistics were used to provide insight into participant demographics. Information on the impressions, perceptions, and opportunities related to clinician involvement in design projects was collected from nurses, architects, interior designers, and construction managers. Qualitative analysis revealed themes of clinician input, organizational dynamics, and a variety of communication strategies to be the most frequently mentioned elements of successful interprofessional collaboration. This study validates the need to include clinician input in the design process, to consider the importance of organizational dynamics on design team functioning, and to incorporate effective communication strategies during design and construction projects.

Sulfur extended asphalt pavement evaluation in the State of Washington: SR 270 highway pavement performance report

NASA Astrophysics Data System (ADS)

Mahoney, J. P.; Terrel, R. L.; Cook, J. C.

1982-11-01

The placement and performance of sulfur extended asphalt (SEA) paving mixtures at a highway test site (SR 270) near Pullman, Washington is summarized. The mixture and structural designs and construction details are included. This is followed by a discussion of the data collection and analysis accomplished over a three year evaluation period (1979-1982). A major experimental feature of the study was the use of 0.100 (conventional asphalt concrete), 30/70 and 40/60 SEA binder ratios (sulfur/asphalt ratios are expressed as weight percents in the experimental paving mixtures.

Shape Optimization of Cylindrical Shell for Interior Noise

NASA Technical Reports Server (NTRS)

Robinson, Jay H.

1999-01-01

In this paper an analytic method is used to solve for the cross spectral density of the interior acoustic response of a cylinder with nonuniform thickness subjected to turbulent boundary layer excitation. The cylinder is of honeycomb core construction with the thickness of the core material expressed as a cosine series in the circumferential direction. The coefficients of this series are used as the design variable in the optimization study. The objective function is the space and frequency averaged acoustic response. Results confirm the presence of multiple local minima as previously reported and demonstrate the potential for modest noise reduction.

10 CFR 4.128 - New construction.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 10 Energy 1 2014-01-01 2014-01-01 false New construction. 4.128 Section 4.128 Energy NUCLEAR... 1973, as Amended Discriminatory Practices § 4.128 New construction. (a) Design, construction, and... Accessibility Standards. (1) Effective as of January 18, 1991, design, construction, or alteration of buildings...

10 CFR 4.128 - New construction.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 10 Energy 1 2013-01-01 2013-01-01 false New construction. 4.128 Section 4.128 Energy NUCLEAR... 1973, as Amended Discriminatory Practices § 4.128 New construction. (a) Design, construction, and... Accessibility Standards. (1) Effective as of January 18, 1991, design, construction, or alteration of buildings...

Virtual design and construction of plumbing systems

NASA Astrophysics Data System (ADS)

Filho, João Bosco P. Dantas; Angelim, Bruno Maciel; Guedes, Joana Pimentel; de Castro, Marcelo Augusto Farias; Neto, José de Paula Barros

2016-12-01

Traditionally, the design coordination process is carried out by overlaying and comparing 2D drawings made by different project participants. Detecting information errors from a composite drawing is especially challenging and error prone. This procedure usually leaves many design errors undetected until construction begins, and typically lead to rework. Correcting conflict issues, which were not identified during design and coordination phase, reduces the overall productivity for everyone involved in the construction process. The identification of construction issues in the field generate Request for Information (RFIs) that is one of delays causes. The application of Virtual Design and Construction (VDC) tools to the coordination processes can bring significant value to architecture, structure, and mechanical, electrical, and plumbing (MEP) designs in terms of a reduced number of errors undetected and requests for information. This paper is focused on evaluating requests for information (RFI) associated with water/sanitary facilities of a BIM model. Thus, it is expected to add improvements of water/sanitary facility designs, as well as to assist the virtual construction team to notice and identify design problems. This is an exploratory and descriptive research. A qualitative methodology is used. This study adopts RFI's classification in six analyzed categories: correction, omission, validation of information, modification, divergence of information and verification. The results demonstrate VDC's contribution improving the plumbing system designs. Recommendations are suggested to identify and avoid these RFI types in plumbing system design process or during virtual construction.

Dental Pulp Stem Cell-Derived, Scaffold-Free Constructs for Bone Regeneration.

PubMed

Tatsuhiro, Fukushima; Seiko, Tatehara; Yusuke, Takebe; Reiko, Tokuyama-Toda; Kazuhito, Satomura

2018-06-22

In the present study, a scaffold-free tissue construct was developed as an approach for the regeneration of tissue defects, which produced good outcomes. We fabricated a scaffold-free tissue construct from human dental pulp stem cells (hDPSCs construct), and examined the characteristics of the construct. For its fabrication, basal sheets prepared by 4-week hDPSCs culturing were subjected to 1-week three-dimensional culture, with or without osteogenic induction, whereas hDPSC sheets (control) were fabricated by 1-week culturing of basal sheets on monolayer culture. The hDPSC constructs formed a spherical structure and calcified matrix that are absent in the control. The expression levels for bone-related genes in the hDPSC constructs were significantly upregulated compared with those in the control. Moreover, the hDPSC constructs with osteogenic induction had a higher degree of calcified matrix formation, and higher expression levels for bone-related genes, than those for the hDPSC constructs without osteogenic induction. These results suggest that the hDPSC constructs with osteogenic induction are composed of cells and extracellular and calcified matrices, and that they can be a possible scaffold-free material for bone regeneration.

Ontological forms of religious meaning and the conflict between science and religion

NASA Astrophysics Data System (ADS)

Hathcoat, John D.; Habashi, Janette

2013-06-01

Epistemological constructions are central considerations in vivisecting an expressed conflict between science and religion. It is argued that the conflict thesis is only meaningful when examined from a specific socio-historical perspective. The dialectical relation between science and religion should therefore be considered at both a macro and micro level. At the macro level broad changes in the meaning of science and religion occur; whereas at the micro level individuals immersed within particular expressions of these concepts socially construct, re-construct, and appropriate meaning. Specific attention is given to expressions of meaning surrounding sacred texts in this dialectical relation. Two ontological forms of meaning are examined through a qualitative content analysis of 16 interviews with individuals from various religious affiliation and academic attainment. A monistic ontology constructs textual meaning as facts that have the qualities of being both self-evident and certain. Potential tension arises with scientific discourse given empirical evidence may either confirm or conflict with scriptural interpretation. The pluralistic ontology constructs textual meaning with multiple categories, which in turn have the qualities of being mediated by human consciousness and uncertain. The science-religion dichotomy appears to be less susceptible to conflict given the uncertainty embedded in this construction of scriptural meaning. This paper implies that truth as correspondence may not necessitate the conflict thesis.

Lateral Support Systems And Underpinning. Volume I: Design And Construction

DOT National Transportation Integrated Search

1976-04-01

This volume is a convenient reference on the design and construction of lateral support systems and underpining which are often required in conjunction with cut-and-cover or soft ground tunneling. The design recommendations and construction methods d...

49 CFR 230.66 - Design, construction, and maintenance.

Code of Federal Regulations, 2010 CFR

2010-10-01

... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION STEAM LOCOMOTIVE INSPECTION AND MAINTENANCE STANDARDS Steam Locomotives and Tenders § 230.66 Design, construction, and maintenance. The steam locomotive owner and operator are responsible for the general design, construction and maintenance of the steam locomotives and...

Public Space in Barcelona (1992-2017) - Evolution and Case Studies

NASA Astrophysics Data System (ADS)

Sanfeliu Arboix, Ignacio; Martín, Estefanía

2017-10-01

The construction of the public space has become in the last decades something so important and fundamental in the architecture of the cities, that requires a specific discipline and a concrete study that evaluates the characteristics and actions on it. Not already from an urbanistic perspective but from its own design and constructive perspective, with its character as a place for everyone and for everything, must gather a series of elements that are unique to this space. Barcelona is one of the densest cities in the world that, since the end of the s. XX until our days, tries to solve the public space with a design of quality and optimum. The shortage of public space, which also hosts more than eight million tourists each year, makes it necessary to propose a type of meticulous intervention in order to accommodate all types of users and activities. From the first Universal Exhibition of 1888 through 1929 to the 1992 Olympics as the most important stimulus for this renewal of urban space, Barcelona has been rethinking and evolving in the modus operandi in terms of its urban space. From our professional experience as architects both in the municipal, private and university spheres, we believe that it is our responsibility to confer the public space, that is to say, the design of the urban infrastructure with the attributes necessary to consolidate it in a space Suitable for all without exception and as a place of expression of citizenship. Through the projects of public space developed in our office we will analyze this change of procedure in the construction of squares, parks and other spaces in the city of Barcelona.

[Zinc-dependent metalloprotease 1 promotes apoptosis of RAW264.7 macrophages].

PubMed

Li, Peng; He, Yonglin; Zhang, Jiming; Fang, Chencheng

2015-12-01

To construct the eukaryotic expression vector of zinc-dependent metalloprotease 1 (zmp1) gene from Bacillus Calmette-Guerin (BCG) and investigate its impact on the apoptosis of RAW264.7 macrophages. Zmp1 gene was amplified from the genome of BCG by PCR. The zmp1 gene fragment was inserted into multiple cloning sites of pEGFP-N1 to construct the eukaryotic expression vector pEGFP-N1-zmp1. The constructed pEGFP-N1-zmp1 was transfected into RAW264.7 cells by Lipofectamine(TM) 2000. The expression of green fluorescent protein (GFP) was observed by fluorescence microscopy. The zmp1 mRNA was detected by quantitative real-time PCR (qR-PCR). The effect of Zmp1 protein on the apoptosis of RAW264.7 macrophages was detected by flow cytometry (FCM). With zmp1 gene amplified by PCR, we successfully constructed the recombinant vector pEGFP-N1-zmp1 as demonstrated by restriction enzyme analysis and sequencing. GFP was seen in RAW264.7 cells 24 hours after transfected with the recombinant plasmid. As qRT-PCR showed, the expression level of zmp1 mRNA was up-regulated. The early apoptotic rate increased 48 hours after transfection. The increased expression of Zmp1 in RAW264.7 cells promotes the apoptosis of RAW264.7 cells.

Efficient gusA transient expression in Porphyra yezoensis protoplasts mediated by endogenous beta-tubulin flanking sequences

NASA Astrophysics Data System (ADS)

Gong, Qianhong; Yu, Wengong; Dai, Jixun; Liu, Hongquan; Xu, Rifu; Guan, Huashi; Pan, Kehou

2007-01-01

Endogenous tubulin promoter has been widely used for expressing foreign genes in green algae, but the efficiency and feasibility of endogenous tubulin promoter in the economically important Porphyra yezoensis (Rhodophyta) are unknown. In this study, the flanking sequences of beta-tubulin gene from P. yezoensis were amplified and two transient expression vectors were constructed to determine their transcription promoting feasibility for foreign gene gusA. The testing vector pATubGUS was constructed by inserting 5'-and 3'-flanking regions ( Tub5' and Tub3') up-and down-stream of β-glucuronidase (GUS) gene ( gusA), respectively, into pA, a derivative of pCAT®3-enhancer vector. The control construct, pAGUSTub3, contains only gusA and Tub3'. These constructs were electroporated into P. yezoensis protoplasts and the GUS activities were quantitatively analyzed by spectrometry. The results demonstrated that gusA gene was efficiently expressed in P. yezoensis protoplasts under the regulation of 5'-flanking sequence of the beta-tubulin gene. More interestingly, the pATubGUS produced stronger GUS activity in P. yezoensis protoplasts when compared to the result from pBI221, in which the gusA gene was directed by a constitutive CaMV 35S promoter. The data suggest that the integration of P. yezoensis protoplast and its endogenous beta-tubulin flanking sequences is a potential novel system for foreign gene expression.

Modification of nucleic acids by azobenzene derivatives and their applications in biotechnology and nanotechnology.

PubMed

Li, Jing; Wang, Xingyu; Liang, Xingguo

2014-12-01

Azobenzene has been widely used as a photoregulator due to its reversible photoisomerization, large structural change between E and Z isomers, high photoisomerization yield, and high chemical stability. On the other hand, some azobenzene derivatives can be used as universal quenchers for many fluorophores. Nucleic acid is a good candidate to be modified because it is not only the template of gene expression but also widely used for building well-organized nanostructures and nanodevices. Because the size and polarity distribution of the azobenzene molecule is similar to a nucleobase pair, the introduction of azobenzene into nucleic acids has been shown to be an ingenious molecular design for constructing light-switching biosystems or light-driven nanomachines. Here we review recent advances in azobenzene-modified nucleic acids and their applications for artificial regulation of gene expression and enzymatic reactions, construction of photoresponsive nanostructures and nanodevices, molecular beacons, as well as obtaining structural information using the introduced azobenzene as an internal probe. In particular, nucleic acids bearing multiple azobenzenes can be used as a novel artificial nanomaterial with merits of high sequence specificity, regular duplex structure, and high photoregulation efficiency. The combination of functional groups with biomolecules may further advance the development of chemical biotechnology and biomolecular engineering. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modeling listeners' emotional response to music.

PubMed

Eerola, Tuomas

2012-10-01

An overview of the computational prediction of emotional responses to music is presented. Communication of emotions by music has received a great deal of attention during the last years and a large number of empirical studies have described the role of individual features (tempo, mode, articulation, timbre) in predicting the emotions suggested or invoked by the music. However, unlike the present work, relatively few studies have attempted to model continua of expressed emotions using a variety of musical features from audio-based representations in a correlation design. The construction of the computational model is divided into four separate phases, with a different focus for evaluation. These phases include the theoretical selection of relevant features, empirical assessment of feature validity, actual feature selection, and overall evaluation of the model. Existing research on music and emotions and extraction of musical features is reviewed in terms of these criteria. Examples drawn from recent studies of emotions within the context of film soundtracks are used to demonstrate each phase in the construction of the model. These models are able to explain the dominant part of the listeners' self-reports of the emotions expressed by music and the models show potential to generalize over different genres within Western music. Possible applications of the computational models of emotions are discussed. Copyright © 2012 Cognitive Science Society, Inc.

RNA interference mediated in human primary cells via recombinant baculoviral vectors.

PubMed

Nicholson, Linda J; Philippe, Marie; Paine, Alan J; Mann, Derek A; Dolphin, Colin T

2005-04-01

The success of RNA interference (RNAi) in mammalian cells, mediated by siRNAs or shRNA-generating plasmids, is dependent, to an extent, upon transfection efficiency. This is a particular problem with primary cells, which are often difficult to transfect using cationic lipid vehicles. Effective RNAi in primary cells is thus best achieved with viral vectors, and retro-, adeno-, and lentivirus RNAi systems have been described. However, the use of such human viral vectors is inherently problematic, e.g., Class 2 status and requirement of secondary helper functions. Although insect cells are their natural host, baculoviruses also transduce a range of vertebrate cell lines and primary cells with high efficiency. The inability of baculoviral vectors to replicate in mammalian cells, their Class 1 status, and the simplicity of their construction make baculovirus an attractive alternative gene delivery vector. We have developed a baculoviral-based RNAi system designed to express shRNAs and GFP from U6 and CMV promoters, respectively. Transduction of Saos2, HepG2, Huh7, and primary human hepatic stellate cells with a baculoviral construct expressing shRNAs targeting lamin A/C resulted in effective knockdown of the corresponding mRNA and protein. Development of this baculoviral-based system provides an additional shRNA delivery option for RNAi-based investigations in mammalian cells.

Improvement of a yeast self-excising integrative vector by prevention of expression leakage of the intronated Cre recombinase gene during plasmid maintenance in Escherichia coli.

PubMed

Agaphonov, Michael O

2017-12-01

The use of plasmids possessing a regulatable gene coding for a site-specific recombinase together with its recognition sequences significantly facilitates genome manipulations since it allows self-excision of the portion of the genetic construct integrated into the host genome. Stable maintenance of such plasmids in Escherichia coli, which is used for plasmid preparation, requires prevention of recombinase synthesis in this host, which can be achieved by interrupting the recombinase gene with an intron. Based on this approach, Saccharomyces cerevisiae and Hansenula polymorpha self-excising vectors possessing intronated gene for Cre recombinase and its recognition sites (LoxP) were previously constructed. However, this work shows instability of the H. polymorpha vectors during plasmid maintenance in E. coli cells. This could be due to recombination between the loxP sites caused by residual expression of the cre gene. Prevention of translation reinitiation on an internal methionine codon completely solved this problem. A similar modification was made in a self-excising vector designed for S. cerevisiae. Apart from substantial improvement of yeast self-excising vectors, the obtained results also narrow down the essential part of Cre sequence. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Construction of doxycycline-mediated BMP-2 transgene combining with APA microcapsules for bone repair.

PubMed

Qian, Dongyang; Bai, Bo; Yan, Guangbin; Zhang, Shujiang; Liu, Qi; Chen, Yi; Tan, Xiaobo; Zeng, Yanjun

2016-01-01

The repairing of large segmental bone defects is difficult for clinical orthopedists at present. Gene therapy is regarded as a promising method for bone defects repair. The present study aimed to construct an effective and controllable Tet-On expression system for transferring hBMP-2 gene into bone marrow mesenchymal progenitor cells (BMSCs). Meanwhile, with combination of alginate-poly-L-lysine-alginate (APA) microencapsulation technology, we attempted to reduce the influence of immunologic rejection and examine the effect of the Tet-On expression system on osteogenesis of BMSCs. The adenovirus encoding hBMP-2 (ADV-hBMP2) was constructed using the means of molecular cloning. The ADV-hBMP2 and Adeno-X Tet-On virus was respectively transfected to the HEK293 for amplification and afterward BMSCs were co-infected with the virus of ADV-hBMP2 and the Adeno-X Tet-On. The expression of hBMP-2 was measured with induction by doxycycline (DOX) at different concentration by means of RT-PCR and ELISA. Combining Tet-On expression system and APA microcapsules with the use of the pulsed high-voltage electrostatic microcapsule instrument, we examined the expression level of hBMP-2 in APA microcapsules by ELISA as well as the osteogenesis by alizarin red S staining. An effective Tet-On expression system for transferring hBMP-2 gene into BMSCs was constructed successfully. Also, the expression of hBMP-2 could be regulated by concentration of DOX. The data exhibited that BMSCs in APA microcapsules maintained the capability of proliferation and differentiation. The combination of Tet-On expression system and APA microcapsules could promote the osteogenesis of BMSCs. According to the results, microencapsulated Tet-On expression system showed the effective characteristics of secreting hBMP-2 and enhancing osteogenesis, which would provide a promising way for bone repair.

Methods and constructs for expression of foreign proteins in photosynthetic organisms

DOEpatents

Laible, Philip D.; Hanson, Deborah K.

2002-01-01

A method for expressing and purifying foreign proteins in photosynthetic organisms comprising the simultaneous expression of both the heterologous protein and a means for compartmentalizing or sequestering of the protein.

10 CFR Appendix N to Part 50 - Standardization of Nuclear Power Plant Designs: Permits To Construct and Licenses To Operate...

Code of Federal Regulations, 2011 CFR

2011-01-01

... Construct and Licenses To Operate Nuclear Power Reactors of Identical Design at Multiple Sites N Appendix N... Construct and Licenses To Operate Nuclear Power Reactors of Identical Design at Multiple Sites Section 101... nuclear power reactors of essentially the same design to be located at different sites. 1 1 If the design...

10 CFR Appendix N to Part 50 - Standardization of Nuclear Power Plant Designs: Permits To Construct and Licenses To Operate...

Code of Federal Regulations, 2012 CFR

2012-01-01

... Construct and Licenses To Operate Nuclear Power Reactors of Identical Design at Multiple Sites N Appendix N... Construct and Licenses To Operate Nuclear Power Reactors of Identical Design at Multiple Sites Section 101... nuclear power reactors of essentially the same design to be located at different sites. 1 1 If the design...

10 CFR Appendix N to Part 50 - Standardization of Nuclear Power Plant Designs: Permits To Construct and Licenses To Operate...

Code of Federal Regulations, 2013 CFR

2013-01-01

... Construct and Licenses To Operate Nuclear Power Reactors of Identical Design at Multiple Sites N Appendix N... Construct and Licenses To Operate Nuclear Power Reactors of Identical Design at Multiple Sites Section 101... nuclear power reactors of essentially the same design to be located at different sites. 1 1 If the design...

Development of inducer-free expression plasmids based on IPTG-inducible promoters for Bacillus subtilis.

PubMed

Tran, Dinh Thi Minh; Phan, Trang Thi Phuong; Huynh, Thanh Kieu; Dang, Ngan Thi Kim; Huynh, Phuong Thi Kim; Nguyen, Tri Minh; Truong, Tuom Thi Tinh; Tran, Thuoc Linh; Schumann, Wolfgang; Nguyen, Hoang Duc

2017-07-25

Besides Escherichia coli, Bacillus subtilis is an important bacterial species for the production of recombinant proteins. Recombinant genes are inserted into shuttle expression vectors which replicate in both E. coli and in B. subtilis. The ligation products are first transformed into E. coli cells, analyzed for correct insertions, and the correct recombinant plasmids are then transformed into B. subtilis. A major problem using E. coli cells can be the strong basal level of expression of the recombinant protein which may interfere with the stability of the cells. To minimize this problem, we developed strong expression vectors being repressed in E. coli and inducer-free in B. subtilis. In general, induction of IPTG-inducible expression vectors is determined by the regulatory lacI gene encoding the LacI repressor in combination with the lacO operator on the promoter. To investigate the inducer-free properties of the vectors, we constructed inducer-free expression plasmids by removing the lacI gene and characterized their properties. First, we examined the ability to repress a reporter gene in E. coli, which is a prominent property facilitating the construction of the expression vectors carrying a target gene. The β-galactosidase (bgaB gene) basal levels expressed from Pgrac01-bgaB could be repressed at least twice in the E. coli cloning strain. Second, the inducer-free production of BgaB from four different plasmids with the Pgrac01 promoter in B. subtilis was investigated. As expected, BgaB expression levels of inducer-free constructs are at least 37 times higher than that of the inducible constructs in the absence of IPTG, and comparable to those in the presence of the inducer. Third, using efficient IPTG-inducible expression vectors containing the strong promoter Pgrac100, we could convert them into inducer-free expression plasmids. The BgaB production levels from the inducer-free plasmid in the absence of the inducer were at least 4.5 times higher than that of the inducible vector using the same promoter. Finally, we used gfp as a reporter gene in combination with the two promoters Pgrac01 and Pgrac100 to test the new vector types. The GFP expression levels could be repressed at least 1.5 times for the Pgrac01-gfp+ inducer-free construct in E. coli. The inducer-free constructs Pgrac01-gfp+ and Pgrac100-gfp+ allowed GFP expression at high levels from 23 × 10 4 to 32 × 10 4 RFU units and 9-13% of total intracellular proteins. We could reconfirm the two major advantages of the new inducer-free expression plasmids: (1) Strong repression of the target gene expression in the E. coli cloning strain, and (2) production of the target protein at high levels in B. subtilis in the absence of the inducer. We propose a general strategy to generate inducer-free expression vector by using IPTG-inducible vectors, and more specifically we developed inducer-free expression plasmids using IPTG-inducible promoters in the absence of the LacI repressor. These plasmids could be an excellent choice for high-level production of recombinant proteins in B. subtilis without the addition of inducer and at the same time maintaining a low basal level of the recombinant proteins in E. coli. The repression of the recombinant gene expression would facilitate cloning of genes that potentially inhibit the growth of E. coli cloning strains. The inducer-free expression plasmids will be extended versions of the current available IPTG-inducible expression vectors for B. subtilis, in which all these vectors use the same cognate promoters. These inducer-free and previously developed IPTG-inducible expression plasmids will be a useful cassette to study gene expression at a small scale up to a larger scale up for the production of recombinant proteins.

Identification of differentially expressed genes in childhood asthma.

PubMed

Zhang, Nian-Zhen; Chen, Xiu-Juan; Mu, Yu-Hua; Wang, Hewen

2018-05-01

Asthma has been the most common chronic disease in children that places a major burden for affected people and their families.An integrated analysis of microarrays studies was performed to identify differentially expressed genes (DEGs) in childhood asthma compared with normal control. We also obtained the differentially methylated genes (DMGs) in childhood asthma according to GEO. The genes that were both differentially expressed and differentially methylated were identified. Functional annotation and protein-protein interaction network construction were performed to interpret biological functions of DEGs. We performed q-RT-PCR to verify the expression of selected DEGs.One DNA methylation and 3 gene expression datasets were obtained. Four hundred forty-one DEGs and 1209 DMGs in childhood asthma were identified. Among which, 16 genes were both differentially expressed and differentially methylated in childhood asthma. Natural killer cell mediated cytotoxicity pathway, Jak-STAT signaling pathway, and Wnt signaling pathway were 3 significantly enriched pathways in childhood asthma according to our KEGG enrichment analysis. The PPI network of top 20 up- and downregulated DEGs consisted of 822 nodes and 904 edges and 2 hub proteins (UBQLN4 and MID2) were identified. The expression of 8 DEGs (GZMB, FGFBP2, CLC, TBX21, ALOX15, IL12RB2, UBQLN4) was verified by qRT-PCR and only the expression of GZMB and FGFBP2 was inconsistent with our integrated analysis.Our finding was helpful to elucidate the underlying mechanism of childhood asthma and develop new potential diagnostic biomarker and provide clues for drug design.

The Methodology of Interactive Parametric Modelling of Construction Site Facilities in BIM Environment

NASA Astrophysics Data System (ADS)

Kozlovská, Mária; Čabala, Jozef; Struková, Zuzana

2014-11-01

Information technology is becoming a strong tool in different industries, including construction. The recent trend of buildings designing is leading up to creation of the most comprehensive virtual building model (Building Information Model) in order to solve all the problems relating to the project as early as in the designing phase. Building information modelling is a new way of approaching to the design of building projects documentation. Currently, the building site layout as a part of the building design documents has a very little support in the BIM environment. Recently, the research of designing the construction process conditions has centred on improvement of general practice in planning and on new approaches to construction site layout planning. The state of art in field of designing the construction process conditions indicated an unexplored problem related to connection of knowledge system with construction site facilities (CSF) layout through interactive modelling. The goal of the paper is to present the methodology for execution of 3D construction site facility allocation model (3D CSF-IAM), based on principles of parametric and interactive modelling.

Predicting adsorptive removal of chlorophenol from aqueous solution using artificial intelligence based modeling approaches.

PubMed

Singh, Kunwar P; Gupta, Shikha; Ojha, Priyanka; Rai, Premanjali

2013-04-01

The research aims to develop artificial intelligence (AI)-based model to predict the adsorptive removal of 2-chlorophenol (CP) in aqueous solution by coconut shell carbon (CSC) using four operational variables (pH of solution, adsorbate concentration, temperature, and contact time), and to investigate their effects on the adsorption process. Accordingly, based on a factorial design, 640 batch experiments were conducted. Nonlinearities in experimental data were checked using Brock-Dechert-Scheimkman (BDS) statistics. Five nonlinear models were constructed to predict the adsorptive removal of CP in aqueous solution by CSC using four variables as input. Performances of the constructed models were evaluated and compared using statistical criteria. BDS statistics revealed strong nonlinearity in experimental data. Performance of all the models constructed here was satisfactory. Radial basis function network (RBFN) and multilayer perceptron network (MLPN) models performed better than generalized regression neural network, support vector machines, and gene expression programming models. Sensitivity analysis revealed that the contact time had highest effect on adsorption followed by the solution pH, temperature, and CP concentration. The study concluded that all the models constructed here were capable of capturing the nonlinearity in data. A better generalization and predictive performance of RBFN and MLPN models suggested that these can be used to predict the adsorption of CP in aqueous solution using CSC.

Regulation of COL1A1 expression in type I collagen producing tissues: identification of a 49 base pair region which is required for transgene expression in bone of transgenic mice

NASA Technical Reports Server (NTRS)

Bedalov, A.; Salvatori, R.; Dodig, M.; Kronenberg, M. S.; Kapural, B.; Bogdanovic, Z.; Kream, B. E.; Woody, C. O.; Clark, S. H.; Mack, K.;

FLP recombinase in transgenic plants: constitutive activity in stably transformed tobacco and generation of marked cell clones in Arabidopsis.

PubMed

Kilby, N J; Davies, G J; Snaith, M R

1995-11-01

FLP site-specific recombinase was expressed in stably transformed tobacco and Arabidopsis. FLP-expressing tobacco lines were crossed with other transformed tobacco lines that contained a stably integrated FLP recognition target construct(s). The target construct consisted of two directly-oriented FLP recognition targets (FRTs), flanking a hygromycin resistance cassette located between a GUS coding region and an upstream 35S CaMV promoter. Excision of the hygromycin resistance cassette by FLP-mediated recombination between FRTs brings the GUS coding region under the transcriptional control of the CaMV 35S promoter. In the absence of FLP-mediated recombination, the GUS gene is transcriptionally silent. GUS activity was observed in the progeny of all crosses made between FLP recombinase-expressing and target-containing tobacco lines, but not in the selfs of parents. The predicted recombination product remaining after excision was confirmed by PCR and Southern analysis. In Arabidopsis, inducible expression of FLP recombinase was achieved from the soybean Gmhsp 17.6L heat-shock promoter. Heat-shock induction of FLP expression in plants containing the target construct led to activation of constitutive GUS expression in a subset of cells, whose progeny, therefore, were GUS-positive. A variety of clonal sectors were produced in plants derived from seed that was heat-shocked during germination. The ability to control the timing of GUS activation was demonstrated by heat-shock of unopened flower heads which produced large sectors. It was concluded that heat-shock-induced expression of FLP recombinase provides a readily controllable method for generating marked clonal sectors in Arabidopsis, the size and distribution of which reflects the timing of applied heat-shock.

10 CFR 433.6 - Sustainable principles for siting, design and construction. [Reserved

Code of Federal Regulations, 2013 CFR

2013-01-01

... 10 Energy 3 2013-01-01 2013-01-01 false Sustainable principles for siting, design and construction... FOR NEW FEDERAL COMMERCIAL AND MULTI-FAMILY HIGH-RISE RESIDENTIAL BUILDINGS § 433.6 Sustainable principles for siting, design and construction. [Reserved] ...

10 CFR 433.6 - Sustainable principles for siting, design and construction. [Reserved

Code of Federal Regulations, 2012 CFR

2012-01-01

... 10 Energy 3 2012-01-01 2012-01-01 false Sustainable principles for siting, design and construction... FOR NEW FEDERAL COMMERCIAL AND MULTI-FAMILY HIGH-RISE RESIDENTIAL BUILDINGS § 433.6 Sustainable principles for siting, design and construction. [Reserved] ...

10 CFR 433.6 - Sustainable principles for siting, design and construction. [Reserved

Code of Federal Regulations, 2014 CFR

2014-01-01

... 10 Energy 3 2014-01-01 2014-01-01 false Sustainable principles for siting, design and construction... FOR NEW FEDERAL COMMERCIAL AND MULTI-FAMILY HIGH-RISE RESIDENTIAL BUILDINGS § 433.6 Sustainable principles for siting, design and construction. [Reserved] ...

Assessment of Attention to Clothing and Impact of Its Restrictive Factors in Iranian Patients with Traumatic Spinal Cord Injury (ACIRF-SCI): Introduction of a New Questionnaire.

PubMed

Laleh, Leila; Latifi, Sahar; Koushki, Davood; Matin, Marzieh; Javidan, Abbas Norouzi; Yekaninejad, Mir Saeed

2015-01-01

Patients with spinal cord injury (SCI) deal with various restrictive factors regarding their clothing, such as disability and difficulty with access to shopping centers. We designed a questionnaire to assess attention to clothing and impact of its restrictive factors among Iranian patients with SCI (ACIRF-SCI). The ACIRF-SCI has 5 domains: functional, medical, attitude, aesthetic, and emotional. The first 3 domains reflect the impact of restrictive factors (factors that restrict attention to clothing), and the last 2 domains reflect attention to clothing and fashion. Functional restrictive factors include disability and dependence. Medical restrictive factors include existence of specific medical conditions that interfere with clothing choice. Construct validity was assessed by factorial analysis, and reliability was expressed by Cronbach's alpha. A total of 100 patients (75 men and 25 women) entered this study. Patients with a lower injury level had a higher total score (P < .0001), and similarly, patients with paraplegia had higher scores than those with tetraplegia (P < .0001), which illustrates an admissible discriminant validity. Postinjury duration was positively associated with total scores (r = 0.21, P = .04). Construct validity was 0.97, and Cronbach's alpha was 0.61. Iranian patients with SCI who have greater ability and independence experience a lower impact of restrictive factors related to clothing. The ACIRF-SCI reveals that this assumption is statistically significant, which shows its admissible discriminant validity. The measured construct validity (0.97) and reliability (internal consistency expressed by alpha = 0.61) are acceptable.

Assessment of Attention to Clothing and Impact of Its Restrictive Factors in Iranian Patients with Traumatic Spinal Cord Injury (ACIRF-SCI): Introduction of a New Questionnaire

PubMed Central

Laleh, Leila; Koushki, Davood; Matin, Marzieh; Javidan, Abbas Norouzi; Yekaninejad, Mir Saeed

2015-01-01

Background: Patients with spinal cord injury (SCI) deal with various restrictive factors regarding their clothing, such as disability and difficulty with access to shopping centers. Objectives: We designed a questionnaire to assess attention to clothing and impact of its restrictive factors among Iranian patients with SCI (ACIRF-SCI). Methods: The ACIRF-SCI has 5 domains: functional, medical, attitude, aesthetic, and emotional. The first 3 domains reflect the impact of restrictive factors (factors that restrict attention to clothing), and the last 2 domains reflect attention to clothing and fashion. Functional restrictive factors include disability and dependence. Medical restrictive factors include existence of specific medical conditions that interfere with clothing choice. Construct validity was assessed by factorial analysis, and reliability was expressed by Cronbach’s alpha. Results: A total of 100 patients (75 men and 25 women) entered this study. Patients with a lower injury level had a higher total score (P < .0001), and similarly, patients with paraplegia had higher scores than those with tetraplegia (P < .0001), which illustrates an admissible discriminant validity. Postinjury duration was positively associated with total scores (r = 0.21, P = .04). Construct validity was 0.97, and Cronbach’s alpha was 0.61. Conclusion: Iranian patients with SCI who have greater ability and independence experience a lower impact of restrictive factors related to clothing. The ACIRF-SCI reveals that this assumption is statistically significant, which shows its admissible discriminant validity. The measured construct validity (0.97) and reliability (internal consistency expressed by alpha = 0.61) are acceptable. PMID:26363593

Biomimetic perfusion and electrical stimulation applied in concert improved the assembly of engineered cardiac tissue.

PubMed

Maidhof, Robert; Tandon, Nina; Lee, Eun Jung; Luo, Jianwen; Duan, Yi; Yeager, Keith; Konofagou, Elisa; Vunjak-Novakovic, Gordana

2012-11-01

Maintenance of normal myocardial function depends intimately on synchronous tissue contraction, driven by electrical activation and on adequate nutrient perfusion in support thereof. Bioreactors have been used to mimic aspects of these factors in vitro to engineer cardiac tissue but, due to design limitations, previous bioreactor systems have yet to simultaneously support nutrient perfusion, electrical stimulation and unconstrained (i.e. not isometric) tissue contraction. To the best of our knowledge, the bioreactor system described herein is the first to integrate these three key factors in concert. We present the design of our bioreactor and characterize its capability in integrated experimental and mathematical modelling studies. We then cultured cardiac cells obtained from neonatal rats in porous, channelled elastomer scaffolds with the simultaneous application of perfusion and electrical stimulation, with controls excluding either one or both of these two conditions. After 8 days of culture, constructs grown with simultaneous perfusion and electrical stimulation exhibited substantially improved functional properties, as evidenced by a significant increase in contraction amplitude (0.23 ± 0.10% vs 0.14 ± 0.05%, 0.13 ± 0.08% or 0.09 ± 0.02% in control constructs grown without stimulation, without perfusion, or either stimulation or perfusion, respectively). Consistently, these constructs had significantly improved DNA contents, cell distribution throughout the scaffold thickness, cardiac protein expression, cell morphology and overall tissue organization compared to control groups. Thus, the simultaneous application of medium perfusion and electrical conditioning enabled by the use of the novel bioreactor system may accelerate the generation of fully functional, clinically sized cardiac tissue constructs. Copyright © 2011 John Wiley & Sons, Ltd.

49 CFR 178.277 - Requirements for the design, construction, inspection and testing of portable tanks intended for...

Code of Federal Regulations, 2011 CFR

2011-10-01

... design pressure in the ASME Code, Section VIII (IBR, see § 171.7 of this subchapter). Holding time is the... 49 Transportation 3 2011-10-01 2011-10-01 false Requirements for the design, construction... FOR PACKAGINGS Specifications for Portable Tanks § 178.277 Requirements for the design, construction...

49 CFR 178.277 - Requirements for the design, construction, inspection and testing of portable tanks intended for...

Code of Federal Regulations, 2012 CFR

2012-10-01

... design pressure in the ASME Code, Section VIII (IBR, see § 171.7 of this subchapter). Holding time is the... 49 Transportation 3 2012-10-01 2012-10-01 false Requirements for the design, construction... FOR PACKAGINGS Specifications for Portable Tanks § 178.277 Requirements for the design, construction...

49 CFR 178.277 - Requirements for the design, construction, inspection and testing of portable tanks intended for...

Code of Federal Regulations, 2014 CFR

2014-10-01

... design pressure in the ASME Code, Section VIII (IBR, see § 171.7 of this subchapter). Holding time is the... 49 Transportation 3 2014-10-01 2014-10-01 false Requirements for the design, construction... FOR PACKAGINGS Specifications for Portable Tanks § 178.277 Requirements for the design, construction...

49 CFR 178.277 - Requirements for the design, construction, inspection and testing of portable tanks intended for...

Code of Federal Regulations, 2010 CFR

2010-10-01

... design pressure in the ASME Code, Section VIII (IBR, see § 171.7 of this subchapter). Holding time is the... 49 Transportation 2 2010-10-01 2010-10-01 false Requirements for the design, construction... FOR PACKAGINGS Specifications for Portable Tanks § 178.277 Requirements for the design, construction...

49 CFR 178.277 - Requirements for the design, construction, inspection and testing of portable tanks intended for...

Code of Federal Regulations, 2013 CFR

2013-10-01

... design pressure in the ASME Code, Section VIII (IBR, see § 171.7 of this subchapter). Holding time is the... 49 Transportation 3 2013-10-01 2013-10-01 false Requirements for the design, construction... FOR PACKAGINGS Specifications for Portable Tanks § 178.277 Requirements for the design, construction...