A rapid method for establishment of a reverse genetics system for canine parvovirus.

PubMed

Yu, Yongle; Su, Jun; Wang, Jigui; Xi, Ji; Mao, Yaping; Hou, Qiang; Zhang, Xiaomei; Liu, Weiquan

2017-12-01

Canine parvovirus (CPV) is an important and highly prevalent pathogen of dogs that causes acute hemorrhagic enteritis disease. Here, we describe a rapid method for the construction and characterization of a full-length infectious clone (rCPV) of CPV. Feline kidney (F81) cells were transfected with rCPV incorporating an engineered EcoR I site that served as a genetic marker. The rescued virus was indistinguishable from that of wild-type virus in its biological properties.

Reverse Engineering a Signaling Network Using Alternative Inputs

PubMed Central

Tanaka, Hiromasa; Yi, Tau-Mu

2009-01-01

One of the goals of systems biology is to reverse engineer in a comprehensive fashion the arrow diagrams of signal transduction systems. An important tool for ordering pathway components is genetic epistasis analysis, and here we present a strategy termed Alternative Inputs (AIs) to perform systematic epistasis analysis. An alternative input is defined as any genetic manipulation that can activate the signaling pathway instead of the natural input. We introduced the concept of an “AIs-Deletions matrix” that summarizes the outputs of all combinations of alternative inputs and deletions. We developed the theory and algorithms to construct a pairwise relationship graph from the AIs-Deletions matrix capturing both functional ordering (upstream, downstream) and logical relationships (AND, OR), and then interpreting these relationships into a standard arrow diagram. As a proof-of-principle, we applied this methodology to a subset of genes involved in yeast mating signaling. This experimental pilot study highlights the robustness of the approach and important technical challenges. In summary, this research formalizes and extends classical epistasis analysis from linear pathways to more complex networks, facilitating computational analysis and reconstruction of signaling arrow diagrams. PMID:19898612

Islet xenotransplantation from genetically engineered pigs.

PubMed

Nagaraju, Santosh; Bottino, Rita; Wijkstrom, Martin; Hara, Hidetaka; Trucco, Massimo; Cooper, David K C

2013-12-01

Pigs have emerged as potential sources of islets for clinical transplantation. Wild-type porcine islets (adult and neonatal) transplanted into the portal vein have successfully reversed diabetes in nonhuman primates. However, there is a rapid loss of the transplanted islets on exposure to blood, known as the instant blood-mediated inflammatory reaction (IBMIR), as well as a T-cell response that leads to rejection of the graft. Genetically modified pig islets offer a number of potential advantages, particularly with regard to reducing the IBMIR-related graft loss and protecting the islets from the primate immune response. Emerging data indicate that transgenes specifically targeted to pig β cells using an insulin promoter (in order to maximize target tissue expression while limiting host effects) can be achieved without significant effects on the pig's glucose metabolism. Experience with the transplantation of islets from genetically engineered pigs into nonhuman primates is steadily increasing, and has involved the deletion of pig antigenic targets to reduce the primate humoral response, the expression of transgenes for human complement-regulatory and coagulation-regulatory proteins, and manipulations to reduce the effect of the T-cell response. There is increasing evidence of the advantages of using genetically engineered pigs as sources of islets for future clinical trials.

Rescue of a Plant Negative-Strand RNA Virus from Cloned cDNA: Insights into Enveloped Plant Virus Movement and Morphogenesis.

PubMed

Wang, Qiang; Ma, Xiaonan; Qian, ShaSha; Zhou, Xin; Sun, Kai; Chen, Xiaolan; Zhou, Xueping; Jackson, Andrew O; Li, Zhenghe

2015-10-01

Reverse genetics systems have been established for all major groups of plant DNA and positive-strand RNA viruses, and our understanding of their infection cycles and pathogenesis has benefitted enormously from use of these approaches. However, technical difficulties have heretofore hampered applications of reverse genetics to plant negative-strand RNA (NSR) viruses. Here, we report recovery of infectious virus from cloned cDNAs of a model plant NSR, Sonchus yellow net rhabdovirus (SYNV). The procedure involves Agrobacterium-mediated transcription of full-length SYNV antigenomic RNA and co-expression of the nucleoprotein (N), phosphoprotein (P), large polymerase core proteins and viral suppressors of RNA silencing in Nicotiana benthamiana plants. Optimization of core protein expression resulted in up to 26% recombinant SYNV (rSYNV) infections of agroinfiltrated plants. A reporter virus, rSYNV-GFP, engineered by inserting a green fluorescence protein (GFP) gene between the N and P genes was able to express GFP during systemic infections and after repeated plant-to-plant mechanical passages. Deletion analyses with rSYNV-GFP demonstrated that SYNV cell-to-cell movement requires the sc4 protein and suggested that uncoiled nucleocapsids are infectious movement entities. Deletion analyses also showed that the glycoprotein is not required for systemic infection, although the glycoprotein mutant was defective in virion morphogenesis. Taken together, we have developed a robust reverse genetics system for SYNV that provides key insights into morphogenesis and movement of an enveloped plant virus. Our study also provides a template for developing analogous systems for reverse genetic analysis of other plant NSR viruses.

Rescue of a Plant Negative-Strand RNA Virus from Cloned cDNA: Insights into Enveloped Plant Virus Movement and Morphogenesis

PubMed Central

Zhou, Xin; Sun, Kai; Chen, Xiaolan; Zhou, Xueping; Jackson, Andrew O.; Li, Zhenghe

2015-01-01

Reverse genetics systems have been established for all major groups of plant DNA and positive-strand RNA viruses, and our understanding of their infection cycles and pathogenesis has benefitted enormously from use of these approaches. However, technical difficulties have heretofore hampered applications of reverse genetics to plant negative-strand RNA (NSR) viruses. Here, we report recovery of infectious virus from cloned cDNAs of a model plant NSR, Sonchus yellow net rhabdovirus (SYNV). The procedure involves Agrobacterium-mediated transcription of full-length SYNV antigenomic RNA and co-expression of the nucleoprotein (N), phosphoprotein (P), large polymerase core proteins and viral suppressors of RNA silencing in Nicotiana benthamiana plants. Optimization of core protein expression resulted in up to 26% recombinant SYNV (rSYNV) infections of agroinfiltrated plants. A reporter virus, rSYNV-GFP, engineered by inserting a green fluorescence protein (GFP) gene between the N and P genes was able to express GFP during systemic infections and after repeated plant-to-plant mechanical passages. Deletion analyses with rSYNV-GFP demonstrated that SYNV cell-to-cell movement requires the sc4 protein and suggested that uncoiled nucleocapsids are infectious movement entities. Deletion analyses also showed that the glycoprotein is not required for systemic infection, although the glycoprotein mutant was defective in virion morphogenesis. Taken together, we have developed a robust reverse genetics system for SYNV that provides key insights into morphogenesis and movement of an enveloped plant virus. Our study also provides a template for developing analogous systems for reverse genetic analysis of other plant NSR viruses. PMID:26484673

Reverse breeding: a novel breeding approach based on engineered meiosis.

PubMed

Dirks, Rob; van Dun, Kees; de Snoo, C Bastiaan; van den Berg, Mark; Lelivelt, Cilia L C; Voermans, William; Woudenberg, Leo; de Wit, Jack P C; Reinink, Kees; Schut, Johan W; van der Zeeuw, Eveline; Vogelaar, Aat; Freymark, Gerald; Gutteling, Evert W; Keppel, Marina N; van Drongelen, Paul; Kieny, Matthieu; Ellul, Philippe; Touraev, Alisher; Ma, Hong; de Jong, Hans; Wijnker, Erik

2009-12-01

Reverse breeding (RB) is a novel plant breeding technique designed to directly produce parental lines for any heterozygous plant, one of the most sought after goals in plant breeding. RB generates perfectly complementing homozygous parental lines through engineered meiosis. The method is based on reducing genetic recombination in the selected heterozygote by eliminating meiotic crossing over. Male or female spores obtained from such plants contain combinations of non-recombinant parental chromosomes which can be cultured in vitro to generate homozygous doubled haploid plants (DHs). From these DHs, complementary parents can be selected and used to reconstitute the heterozygote in perpetuity. Since the fixation of unknown heterozygous genotypes is impossible in traditional plant breeding, RB could fundamentally change future plant breeding. In this review, we discuss various other applications of RB, including breeding per chromosome.

Reverse breeding: a novel breeding approach based on engineered meiosis

PubMed Central

Dirks, Rob; van Dun, Kees; de Snoo, C Bastiaan; van den Berg, Mark; Lelivelt, Cilia L C; Voermans, William; Woudenberg, Leo; de Wit, Jack P C; Reinink, Kees; Schut, Johan W; van der Zeeuw, Eveline; Vogelaar, Aat; Freymark, Gerald; Gutteling, Evert W; Keppel, Marina N; van Drongelen, Paul; Kieny, Matthieu; Ellul, Philippe; Touraev, Alisher; Ma, Hong; de Jong, Hans; Wijnker, Erik

2009-01-01

Reverse breeding (RB) is a novel plant breeding technique designed to directly produce parental lines for any heterozygous plant, one of the most sought after goals in plant breeding. RB generates perfectly complementing homozygous parental lines through engineered meiosis. The method is based on reducing genetic recombination in the selected heterozygote by eliminating meiotic crossing over. Male or female spores obtained from such plants contain combinations of non-recombinant parental chromosomes which can be cultured in vitro to generate homozygous doubled haploid plants (DHs). From these DHs, complementary parents can be selected and used to reconstitute the heterozygote in perpetuity. Since the fixation of unknown heterozygous genotypes is impossible in traditional plant breeding, RB could fundamentally change future plant breeding. In this review, we discuss various other applications of RB, including breeding per chromosome. PMID:19811618

[Transcription activator-like effectors(TALEs)based genome engineering].

PubMed

Zhao, Mei-Wei; Duan, Cheng-Li; Liu, Jiang

2013-10-01

Systematic reverse-engineering of functional genome architecture requires precise modifications of gene sequences and transcription levels. The development and application of transcription activator-like effectors(TALEs) has created a wealth of genome engineering possibilities. TALEs are a class of naturally occurring DNA-binding proteins found in the plant pathogen Xanthomonas species. The DNA-binding domain of each TALE typically consists of tandem 34-amino acid repeat modules rearranged according to a simple cipher to target new DNA sequences. Customized TALEs can be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Such "genome engineering" has now been established in human cells and a number of model organisms, thus opening the door to better understanding gene function in model organisms, improving traits in crop plants and treating human genetic disorders.

Site-specific selfish genes as tools for the control and genetic engineering of natural populations.

PubMed

Burt, Austin

2003-05-07

Site-specific selfish genes exploit host functions to copy themselves into a defined target DNA sequence, and include homing endonuclease genes, group II introns and some LINE-like transposable elements. If such genes can be engineered to target new host sequences, then they can be used to manipulate natural populations, even if the number of individuals released is a small fraction of the entire population. For example, a genetic load sufficient to eradicate a population can be imposed in fewer than 20 generations, if the target is an essential host gene, the knockout is recessive and the selfish gene has an appropriate promoter. There will be selection for resistance, but several strategies are available for reducing the likelihood of it evolving. These genes may also be used to genetically engineer natural populations, by means of population-wide gene knockouts, gene replacements and genetic transformations. By targeting sex-linked loci just prior to meiosis one may skew the population sex ratio, and by changing the promoter one may limit the spread of the gene to neighbouring populations. The proposed constructs are evolutionarily stable in the face of the mutations most likely to arise during their spread, and strategies are also available for reversing the manipulations.

Production and Characterization of Chemically Inactivated Genetically Engineered Clostridium difficile Toxoids.

PubMed

Vidunas, Eugene; Mathews, Antony; Weaver, Michele; Cai, Ping; Koh, Eun Hee; Patel-Brown, Sujata; Yuan, Hailey; Zheng, Zi-Rong; Carriere, Marjolaine; Johnson, J Erik; Lotvin, Jason; Moran, Justin

2016-07-01

A recombinant Clostridium difficile expression system was used to produce genetically engineered toxoids A and B as immunogens for a prophylactic vaccine against C. difficile-associated disease. Although all known enzymatic activities responsible for cytotoxicity were genetically abrogated, the toxoids exhibited residual cytotoxic activity as measured in an in vitro cell-based cytotoxicity assay. The residual cytotoxicity was eliminated by treating the toxoids with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide. Mass spectrometry and amino acid analysis of the EDC-inactivated toxoids identified crosslinks, glycine adducts, and β-alanine adducts. Surface plasmon resonance analysis demonstrated that modifications resulting from the chemical treatment did not appreciably affect recognition of epitopes by both toxin A- and B-specific neutralizing monoclonal antibodies. Compared to formaldehyde-inactivated toxoids, the EDC/N-hydroxysuccinimide-inactivated toxoids exhibited superior stability in solution with respect to reversion of cytotoxic activity. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Sequences in Influenza A Virus PB2 Protein That Determine Productive Infection for an Avian Influenza Virus in Mouse and Human Cell Lines

PubMed Central

Yao, Yongxiu; Mingay, Louise J.; McCauley, John W.; Barclay, Wendy S.

2001-01-01

Reverse genetics was used to analyze the host range of two avian influenza viruses which differ in their ability to replicate in mouse and human cells in culture. Engineered viruses carrying sequences encoding amino acids 362 to 581 of PB2 from a host range variant productively infect mouse and human cells. PMID:11333926

Rational Design of an Ultrasensitive Quorum-Sensing Switch.

PubMed

Zeng, Weiqian; Du, Pei; Lou, Qiuli; Wu, Lili; Zhang, Haoqian M; Lou, Chunbo; Wang, Hongli; Ouyang, Qi

2017-08-18

One of the purposes of synthetic biology is to develop rational methods that accelerate the design of genetic circuits, saving time and effort spent on experiments and providing reliably predictable circuit performance. We applied a reverse engineering approach to design an ultrasensitive transcriptional quorum-sensing switch. We want to explore how systems biology can guide synthetic biology in the choice of specific DNA sequences and their regulatory relations to achieve a targeted function. The workflow comprises network enumeration that achieves the target function robustly, experimental restriction of the obtained candidate networks, global parameter optimization via mathematical analysis, selection and engineering of parts based on these calculations, and finally, circuit construction based on the principles of standardization and modularization. The performance of realized quorum-sensing switches was in good qualitative agreement with the computational predictions. This study provides practical principles for the rational design of genetic circuits with targeted functions.

Under pressure: evolutionary engineering of yeast strains for improved performance in fuels and chemicals production.

PubMed

Mans, Robert; Daran, Jean-Marc G; Pronk, Jack T

2018-04-01

Evolutionary engineering, which uses laboratory evolution to select for industrially relevant traits, is a popular strategy in the development of high-performing yeast strains for industrial production of fuels and chemicals. By integrating whole-genome sequencing, bioinformatics, classical genetics and genome-editing techniques, evolutionary engineering has also become a powerful approach for identification and reverse engineering of molecular mechanisms that underlie industrially relevant traits. New techniques enable acceleration of in vivo mutation rates, both across yeast genomes and at specific loci. Recent studies indicate that phenotypic trade-offs, which are often observed after evolution under constant conditions, can be mitigated by using dynamic cultivation regimes. Advances in research on synthetic regulatory circuits offer exciting possibilities to extend the applicability of evolutionary engineering to products of yeasts whose synthesis requires a net input of cellular energy. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

14 CFR 25.934 - Turbojet engine thrust reverser system tests.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Turbojet engine thrust reverser system... TRANSPORTATION AIRCRAFT AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY AIRPLANES Powerplant General § 25.934 Turbojet engine thrust reverser system tests. Thrust reversers installed on turbojet engines must meet the...

Utilisation of ISA Reverse Genetics and Large-Scale Random Codon Re-Encoding to Produce Attenuated Strains of Tick-Borne Encephalitis Virus within Days.

PubMed

de Fabritus, Lauriane; Nougairède, Antoine; Aubry, Fabien; Gould, Ernest A; de Lamballerie, Xavier

2016-01-01

Large-scale codon re-encoding is a new method of attenuating RNA viruses. However, the use of infectious clones to generate attenuated viruses has inherent technical problems. We previously developed a bacterium-free reverse genetics protocol, designated ISA, and now combined it with large-scale random codon-re-encoding method to produce attenuated tick-borne encephalitis virus (TBEV), a pathogenic flavivirus which causes febrile illness and encephalitis in humans. We produced wild-type (WT) and two re-encoded TBEVs, containing 273 or 273+284 synonymous mutations in the NS5 and NS5+NS3 coding regions respectively. Both re-encoded viruses were attenuated when compared with WT virus using a laboratory mouse model and the relative level of attenuation increased with the degree of re-encoding. Moreover, all infected animals produced neutralizing antibodies. This novel, rapid and efficient approach to engineering attenuated viruses could potentially expedite the development of safe and effective new-generation live attenuated vaccines.

Reverse Genetics for Mammalian Orthoreovirus.

PubMed

Stuart, Johnasha D; Phillips, Matthew B; Boehme, Karl W

2017-01-01

Reverse genetics allows introduction of specific alterations into a viral genome. Studies performed with mutant viruses generated using reverse genetics approaches have contributed immeasurably to our understanding of viral replication and pathogenesis, and also have led to development of novel vaccines and virus-based vectors. Here, we describe the reverse genetics system that allows for production and recovery of mammalian orthoreovirus, a double-stranded (ds) RNA virus, from plasmids that encode the viral genome.

Universal Influenza B Virus Genomic Amplification Facilitates Sequencing, Diagnostics, and Reverse Genetics

PubMed Central

Zhou, Bin; Lin, Xudong; Wang, Wei; Halpin, Rebecca A.; Bera, Jayati; Stockwell, Timothy B.; Barr, Ian G.

2014-01-01

Although human influenza B virus (IBV) is a significant human pathogen, its great genetic diversity has limited our ability to universally amplify the entire genome for subsequent sequencing or vaccine production. The generation of sequence data via next-generation approaches and the rapid cloning of viral genes are critical for basic research, diagnostics, antiviral drugs, and vaccines to combat IBV. To overcome the difficulty of amplifying the diverse and ever-changing IBV genome, we developed and optimized techniques that amplify the complete segmented negative-sense RNA genome from any IBV strain in a single tube/well (IBV genomic amplification [IBV-GA]). Amplicons for >1,000 diverse IBV genomes from different sample types (e.g., clinical specimens) were generated and sequenced using this robust technology. These approaches are sensitive, robust, and sequence independent (i.e., universally amplify past, present, and future IBVs), which facilitates next-generation sequencing and advanced genomic diagnostics. Importantly, special terminal sequences engineered into the optimized IBV-GA2 products also enable ligation-free cloning to rapidly generate reverse-genetics plasmids, which can be used for the rescue of recombinant viruses and/or the creation of vaccine seed stock. PMID:24501036

Reverse Genetics System Demonstrates that Rotavirus Nonstructural Protein NSP6 Is Not Essential for Viral Replication in Cell Culture.

PubMed

Komoto, Satoshi; Kanai, Yuta; Fukuda, Saori; Kugita, Masanori; Kawagishi, Takahiro; Ito, Naoto; Sugiyama, Makoto; Matsuura, Yoshiharu; Kobayashi, Takeshi; Taniguchi, Koki

2017-11-01

The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two nonstructural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid-only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6-deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture, although the NSP6 protein seemed to promote efficient virus growth. IMPORTANCE Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmid-only-based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can hardly be addressed by conventional approaches. Copyright © 2017 American Society for Microbiology.

Reveal, A General Reverse Engineering Algorithm for Inference of Genetic Network Architectures

NASA Technical Reports Server (NTRS)

Liang, Shoudan; Fuhrman, Stefanie; Somogyi, Roland

1998-01-01

Given the immanent gene expression mapping covering whole genomes during development, health and disease, we seek computational methods to maximize functional inference from such large data sets. Is it possible, in principle, to completely infer a complex regulatory network architecture from input/output patterns of its variables? We investigated this possibility using binary models of genetic networks. Trajectories, or state transition tables of Boolean nets, resemble time series of gene expression. By systematically analyzing the mutual information between input states and output states, one is able to infer the sets of input elements controlling each element or gene in the network. This process is unequivocal and exact for complete state transition tables. We implemented this REVerse Engineering ALgorithm (REVEAL) in a C program, and found the problem to be tractable within the conditions tested so far. For n = 50 (elements) and k = 3 (inputs per element), the analysis of incomplete state transition tables (100 state transition pairs out of a possible 10(exp 15)) reliably produced the original rule and wiring sets. While this study is limited to synchronous Boolean networks, the algorithm is generalizable to include multi-state models, essentially allowing direct application to realistic biological data sets. The ability to adequately solve the inverse problem may enable in-depth analysis of complex dynamic systems in biology and other fields.

Engineering Foot-and-Mouth Disease Viruses with Improved Growth Properties for Vaccine Development

PubMed Central

Zheng, Haixue; Guo, Jianhong; Jin, Ye; Yang, Fan; He, Jijun; Lv, Lv; Zhang, Kesan; Wu, Qiong; Liu, Xiangtao; Cai, Xuepeng

2013-01-01

Background No licensed vaccine is currently available against serotype A foot-and-mouth disease (FMD) in China, despite the isolation of A/WH/CHA/09 in 2009, partly because this strain does not replicate well in baby hamster kidney (BHK) cells. Methodology/Principal Findings A novel plasmid-based reverse genetics system was used to construct a chimeric strain by replacing the P1 gene in the vaccine strain O/CHA/99 with that from the epidemic stain A/WH/CHA/09. The chimeric virus displayed growth kinetics similar to those of O/CHA/99 and was selected for use as a candidate vaccine strain after 12 passages in BHK cells. Cattle were vaccinated with the inactivated vaccine and humoral immune responses were induced in most of the animals on day 7. A challenge infection with A/WH/CHA/09 on day 28 indicated that the group given a 4-µg dose was fully protected and neither developed viremia nor seroconverted to a 3ABC antigen. Conclusions/Significance Our data demonstrate that the chimeric virus not only propagates well in BHK cells and has excellent antigenic matching against serotype A FMD, but is also a potential marker vaccine to distinguish infection from vaccination. These results suggest that reverse genetics technology is a useful tool for engineering vaccines for the prevention and control of FMD. PMID:23372840

A swarm intelligence framework for reconstructing gene networks: searching for biologically plausible architectures.

PubMed

Kentzoglanakis, Kyriakos; Poole, Matthew

2012-01-01

In this paper, we investigate the problem of reverse engineering the topology of gene regulatory networks from temporal gene expression data. We adopt a computational intelligence approach comprising swarm intelligence techniques, namely particle swarm optimization (PSO) and ant colony optimization (ACO). In addition, the recurrent neural network (RNN) formalism is employed for modeling the dynamical behavior of gene regulatory systems. More specifically, ACO is used for searching the discrete space of network architectures and PSO for searching the corresponding continuous space of RNN model parameters. We propose a novel solution construction process in the context of ACO for generating biologically plausible candidate architectures. The objective is to concentrate the search effort into areas of the structure space that contain architectures which are feasible in terms of their topological resemblance to real-world networks. The proposed framework is initially applied to the reconstruction of a small artificial network that has previously been studied in the context of gene network reverse engineering. Subsequently, we consider an artificial data set with added noise for reconstructing a subnetwork of the genetic interaction network of S. cerevisiae (yeast). Finally, the framework is applied to a real-world data set for reverse engineering the SOS response system of the bacterium Escherichia coli. Results demonstrate the relative advantage of utilizing problem-specific knowledge regarding biologically plausible structural properties of gene networks over conducting a problem-agnostic search in the vast space of network architectures.

Next-generation AAV vectors for clinical use: an ever-accelerating race.

PubMed

Weinmann, Jonas; Grimm, Dirk

2017-10-01

During the past five decades, it has become evident that Adeno-associated virus (AAV) represents one of the most potent, most versatile, and thus most auspicious platforms available for gene delivery into cells, animals and, ultimately, humans. Particularly attractive is the ease with which the viral capsid-the major determinant of virus-host interaction including cell specificity and antibody recognition-can be modified and optimized at will. This has motivated countless researchers to develop high-throughput technologies in which genetically engineered AAV capsid libraries are subjected to a vastly hastened emulation of natural evolution, with the aim to enrich novel synthetic AAV capsids displaying superior features for clinical application. While the power and potential of these forward genetics approaches is undisputed, they are also inherently challenging as success depends on a combination of library quality, fidelity, and complexity. Here, we will describe and discuss two original, very exciting strategies that have emerged over the last three years and that promise to alleviate at least some of these concerns, namely, (i) a reverse genetics approach termed "ancestral AAV sequence reconstruction," and (ii) AAV genome barcoding as a technology that can advance both, forward and reverse genetics stratagems. Notably, despite the conceptual differences of these two technologies, they pursue the same goal which is tailored acceleration of AAV evolution and thus winning the race for the next-generation AAV vectors for clinical use.

Genetically Programmable Thermoresponsive Plasmonic Gold/Silk-Elastin Protein Core/Shell Nanoparticles

PubMed Central

2015-01-01

The design and development of future molecular photonic/electronic systems pose the challenge of integrating functional molecular building blocks in a controlled, tunable, and reproducible manner. The modular nature and fidelity of the biosynthesis method provides a unique chemistry approach to one-pot synthesis of environmental factor-responsive chimeric proteins capable of energy conversion between the desired forms. In this work, facile tuning of dynamic thermal response in plasmonic nanoparticles was facilitated by genetic engineering of the structure, size, and self-assembly of the shell silk-elastin-like protein polymers (SELPs). Recombinant DNA techniques were implemented to synthesize a new family of SELPs, S4E8Gs, with amino acid repeats of [(GVGVP)4(GGGVP)(GVGVP)3(GAGAGS)4] and tunable molecular weight. The temperature-reversible conformational switching between the hydrophilic random coils and the hydrophobic β-turns in the elastin blocks were programmed to between 50 and 60 °C by site-specific glycine mutation, as confirmed by variable-temperature proton NMR and circular dichroism (CD) spectroscopy, to trigger the nanoparticle aggregation. The dynamic self-aggregation/disaggregation of the Au-SELPs nanoparticles was regulated in size and pattern by the β-sheet-forming, thermally stable silk blocks, as revealed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The thermally reversible, shell dimension dependent, interparticle plasmon coupling was investigated by both variable-temperature UV–vis spectroscopy and finite-difference time-domain (FDTD)-based simulations. Good agreement between the calculated and measured spectra sheds light on design and synthesis of responsive plasmonic nanostructures by independently tuning the refractive index and size of the SELPs through genetic engineering. PMID:24712906

Genetically Engineered Virulent Phage Banks in the Detection and Control of Emergent Pathogenic Bacteria

PubMed Central

Blois, Hélène; Iris, François

2010-01-01

Natural outbreaks of multidrug-resistant microorganisms can cause widespread devastation, and several can be used or engineered as agents of bioterrorism. From a biosecurity standpoint, the capacity to detect and then efficiently control, within hours, the spread and the potential pathological effects of an emergent outbreak, for which there may be no effective antibiotics or vaccines, become key challenges that must be met. We turned to phage engineering as a potentially highly flexible and effective means to both detect and eradicate threats originating from emergent (uncharacterized) bacterial strains. To this end, we developed technologies allowing us to (1) concurrently modify multiple regions within the coding sequence of a gene while conserving intact the remainder of the gene, (2) reversibly interrupt the lytic cycle of an obligate virulent phage (T4) within its host, (3) carry out efficient insertion, by homologous recombination, of any number of engineered genes into the deactivated genomes of a T4 wild-type phage population, and (4) reactivate the lytic cycle, leading to the production of engineered infective virulent recombinant progeny. This allows the production of very large, genetically engineered lytic phage banks containing, in an E. coli host, a very wide spectrum of variants for any chosen phage-associated function, including phage host-range. Screening of such a bank should allow the rapid isolation of recombinant T4 particles capable of detecting (ie, diagnosing), infecting, and destroying hosts belonging to gram-negative bacterial species far removed from the original E. coli host. PMID:20569057

An internal deletion in MTH1 enables growth on glucose of pyruvate-decarboxylase negative, non-fermentative Saccharomyces cerevisiae

PubMed Central

2012-01-01

Background Pyruvate-decarboxylase negative (Pdc-) strains of Saccharomyces cerevisiae combine the robustness and high glycolytic capacity of this yeast with the absence of alcoholic fermentation. This makes Pdc-S. cerevisiae an interesting platform for efficient conversion of glucose towards pyruvate-derived products without formation of ethanol as a by-product. However, Pdc- strains cannot grow on high glucose concentrations and require C2-compounds (ethanol or acetate) for growth under conditions with low glucose concentrations, which hitherto has limited application in industry. Results Genetic analysis of a Pdc- strain previously evolved to overcome these deficiencies revealed a 225bp in-frame internal deletion in MTH1, encoding a transcriptional regulator involved in glucose sensing. This internal deletion contains a phosphorylation site required for degradation, thereby hypothetically resulting in increased stability of the protein. Reverse engineering of this alternative MTH1 allele into a non-evolved Pdc- strain enabled growth on 20 g l-1 glucose and 0.3% (v/v) ethanol at a maximum specific growth rate (0.24 h-1) similar to that of the evolved Pdc- strain (0.23 h-1). Furthermore, the reverse engineered Pdc- strain grew on glucose as sole carbon source, albeit at a lower specific growth rate (0.10 h-1) than the evolved strain (0.20 h-1). The observation that overexpression of the wild-type MTH1 allele also restored growth of Pdc-S. cerevisiae on glucose is consistent with the hypothesis that the internal deletion results in decreased degradation of Mth1. Reduced degradation of Mth1 has been shown to result in deregulation of hexose transport. In Pdc- strains, reduced glucose uptake may prevent intracellular accumulation of pyruvate and/or redox problems, while release of glucose repression due to the MTH1 internal deletion may contribute to alleviation of the C2-compound auxotrophy. Conclusions In this study we have discovered and characterised a mutation in MTH1 enabling Pdc- strains to grow on glucose as the sole carbon source. This successful example of reverse engineering not only increases the understanding of the glucose tolerance of evolved Pdc-S. cerevisiae, but also allows introduction of this portable genetic element into various industrial yeast strains, thereby simplifying metabolic engineering strategies. PMID:22978798

14 CFR 23.934 - Turbojet and turbofan engine thrust reverser systems tests.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Turbojet and turbofan engine thrust... CATEGORY AIRPLANES Powerplant General § 23.934 Turbojet and turbofan engine thrust reverser systems tests. Thrust reverser systems of turbojet or turbofan engines must meet the requirements of § 33.97 of this...

14 CFR 23.1155 - Turbine engine reverse thrust and propeller pitch settings below the flight regime.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Turbine engine reverse thrust and propeller... COMMUTER CATEGORY AIRPLANES Powerplant Powerplant Controls and Accessories § 23.1155 Turbine engine reverse thrust and propeller pitch settings below the flight regime. For turbine engine installations, each...

14 CFR 23.1155 - Turbine engine reverse thrust and propeller pitch settings below the flight regime.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Turbine engine reverse thrust and propeller... COMMUTER CATEGORY AIRPLANES Powerplant Powerplant Controls and Accessories § 23.1155 Turbine engine reverse thrust and propeller pitch settings below the flight regime. For turbine engine installations, each...

14 CFR 23.1155 - Turbine engine reverse thrust and propeller pitch settings below the flight regime.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 14 Aeronautics and Space 1 2014-01-01 2014-01-01 false Turbine engine reverse thrust and propeller... COMMUTER CATEGORY AIRPLANES Powerplant Powerplant Controls and Accessories § 23.1155 Turbine engine reverse thrust and propeller pitch settings below the flight regime. For turbine engine installations, each...

14 CFR 23.1155 - Turbine engine reverse thrust and propeller pitch settings below the flight regime.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 14 Aeronautics and Space 1 2013-01-01 2013-01-01 false Turbine engine reverse thrust and propeller... COMMUTER CATEGORY AIRPLANES Powerplant Powerplant Controls and Accessories § 23.1155 Turbine engine reverse thrust and propeller pitch settings below the flight regime. For turbine engine installations, each...

14 CFR 23.1155 - Turbine engine reverse thrust and propeller pitch settings below the flight regime.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 14 Aeronautics and Space 1 2012-01-01 2012-01-01 false Turbine engine reverse thrust and propeller... COMMUTER CATEGORY AIRPLANES Powerplant Powerplant Controls and Accessories § 23.1155 Turbine engine reverse thrust and propeller pitch settings below the flight regime. For turbine engine installations, each...

Expression of type 2 diacylglycerol acyltransferse gene DGTT1 from Chlamydomonas reinhardtii enhances lipid production in Scenedesmus obliquus.

PubMed

Chen, Chun-Yen; Kao, Ai-Ling; Tsai, Zheng-Chia; Chow, Te-Jin; Chang, Hsin-Yueh; Zhao, Xin-Qing; Chen, Po-Ting; Su, Hsiang-Yen; Chang, Jo-Shu

2016-03-01

Microalgal strains of Scenedesmus obliquus have the great potential for the production of biofuels, CO2 fixation, and bioremediation. However, metabolic engineering of S. obliquus to improve their useful phenotypes are still not fully developed. In this study, S. obliquus strain CPC2 was genetically engineered to promote the autotrophic growth and lipid productivity. The overexpression plasmid containing the type 2 diacylglycerol acyltransferse (DGAT) gene DGTT1 from Chlamydomonas reinhardtii was constructed and transformed into S. obliquus CPC2, and the positive transformants were obtained. The expression of DGTT1 gene was confirmed by reverse transcription PCR analysis. Enhanced lipid content of the transformant S. obliquus CPC2-G1 by nearly two-fold was observed. The biomass concentration of the recombinant strains was also 29% higher than that of the wild-type strain. Furthermore, the recombinant strain CPC2-G1 was successfully grown in 40 L tubular type photobioreactor and open pond system in an outdoor environment. The lipid content, biomass concentration, and biomass productivity obtained from 40 L tubular PBR were 127.8% 20.0%, and 232.6% higher than those obtained from the wild-type strain. The major aim of this work is to develop a tool to genetically engineer an isolated S. obliquus strain for the desired purpose. This is the first report that genetic engineering of S. obliquus has been successful employed to improve both the microalgal cell growth and the lipid production. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

New Generation Live Vaccines against Human Respiratory Syncytial Virus Designed by Reverse Genetics

PubMed Central

Collins, Peter L.; Murphy, Brian R.

2005-01-01

Development of a live pediatric vaccine against human respiratory syncytial virus (RSV) is complicated by the need to immunize young infants and the difficulty in balancing attenuation and immunogenicity. The ability to introduce desired mutations into infectious virus by reverse genetics provides a method for identifying and designing highly defined attenuating mutations. These can be introduced in combinations as desired to achieve gradations of attenuation. Attenuation is based on several strategies: multiple independent temperature-sensitive point mutations in the polymerase, a temperature-sensitive point mutation in a transcription signal, a set of non–temperature-sensitive mutations involving several genes, deletion of a viral RNA synthesis regulatory protein, and deletion of viral IFN α/β antagonists. The genetic stability of the live vaccine can be increased by judicious choice of mutations. The virus also can be engineered to increase the level of expression of the protective antigens. Protective antigens from antigenically distinct RSV strains can be added or swapped to increase the breadth of coverage. Alternatively, the major RSV protective antigens can be expressed from transcription units added to an attenuated parainfluenza vaccine virus, making a bivalent vaccine. This would obviate the difficulties inherent in the fragility and inefficient in vitro growth of RSV, simplifying vaccine design and use. PMID:16113487

Tamavidin 2-REV: an engineered tamavidin with reversible biotin-binding capability.

PubMed

Takakura, Yoshimitsu; Sofuku, Kozue; Tsunashima, Masako

2013-03-10

A biotin-binding protein with reversible biotin-binding capability is of great technical value in the affinity purification of biotinylated biomolecules. Although several proteins, chemically or genetically modified from avidin or streptavidin, with reversible biotin-binding have been reported, they have been problematic in one way or another. Tamavidin 2 is a fungal protein similar to avidin and streptavidin in biotin-binding. Here, a mutein, tamavidin 2-REV, was engineered from tamavidin 2 by replacing the serine at position 36 (S36) with alanine. S36 is thought to form a hydrogen bond with biotin in tamavidin 2/biotin complexes and two hydrogen bonds with V38 within the protein. Tamavidin 2-REV bound to biotin-agarose and was eluted with excess free biotin at a neutral pH. In addition, the model substrate biotinylated bovine serum albumin was efficiently purified from a crude extract from Escherichia coli by means of single-step affinity chromatography with tamavidin 2-REV-immobilized resin. Tamavidin 2-REV thus demonstrated reversible biotin-binding capability. The Kd value of tamavidin 2-REV to biotin was 2.8-4.4×10(-7)M.Tamavidin 2-REV retained other convenient characteristics of tamavidin 2, such as high-level expression in E. coli, resistance to proteases, and a neutral isoelectric point, demonstrating that tamavidin 2-REV is a powerful tool for the purification of biotinylated biomolecules. Copyright © 2013 Elsevier B.V. All rights reserved.

Reversible immortalisation enables genetic correction of human muscle progenitors and engineering of next-generation human artificial chromosomes for Duchenne muscular dystrophy.

PubMed

Benedetti, Sara; Uno, Narumi; Hoshiya, Hidetoshi; Ragazzi, Martina; Ferrari, Giulia; Kazuki, Yasuhiro; Moyle, Louise Anne; Tonlorenzi, Rossana; Lombardo, Angelo; Chaouch, Soraya; Mouly, Vincent; Moore, Marc; Popplewell, Linda; Kazuki, Kanako; Katoh, Motonobu; Naldini, Luigi; Dickson, George; Messina, Graziella; Oshimura, Mitsuo; Cossu, Giulio; Tedesco, Francesco Saverio

2018-02-01

Transferring large or multiple genes into primary human stem/progenitor cells is challenged by restrictions in vector capacity, and this hurdle limits the success of gene therapy. A paradigm is Duchenne muscular dystrophy (DMD), an incurable disorder caused by mutations in the largest human gene: dystrophin. The combination of large-capacity vectors, such as human artificial chromosomes (HACs), with stem/progenitor cells may overcome this limitation. We previously reported amelioration of the dystrophic phenotype in mice transplanted with murine muscle progenitors containing a HAC with the entire dystrophin locus (DYS-HAC). However, translation of this strategy to human muscle progenitors requires extension of their proliferative potential to withstand clonal cell expansion after HAC transfer. Here, we show that reversible cell immortalisation mediated by lentivirally delivered excisable hTERT and Bmi1 transgenes extended cell proliferation, enabling transfer of a novel DYS-HAC into DMD satellite cell-derived myoblasts and perivascular cell-derived mesoangioblasts. Genetically corrected cells maintained a stable karyotype, did not undergo tumorigenic transformation and retained their migration ability. Cells remained myogenic in vitro (spontaneously or upon MyoD induction) and engrafted murine skeletal muscle upon transplantation. Finally, we combined the aforementioned functions into a next-generation HAC capable of delivering reversible immortalisation, complete genetic correction, additional dystrophin expression, inducible differentiation and controllable cell death. This work establishes a novel platform for complex gene transfer into clinically relevant human muscle progenitors for DMD gene therapy. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Extending the boundaries of reverse engineering

NASA Astrophysics Data System (ADS)

Lawrie, Chris

2002-04-01

In today's market place the potential of Reverse Engineering as a time compression tool is commonly lost under its traditional definition. The term Reverse Engineering was coined way back at the advent of CMM machines and 3D CAD systems to describe the process of fitting surfaces to captured point data. Since these early beginnings, downstream hardware scanning and digitising systems have evolved in parallel with an upstream demand, greatly increasing the potential of a point cloud data set within engineering design and manufacturing processes. The paper will discuss the issues surrounding Reverse Engineering at the turn of the millennium.

Genetic networks and soft computing.

PubMed

Mitra, Sushmita; Das, Ranajit; Hayashi, Yoichi

2011-01-01

The analysis of gene regulatory networks provides enormous information on various fundamental cellular processes involving growth, development, hormone secretion, and cellular communication. Their extraction from available gene expression profiles is a challenging problem. Such reverse engineering of genetic networks offers insight into cellular activity toward prediction of adverse effects of new drugs or possible identification of new drug targets. Tasks such as classification, clustering, and feature selection enable efficient mining of knowledge about gene interactions in the form of networks. It is known that biological data is prone to different kinds of noise and ambiguity. Soft computing tools, such as fuzzy sets, evolutionary strategies, and neurocomputing, have been found to be helpful in providing low-cost, acceptable solutions in the presence of various types of uncertainties. In this paper, we survey the role of these soft methodologies and their hybridizations, for the purpose of generating genetic networks.

Engineering and Functional Analysis of Mitotic Kinases Through Chemical Genetics.

PubMed

Jones, Mathew J K; Jallepalli, Prasad V

2016-01-01

During mitosis, multiple protein kinases transform the cytoskeleton and chromosomes into new and highly dynamic structures that mediate the faithful transmission of genetic information and cell division. However, the large number and strong conservation of mammalian kinases in general pose significant obstacles to interrogating them with small molecules, due to the difficulty in identifying and validating those which are truly selective. To overcome this problem, a steric complementation strategy has been developed, in which a bulky "gatekeeper" residue within the active site of the kinase of interest is replaced with a smaller amino acid, such as glycine or alanine. The enlarged catalytic pocket can then be targeted in an allele-specific manner with bulky purine analogs. This strategy provides a general framework for dissecting kinase function with high selectivity, rapid kinetics, and reversibility. In this chapter we discuss the principles and techniques needed to implement this chemical genetic approach in mammalian cells.

An efficient and rapid influenza gene cloning strategy for reverse genetics system.

PubMed

Shao, Hongxia; Fan, Zhonglei; Wan, Zhimin; Tian, Xiaoyan; Chen, Hongjun; Perez, Daniel R; Qin, Aijian; Ye, Jianqiang

2015-09-15

Influenza reverse genetics plays vital roles in understanding influenza molecular characteristics and vaccine development. However, current influenza reverse genetics heavily depends on restriction enzyme and ligation for gene cloning. The traditional cloning process of influenza eight fragments for virus rescuing generally requires considerable work. To simplify and increase the pace of gene cloning for influenza reverse genetics system, we developed a rapid restriction enzyme-free ExnaseTM II-based in vitro recombination approach for influenza gene cloning. We used this strategy rapidly and successfully to clone influenza eight genes both from viruses PR8 and H9N2 for virus rescuing. Our data demonstrate that the strategy developed here can accelerate the process of influenza gene cloning into reverse genetics system, and shows high potential for applications in both influenza basic and applied research. Copyright © 2015 Elsevier B.V. All rights reserved.

Large-Scale Wind-Tunnel Tests of Exhaust Ingestion Due to Thrust Reversal on a Four-Engine Jet Transport during Ground Roll

NASA Technical Reports Server (NTRS)

Tolhurst, William H., Jr.; Hickey, David H.; Aoyagi, Kiyoshi

1961-01-01

Wind-tunnel tests have been conducted on a large-scale model of a swept-wing jet transport type airplane to study the factors affecting exhaust gas ingestion into the engine inlets when thrust reversal is used during ground roll. The model was equipped with four small jet engines mounted in nacelles beneath the wing. The tests included studies of both cascade and target type reversers. The data obtained included the free-stream velocity at the occurrence of exhaust gas ingestion in the outboard engine and the increment of drag due to thrust reversal for various modifications of thrust reverser configuration. Motion picture films of smoke flow studies were also obtained to supplement the data. The results show that the free-stream velocity at which ingestion occurred in the outboard engines could be reduced considerably, by simple modifications to the reversers, without reducing the effective drag due to reversed thrust.

Reverse thrust performance of the QCSEE variable pitch turbofan engine

NASA Technical Reports Server (NTRS)

Samanich, N. E.; Reemsnyder, D. C.; Blodmer, H. E.

1980-01-01

Results of steady state reverse and forward to reverse thrust transient performance tests are presented. The original quiet, clean, short haul, experimental engine four segment variable fan nozzle was retested in reverse and compared with a continuous, 30 deg half angle conical exlet. Data indicated that the significantly more stable, higher pressure recovery flow with the fixed 30 deg exlet resulted in lower engine vibrations, lower fan blade stress, and approximately a 20 percent improvement in reverse thrust. Objective reverse thrust of 35 percent of takeoff thrust was reached. Thrust response of less than 1.5 sec was achieved for the approach and the takeoff to reverse thrust transients.

Engineering Translational Activators with CRISPR-Cas System.

PubMed

Du, Pei; Miao, Chensi; Lou, Qiuli; Wang, Zefeng; Lou, Chunbo

2016-01-15

RNA parts often serve as critical components in genetic engineering. Here we report a design of translational activators which is composed of an RNA endoribonuclease (Csy4) and two exchangeable RNA modules. Csy4, a member of Cas endoribonuclease, cleaves at a specific recognition site; this cleavage releases a cis-repressive RNA module (crRNA) from the masked ribosome binding site (RBS), which subsequently allows the downstream translation initiation. Unlike small RNA as a translational activator, the endoribonuclease-based activator is able to efficiently unfold the perfect RBS-crRNA pairing. As an exchangeable module, the crRNA-RBS duplex was forwardly and reversely engineered to modulate the dynamic range of translational activity. We further showed that Csy4 and its recognition site, together as a module, can also be replaced by orthogonal endoribonuclease-recognition site homologues. These modularly structured, high-performance translational activators would endow the programming of gene expression in the translation level with higher feasibility.

14 CFR 23.933 - Reversing systems.

Code of Federal Regulations, 2013 CFR

2013-01-01

... analysis and testing completed by the engine and propeller manufacturers. [Doc. No. 26344, 58 FR 18971, Apr... only must be designed so that, during any reversal in flight, the engine will produce no more than... engine from producing more than idle thrust when the reversing system malfunctions; except that it may...

14 CFR 23.933 - Reversing systems.

Code of Federal Regulations, 2014 CFR

2014-01-01

... analysis and testing completed by the engine and propeller manufacturers. [Doc. No. 26344, 58 FR 18971, Apr... only must be designed so that, during any reversal in flight, the engine will produce no more than... engine from producing more than idle thrust when the reversing system malfunctions; except that it may...

14 CFR 23.933 - Reversing systems.

Code of Federal Regulations, 2012 CFR

2012-01-01

... analysis and testing completed by the engine and propeller manufacturers. [Doc. No. 26344, 58 FR 18971, Apr... only must be designed so that, during any reversal in flight, the engine will produce no more than... engine from producing more than idle thrust when the reversing system malfunctions; except that it may...

14 CFR 23.933 - Reversing systems.

Code of Federal Regulations, 2011 CFR

2011-01-01

... analysis and testing completed by the engine and propeller manufacturers. [Doc. No. 26344, 58 FR 18971, Apr... only must be designed so that, during any reversal in flight, the engine will produce no more than... engine from producing more than idle thrust when the reversing system malfunctions; except that it may...

14 CFR 23.933 - Reversing systems.

Code of Federal Regulations, 2010 CFR

2010-01-01

... analysis and testing completed by the engine and propeller manufacturers. [Doc. No. 26344, 58 FR 18971, Apr... only must be designed so that, during any reversal in flight, the engine will produce no more than... engine from producing more than idle thrust when the reversing system malfunctions; except that it may...

Bioterrorism and the Fermi Paradox

NASA Astrophysics Data System (ADS)

Cooper, Joshua

2013-04-01

We proffer a contemporary solution to the so-called Fermi Paradox, which is concerned with conflict between Copernicanism and the apparent paucity of evidence for intelligent alien civilizations. In particular, we argue that every community of organisms that reaches its space-faring age will (1) almost immediately use its rocket-building computers to reverse-engineer its genetic chemistry and (2) self-destruct when some individual uses said technology to design an omnicidal pathogen. We discuss some of the possible approaches to prevention with regard to Homo sapiens' vulnerability to bioterrorism, particularly on a short-term basis.

Construction and characterization of VL-VH tail-parallel genetically engineered antibodies against staphylococcal enterotoxins.

PubMed

He, Xianzhi; Zhang, Lei; Liu, Pengchong; Liu, Li; Deng, Hui; Huang, Jinhai

2015-03-01

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus have increasingly given rise to human health and food safety. Genetically engineered small molecular antibody is a useful tool in immuno-detection and treatment for clinical illness caused by SEs. In this study, we constructed the V(L)-V(H) tail-parallel genetically engineered antibody against SEs by using the repertoire of rearranged germ-line immunoglobulin variable region genes. Total RNA were extracted from six hybridoma cell lines that stably express anti-SEs antibodies. The variable region genes of light chain (V(L)) and heavy chain (V(H)) were cloned by reverse transcription PCR, and their classical murine antibody structure and functional V(D)J gene rearrangement were analyzed. To construct the eukaryotic V(H)-V(L) tail-parallel co-expression vectors based on the "5'-V(H)-ivs-IRES-V(L)-3'" mode, the ivs-IRES fragment and V(L) genes were spliced by two-step overlap extension PCR, and then, the recombined gene fragment and V(H) genes were inserted into the pcDNA3.1(+) expression vector sequentially. And then the constructed eukaryotic expression clones termed as p2C2HILO and p5C12HILO were transfected into baby hamster kidney 21 cell line, respectively. Two clonal cell lines stably expressing V(L)-V(H) tail-parallel antibodies against SEs were obtained, and the antibodies that expressed intracytoplasma were evaluated by enzyme-linked immunosorbent assay, immunofluorescence assay, and flow cytometry. SEs can stimulate the expression of some chemokines and chemokine receptors in porcine IPEC-J2 cells; mRNA transcription level of four chemokines and chemokine receptors can be blocked by the recombinant SE antibody prepared in this study. Our results showed that it is possible to get functional V(L)-V(H) tail-parallel genetically engineered antibodies in same vector using eukaryotic expression system.

Kyolic and Pycnogenol increase human growth hormone secretion in genetically-engineered keratinocytes.

PubMed

Buz'Zard, Amber R; Peng, Qiaoling; Lau, Benjamin H S

2002-02-01

The amount of human growth hormone (HGH) decreases significantly after the age of 30. This decrease has been implicated as one of the major causes in the signs of aging, such as thinning of the skin and bones, a decrease in lean muscle mass and an increase in adipose tissue. Supplementing the body's dwindling supply with recombinant human growth hormone (rHGH) has been shown to reverse the signs and symptoms of aging. However, drawbacks in rHGH replacement therapy include prohibitively high cost, the need for repeated injection and side effects such as carpel tunnel syndrome, gynecomastia and insulin resistance. The purpose of this study was to establish an in vitro model using genetically-engineered keratinocytes to screen natural compounds for the ability to stimulate HGH secretion. We now report that a combination of equal amounts of L-arginine and L-lysine, aged garlic extract (Kyolic), S-allyl cysteine and Pycnogenol significantly increased secretion of HGH in this in vitro model. The data indicate that this in vitro model may be used to screen for other secretagogues.

Reconstructing the regulatory network controlling commitment and sporulation in Physarum polycephalum based on hierarchical Petri Net modelling and simulation.

PubMed

Marwan, Wolfgang; Sujatha, Arumugam; Starostzik, Christine

2005-10-21

We reconstruct the regulatory network controlling commitment and sporulation of Physarum polycephalum from experimental results using a hierarchical Petri Net-based modelling and simulation framework. The stochastic Petri Net consistently describes the structure and simulates the dynamics of the molecular network as analysed by genetic, biochemical and physiological experiments within a single coherent model. The Petri Net then is extended to simulate time-resolved somatic complementation experiments performed by mixing the cytoplasms of mutants altered in the sporulation response, to systematically explore the network structure and to probe its dynamics. This reverse engineering approach presumably can be employed to explore other molecular or genetic signalling systems where the activity of genes or their products can be experimentally controlled in a time-resolved manner.

Harnessing the hygroscopic and biofluorescent behaviors of genetically tractable microbial cells to design biohybrid wearables.

PubMed

Wang, Wen; Yao, Lining; Cheng, Chin-Yi; Zhang, Teng; Atsumi, Hiroshi; Wang, Luda; Wang, Guanyun; Anilionyte, Oksana; Steiner, Helene; Ou, Jifei; Zhou, Kang; Wawrousek, Chris; Petrecca, Katherine; Belcher, Angela M; Karnik, Rohit; Zhao, Xuanhe; Wang, Daniel I C; Ishii, Hiroshi

2017-05-01

Cells' biomechanical responses to external stimuli have been intensively studied but rarely implemented into devices that interact with the human body. We demonstrate that the hygroscopic and biofluorescent behaviors of living cells can be engineered to design biohybrid wearables, which give multifunctional responsiveness to human sweat. By depositing genetically tractable microbes on a humidity-inert material to form a heterogeneous multilayered structure, we obtained biohybrid films that can reversibly change shape and biofluorescence intensity within a few seconds in response to environmental humidity gradients. Experimental characterization and mechanical modeling of the film were performed to guide the design of a wearable running suit and a fluorescent shoe prototype with bio-flaps that dynamically modulates ventilation in synergy with the body's need for cooling.

Reverse genetics of Newcastle disease virus

USDA-ARS?s Scientific Manuscript database

Reverse genetics allows the generation of recombinant viruses or vectors used in functional studies, vaccine development, and gene therapy. This technique allows genetic manipulation and cloning of viral genomes, mutation through site-directed mutagenesis, and gene insertion or deletion, among othe...

RNA Virus Reverse Genetics and Vaccine Design

PubMed Central

Stobart, Christopher C.; Moore, Martin L.

2014-01-01

RNA viruses are capable of rapid spread and severe or potentially lethal disease in both animals and humans. The development of reverse genetics systems for manipulation and study of RNA virus genomes has provided platforms for designing and optimizing viral mutants for vaccine development. Here, we review the impact of RNA virus reverse genetics systems on past and current efforts to design effective and safe viral therapeutics and vaccines. PMID:24967693

Cloned cDNA of A/swine/Iowa/15/1930 internal genes as a candidate backbone for reverse genetics vaccine against influenza A viruses

PubMed Central

Lekcharoensuk, Porntippa; Wiriyarat, Witthawat; Petcharat, Nuntawan; Lekcharoensuk, Chalermpol; Auewarakul, Prasert; Richt, Juergen A

2012-01-01

Reverse genetics viruses for influenza vaccine production usually utilize the internal genes of the egg-adapted A/Puerto Rico/8/34 (PR8) strain. This egg-adapted strain provides high production yield in embryonated eggs but does not necessarily give the best yield in mammalian cell culture. In order to generate a reverse genetics viral backbone that is well-adapted to high growth in mammalian cell culture, a swine influenza isolate (A/swine/Iowa/15/30 (H1N1) (rg1930) that was shown to give high yield in Madin-Darby Canine Kidney (MDCK) cells was used as the internal gene donor for reverse genetics plasmids. In this report, the internal genes from rg1930 were used for construction of reverse genetics viruses carrying a cleavage site-modified hemagglutinin (HA) gene and neuraminidase (NA) gene from a highly pathogenic H5N1 virus. The resulting virus (rg1930H5N1) was low pathogenic in vivo. Inactivated rg1930H5N1 vaccine completely protected chickens from morbidity and mortality after challenge with highly pathogenic H5N1. Protective immunity was obtained when chickens were immunized with an inactivated vaccine consisting of at least 29 HA units of the rg1930H5N1 virus. In comparison to the PR8-based reverse genetics viruses carrying the same HA and NA genes from an H5N1 virus, rg1930 based viruses yielded higher viral titers in MDCK and Vero cells. In addition, the reverse genetics derived H3N2 and H5N2 viruses with the rg1930 backbone replicated in MDCK cells better than the cognate viruses with the rgPR8 backbone. It is concluded that this newly established reverse genetics backbone system could serve as a candidate for a master donor strain for development of inactivated influenza vaccines in cell-based systems. PMID:22230579

14 CFR 25.933 - Reversing systems.

Code of Federal Regulations, 2014 CFR

2014-01-01

... reversal in flight the engine will produce no more than flight idle thrust. In addition, it must be shown... kind of failure is extremely remote. (3) Each system must have means to prevent the engine from... alone, under the most critical reversing condition expected in operation. (b) For propeller reversing...

14 CFR 25.933 - Reversing systems.

Code of Federal Regulations, 2012 CFR

2012-01-01

... reversal in flight the engine will produce no more than flight idle thrust. In addition, it must be shown... kind of failure is extremely remote. (3) Each system must have means to prevent the engine from... alone, under the most critical reversing condition expected in operation. (b) For propeller reversing...

14 CFR 25.933 - Reversing systems.

Code of Federal Regulations, 2011 CFR

2011-01-01

... reversal in flight the engine will produce no more than flight idle thrust. In addition, it must be shown... kind of failure is extremely remote. (3) Each system must have means to prevent the engine from... alone, under the most critical reversing condition expected in operation. (b) For propeller reversing...

Biological safety concepts of genetically modified live bacterial vaccines.

PubMed

Frey, Joachim

2007-07-26

Live vaccines possess the advantage of having access to induce cell-mediated and antibody-mediated immunity; thus in certain cases they are able to prevent infection, and not only disease. Furthermore, live vaccines, particularly bacterial live vaccines, are relatively cheap to produce and easy to apply. Hence they are suitable to immunize large communities or herds. The induction of both cell-mediated immunity as well as antibody-mediated immunity, which is particularly beneficial in inducing mucosal immune responses, is obtained by the vaccine-strain's ability to colonize and multiply in the host without causing disease. For this reason, live vaccines require attenuation of virulence of the bacterium to which immunity must be induced. Traditionally attenuation was achieved simply by multiple passages of the microorganism on growth medium, in animals, eggs or cell cultures or by chemical or physical mutagenesis, which resulted in random mutations that lead to attenuation. In contrast, novel molecular methods enable the development of genetically modified organisms (GMOs) targeted to specific genes that are particularly suited to induce attenuation or to reduce undesirable effects in the tissue in which the vaccine strains can multiply and survive. Since live vaccine strains (attenuated by natural selection or genetic engineering) are potentially released into the environment by the vaccinees, safety issues concerning the medical as well as environmental aspects must be considered. These involve (i) changes in cell, tissue and host tropism, (ii) virulence of the carrier through the incorporation of foreign genes, (iii) reversion to virulence by acquisition of complementation genes, (iv) exchange of genetic information with other vaccine or wild-type strains of the carrier organism and (v) spread of undesired genes such as antibiotic resistance genes. Before live vaccines are applied, the safety issues must be thoroughly evaluated case-by-case. Safety assessment includes knowledge of the precise function and genetic location of the genes to be mutated, their genetic stability, potential reversion mechanisms, possible recombination events with dormant genes, gene transfer to other organisms as well as gene acquisition from other organisms by phage transduction, transposition or plasmid transfer and cis- or trans-complementation. For this, GMOs that are constructed with modern techniques of genetic engineering display a significant advantage over random mutagenesis derived live organisms. The selection of suitable GMO candidate strains can be made under in vitro conditions using basic knowledge on molecular mechanisms of pathogenicity of the corresponding bacterial species rather than by in vivo testing of large numbers of random mutants. This leads to a more targeted safety testing on volunteers and to a reduction in the use of animal experimentation.

Defragged Binary I Ching Genetic Code Chromosomes Compared to Nirenberg’s and Transformed into Rotating 2D Circles and Squares and into a 3D 100% Symmetrical Tetrahedron Coupled to a Functional One to Discern Start From Non-Start Methionines through a Stella Octangula

PubMed Central

Castro-Chavez, Fernando

2012-01-01

Background Three binary representations of the genetic code according to the ancient I Ching of Fu-Xi will be presented, depending on their defragging capabilities by pairing based on three biochemical properties of the nucleic acids: H-bonds, Purine/Pyrimidine rings, and the Keto-enol/Amino-imino tautomerism, yielding the last pair a 32/32 single-strand self-annealed genetic code and I Ching tables. Methods Our working tool is the ancient binary I Ching's resulting genetic code chromosomes defragged by vertical and by horizontal pairing, reverse engineered into non-binaries of 2D rotating 4×4×4 circles and 8×8 squares and into one 3D 100% symmetrical 16×4 tetrahedron coupled to a functional tetrahedron with apical signaling and central hydrophobicity (codon formula: 4[1(1)+1(3)+1(4)+4(2)]; 5:5, 6:6 in man) forming a stella octangula, and compared to Nirenberg's 16×4 codon table (1965) pairing the first two nucleotides of the 64 codons in axis y. Results One horizontal and one vertical defragging had the start Met at the center. Two, both horizontal and vertical pairings produced two pairs of 2×8×4 genetic code chromosomes naturally arranged (M and I), rearranged by semi-introversion of central purines or pyrimidines (M' and I') and by clustering hydrophobic amino acids; their quasi-identity was disrupted by amino acids with odd codons (Met and Tyr pairing to Ile and TGA Stop); in all instances, the 64-grid 90° rotational ability was restored. Conclusions We defragged three I Ching representations of the genetic code while emphasizing Nirenberg's historical finding. The synthetic genetic code chromosomes obtained reflect the protective strategy of enzymes with a similar function, having both humans and mammals a biased G-C dominance of three H-bonds in the third nucleotide of their most used codons per amino acid, as seen in one chromosome of the i, M and M' genetic codes, while a two H-bond A-T dominance was found in their complementary chromosome, as seen in invertebrates and plants. The reverse engineering of chromosome I' into 2D rotating circles and squares was undertaken, yielding a 100% symmetrical 3D geometry which was coupled to a previously obtained genetic code tetrahedron in order to differentiate the start methionine from the methionine that is acting as a codifying non-start codon. PMID:23431415

Teacher-to-Teacher: An Annotated Bibliography on DNA and Genetic Engineering.

ERIC Educational Resources Information Center

Mertens, Thomas R., Comp.

1984-01-01

Presented is an annotated bibliography of 24 books on DNA and genetic engineering. Areas considered in these books include: basic biological concepts to help understand advances in genetic engineering; applications of genetic engineering; social, legal, and moral issues of genetic engineering; and historical aspects leading to advances in…

DIC-CAM recipe for reverse engineering

NASA Astrophysics Data System (ADS)

Romero-Carrillo, P.; Lopez-Alba, E.; Dorado, R.; Diaz-Garrido, F. A.

2012-04-01

Reverse engineering (RE) tries to model and manufacture an object from measurements one of a reference object. Modern optical measurement systems and computer aided engineering software have improved reverse engineering procedures. We detail the main RE steps from 3D digitalization by Digital Image Correlation to manufacturing. The previous description is complemented with an application example, which portrays the performance of RE. The differences between original and manufactured objects are less than 2 mm (close to the tool radius).

Flight Measurements of the Effect of a Controllable Thrust Reverser on the Flight Characteristics of a Single-Engine Jet Airplane

NASA Technical Reports Server (NTRS)

Anderson, Seth B.; Cooper, George E.; Faye, Alan E., Jr.

1959-01-01

A flight investigation was undertaken to determine the effect of a fully controllable thrust reverser on the flight characteristics of a single-engine jet airplane. Tests were made using a cylindrical target-type reverser actuated by a hydraulic cylinder through a "beep-type" cockpit control mounted at the base of the throttle. The thrust reverser was evaluated as an in-flight decelerating device, as a flight path control and airspeed control in landing approach, and as a braking device during the ground roll. Full deflection of the reverser for one reverser configuration resulted in a reverse thrust ratio of as much as 85 percent, which at maximum engine power corresponded to a reversed thrust of 5100 pounds. Use of the reverser in landing approach made possible a wide selection of approach angles, a large reduction in approach speed at steep approach angles, improved control of flight path angle, and more accuracy in hitting a given touchdown point. The use of the reverser as a speed brake at lower airspeeds was compromised by a longitudinal trim change. At the lower airspeeds and higher engine powers there was insufficient elevator power to overcome the nose-down trim change at full reverser deflection.

A novel reverse genetics system for production of infectious West Nile virus using homologous recombination in mammalian cells.

PubMed

Kobayashi, Shintaro; Yoshii, Kentaro; Hirano, Minato; Muto, Memi; Kariwa, Hiroaki

2017-02-01

Reverse genetics systems facilitate investigation of many aspects of the life cycle and pathogenesis of viruses. However, genetic instability in Escherichia coli has hampered development of a reverse genetics system for West Nile virus (WNV). In this study, we developed a novel reverse genetics system for WNV based on homologous recombination in mammalian cells. Introduction of the DNA fragment coding for the WNV structural protein together with a DNA-based replicon resulted in the release of infectious WNV. The growth rate and plaque size of the recombinant virus were almost identical to those of the parent WNV. Furthermore, chimeric WNV was produced by introducing the DNA fragment coding for the structural protein and replicon plasmid derived from various strains. Here, we report development of a novel system that will facilitate research into WNV infection. Copyright © 2016 Elsevier B.V. All rights reserved.

Engineering Encounters: Reverse Engineering

ERIC Educational Resources Information Center

McGowan, Veronica Cassone; Ventura, Marcia; Bell, Philip

2017-01-01

This column presents ideas and techniques to enhance your science teaching. This month's issue shares information on how students' everyday experiences can support science learning through engineering design. In this article, the authors outline a reverse-engineering model of instruction and describe one example of how it looked in our fifth-grade…

Strains and stressors: an analysis of touchscreen learning in genetically diverse mouse strains.

PubMed

Graybeal, Carolyn; Bachu, Munisa; Mozhui, Khyobeni; Saksida, Lisa M; Bussey, Timothy J; Sagalyn, Erica; Williams, Robert W; Holmes, Andrew

2014-01-01

Touchscreen-based systems are growing in popularity as a tractable, translational approach for studying learning and cognition in rodents. However, while mouse strains are well known to differ in learning across various settings, performance variation between strains in touchscreen learning has not been well described. The selection of appropriate genetic strains and backgrounds is critical to the design of touchscreen-based studies and provides a basis for elucidating genetic factors moderating behavior. Here we provide a quantitative foundation for visual discrimination and reversal learning using touchscreen assays across a total of 35 genotypes. We found significant differences in operant performance and learning, including faster reversal learning in DBA/2J compared to C57BL/6J mice. We then assessed DBA/2J and C57BL/6J for differential sensitivity to an environmental insult by testing for alterations in reversal learning following exposure to repeated swim stress. Stress facilitated reversal learning (selectively during the late stage of reversal) in C57BL/6J, but did not affect learning in DBA/2J. To dissect genetic factors underlying these differences, we phenotyped a family of 27 BXD strains generated by crossing C57BL/6J and DBA/2J. There was marked variation in discrimination, reversal and extinction learning across the BXD strains, suggesting this task may be useful for identifying underlying genetic differences. Moreover, different measures of touchscreen learning were only modestly correlated in the BXD strains, indicating that these processes are comparatively independent at both genetic and phenotypic levels. Finally, we examined the behavioral structure of learning via principal component analysis of the current data, plus an archival dataset, totaling 765 mice. This revealed 5 independent factors suggestive of "reversal learning," "motivation-related late reversal learning," "discrimination learning," "speed to respond," and "motivation during discrimination." Together, these findings provide a valuable reference to inform the choice of strains and genetic backgrounds in future studies using touchscreen-based tasks.

Manipulations in Maternal Environment Reverse Periodontitis in Genetically Predisposed Rats

PubMed Central

Sluyter, Frans; Breivik, Torbjørn; Cools, Alexander

2002-01-01

The predisposition to develop periodontitis is partly genetically determined in humans as well as in animals. Here we demonstrate, however, that early manipulations in the maternal environment of an animal (rat) model of periodontitis can fully reverse the genetic predisposition to develop periodontitis at adult age. PMID:12093700

The behavioural consequences of sex reversal in dragons

PubMed Central

Li, Hong; Holleley, Clare E.; Elphick, Melanie; Georges, Arthur

2016-01-01

Sex differences in morphology, physiology, and behaviour are caused by sex-linked genes, as well as by circulating sex-steroid levels. Thus, a shift from genotypic to environmental sex determination may create an organism that exhibits a mixture of male-like and female-like traits. We studied a lizard species (Central Bearded Dragon, Pogona vitticeps), in which the high-temperature incubation of eggs transforms genetically male individuals into functional females. Although they are reproductively female, sex-reversed dragons (individuals with ZZ genotype reversed to female phenotype) resemble genetic males rather than females in morphology (relative tail length), general behaviour (boldness and activity level), and thermoregulatory tactics. Indeed, sex-reversed ‘females’ are more male-like in some behavioural traits than are genetic males. This novel phenotype may impose strong selection on the frequency of sex reversal within natural populations, facilitating rapid shifts in sex-determining systems. A single period of high incubation temperatures (generating thermally induced sex reversal) can produce functionally female individuals with male-like (or novel) traits that enhance individual fitness, allowing the new temperature-dependent sex-determining system to rapidly replace the previous genetically based one.

Marburg Virus Reverse Genetics Systems

PubMed Central

Schmidt, Kristina Maria; Mühlberger, Elke

2016-01-01

The highly pathogenic Marburg virus (MARV) is a member of the Filoviridae family and belongs to the group of nonsegmented negative-strand RNA viruses. Reverse genetics systems established for MARV have been used to study various aspects of the viral replication cycle, analyze host responses, image viral infection, and screen for antivirals. This article provides an overview of the currently established MARV reverse genetic systems based on minigenomes, infectious virus-like particles and full-length clones, and the research that has been conducted using these systems. PMID:27338448

Marburg Virus Reverse Genetics Systems.

PubMed

Schmidt, Kristina Maria; Mühlberger, Elke

2016-06-22

The highly pathogenic Marburg virus (MARV) is a member of the Filoviridae family and belongs to the group of nonsegmented negative-strand RNA viruses. Reverse genetics systems established for MARV have been used to study various aspects of the viral replication cycle, analyze host responses, image viral infection, and screen for antivirals. This article provides an overview of the currently established MARV reverse genetic systems based on minigenomes, infectious virus-like particles and full-length clones, and the research that has been conducted using these systems.

Human telomerase reverse transcriptase is a promising target for cancer inhibition in squamous cell carcinomas.

PubMed

Park, Young-Jin; Kim, Eun-Kyoung; Moon, Sook; Hong, Doo-Pyo; Bae, Jung Yoon; Kim, Jin

2014-11-01

The present study aimed to investigate whether the down-regulation of human telomerase reverse transcriptase (hTERT) may induce an anti-invasive effect in oral squamous cell cancer cell lines. A genetically-engineered squamous carcinoma cell line overexpressing hTERT in immortalized oral keratinocytes transfected by human papilloma virus (HPV)-16 E6/E7 (IHOK) was used. In vivo tumorigenicity was examined using an orthotopic xenograft model of nude mice. For evaluating anti-invasive activity by knockdown of hTERT expression, transwell invasion assay and real-time polymerase chain reaction (PCR) for matrix metalloproteinases (MMP) were employed. The down-regulation of hTERT expression reduced the invasive activity and MMP expression. This result was re-confirmed in the HSC3 oral squamous carcinoma cell line. Targeting hTERT may lead to novel therapeutic approaches. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Cloned cDNA of A/swine/Iowa/15/1930 internal genes as a candidate backbone for reverse genetics vaccine against influenza A viruses.

PubMed

Lekcharoensuk, Porntippa; Wiriyarat, Witthawat; Petcharat, Nantawan; Lekcharoensuk, Chalermpol; Auewarakul, Prasert; Richt, Juergen A

2012-02-14

Reverse genetics viruses for influenza vaccine production usually utilize the internal genes of the egg-adapted A/Puerto Rico/8/34 (PR8) strain. This egg-adapted strain provides high production yield in embryonated eggs but does not necessarily give the best yield in mammalian cell culture. In order to generate a reverse genetics viral backbone that is well-adapted to high growth in mammalian cell culture, a swine influenza isolate A/swine/Iowa/15/30 (H1N1) (rg1930) that was shown to give high yield in Madin-Darby canine kidney (MDCK) cells was used as the internal gene donor for reverse genetics plasmids. In this report, the internal genes from rg1930 were used for construction of reverse genetics viruses carrying a cleavage site-modified hemagglutinin (HA) gene and neuraminidase (NA) gene from a highly pathogenic H5N1 virus. The resulting virus (rg1930H5N1) was low pathogenic in vivo. Inactivated rg1930H5N1 vaccine completely protected chickens from morbidity and mortality after challenge with highly pathogenic H5N1. Protective immunity was obtained when chickens were immunized with an inactivated vaccine consisting of at least 2(9) HA units of the rg1930H5N1 virus. In comparison to the PR8-based reverse genetics viruses carrying the same HA and NA genes from an H5N1 virus, rg1930 based viruses yielded higher viral titers in MDCK and Vero cells. In addition, the reverse genetics derived H3N2 and H5N2 viruses with the rg1930 backbone replicated in MDCK cells better than the cognate viruses with the rgPR8 backbone. It is concluded that this newly established reverse genetics backbone system could serve as a candidate for a master donor strain for development of inactivated influenza vaccines in cell-based systems. Copyright © 2011 Elsevier Ltd. All rights reserved.

Un-Building Blocks: A Model of Reverse Engineering and Applicable Heuristics

DTIC Science & Technology

2015-12-01

CONCLUSIONS The machine does not isolate man from the great problems of nature but plunges him more deeply into them. Antoine de Saint-Exupery— Wind ...DISTRIBUTION CODE 13. ABSTRACT (maximum 200 words) Reverse engineering is the problem -solving activity that ensues when one takes a...Douglas Moses, Vice Provost for Academic Affairs iv THIS PAGE INTENTIONALLY LEFT BLANK v ABSTRACT Reverse engineering is the problem -solving

Reverse engineering of aircraft wing data using a partial differential equation surface model

NASA Astrophysics Data System (ADS)

Huband, Jacalyn Mann

Reverse engineering is a multi-step process used in industry to determine a production representation of an existing physical object. This representation is in the form of mathematical equations that are compatible with computer-aided design and computer-aided manufacturing (CAD/CAM) equipment. The four basic steps to the reverse engineering process are data acquisition, data separation, surface or curve fitting, and CAD/CAM production. The surface fitting step determines the design representation of the object, and thus is critical to the success or failure of the reverse engineering process. Although surface fitting methods described in the literature are used to model a variety of surfaces, they are not suitable for reversing aircraft wings. In this dissertation, we develop and demonstrate a new strategy for reversing a mathematical representation of an aircraft wing. The basis of our strategy is to take an aircraft design model and determine if an inverse model can be derived. A candidate design model for this research is the partial differential equation (PDE) surface model, proposed by Bloor and Wilson and used in the Rapid Airplane Parameter Input Design (RAPID) tool at the NASA-LaRC Geolab. There are several basic mathematical problems involved in reversing the PDE surface model: (i) deriving a computational approximation of the surface function; (ii) determining a radial parametrization of the wing; (iii) choosing mathematical models or classes of functions for representation of the boundary functions; (iv) fitting the boundary data points by the chosen boundary functions; and (v) simultaneously solving for the axial parameterization and the derivative boundary functions. The study of the techniques to solve the above mathematical problems has culminated in a reverse PDE surface model and two reverse PDE surface algorithms. One reverse PDE surface algorithm recovers engineering design parameters for the RAPID tool from aircraft wing data and the other generates a PDE surface model with spline boundary functions from an arbitrary set of grid points. Our numerical tests show that the reverse PDE surface model and the reverse PDE surface algorithms can be used for the reverse engineering of aircraft wing data.

14 CFR 33.97 - Thrust reversers.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 14 Aeronautics and Space 1 2013-01-01 2013-01-01 false Thrust reversers. 33.97 Section 33.97 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT AIRWORTHINESS STANDARDS: AIRCRAFT ENGINES Block Tests; Turbine Aircraft Engines § 33.97 Thrust reversers. (a) If the...

14 CFR 33.97 - Thrust reversers.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Thrust reversers. 33.97 Section 33.97 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT AIRWORTHINESS STANDARDS: AIRCRAFT ENGINES Block Tests; Turbine Aircraft Engines § 33.97 Thrust reversers. (a) If the...

14 CFR 33.97 - Thrust reversers.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 14 Aeronautics and Space 1 2014-01-01 2014-01-01 false Thrust reversers. 33.97 Section 33.97 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION AIRCRAFT AIRWORTHINESS STANDARDS: AIRCRAFT ENGINES Block Tests; Turbine Aircraft Engines § 33.97 Thrust reversers. (a) If the...

14 CFR 33.97 - Thrust reversers.

Code of Federal Regulations, 2012 CFR

2012-01-01

... STANDARDS: AIRCRAFT ENGINES Block Tests; Turbine Aircraft Engines § 33.97 Thrust reversers. (a) If the... this subpart must be run with the reverser installed. In complying with this section, the power control... regimes of control operations are incorporated necessitating scheduling of the power-control lever motion...

14 CFR 33.97 - Thrust reversers.

Code of Federal Regulations, 2010 CFR

2010-01-01

... STANDARDS: AIRCRAFT ENGINES Block Tests; Turbine Aircraft Engines § 33.97 Thrust reversers. (a) If the... this subpart must be run with the reverser installed. In complying with this section, the power control... regimes of control operations are incorporated necessitating scheduling of the power-control lever motion...

Genetic dissection of behavioral flexibility: reversal learning in mice.

PubMed

Laughlin, Rick E; Grant, Tara L; Williams, Robert W; Jentsch, J David

2011-06-01

Behavioral inflexibility is a feature of schizophrenia, attention-deficit/hyperactivity disorder, and behavior addictions that likely results from heritable deficits in the inhibitory control over behavior. Here, we investigate the genetic basis of individual differences in flexibility, measured using an operant reversal learning task. We quantified discrimination acquisition and subsequent reversal learning in a cohort of 51 BXD strains of mice (2-5 mice/strain, n = 176) for which we have matched data on sequence, gene expression in key central nervous system regions, and neuroreceptor levels. Strain variation in trials to criterion on acquisition and reversal was high, with moderate heritability (∼.3). Acquisition and reversal learning phenotypes did not covary at the strain level, suggesting that these traits are effectively under independent genetic control. Reversal performance did covary with dopamine D2 receptor levels in the ventral midbrain, consistent with a similar observed relationship between impulsivity and D2 receptors in humans. Reversal, but not acquisition, is linked to a locus on mouse chromosome 10 with a peak likelihood ratio statistic at 86.2 megabase (p < .05 genome-wide). Variance in messenger RNA levels of select transcripts expressed in neocortex, hippocampus, and striatum correlated with the reversal learning phenotype, including Syn3, Nt5dc3, and Hcfc2. This work demonstrates the clear trait independence between, and genetic control of, discrimination acquisition and reversal and illustrates how globally coherent data sets for a single panel of highly related strains can be interrogated and integrated to uncover genetic sources and molecular and neuropharmacological candidates of complex behavioral traits relevant to human psychopathology. Copyright © 2011 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

M13 bacteriophage-activated superparamagnetic beads for affinity separation.

PubMed

Muzard, Julien; Platt, Mark; Lee, Gil U

2012-08-06

The growth of the biopharmaceutical industry has created a demand for new technologies for the purification of genetically engineered proteins.The efficiency of large-scale, high-gradient magnetic fishing could be improved if magnetic particles offering higher binding capacity and magnetization were available. This article describes several strategies for synthesizing microbeads that are composed of a M13 bacteriophage layer assembled on a superparamagnetic core. Chemical cross-linking of the pVIII proteins to a carboxyl-functionalized bead produces highly responsive superparamagnetic particles (SPM) with a side-on oriented, adherent virus monolayer. Also, the genetic manipulation of the pIII proteins with a His(6) peptide sequence allows reversible assembly of the bacteriophage on a nitrilotriacetic-acid-functionalized core in an end-on configuration. These phage-magnetic particles are successfully used to separate antibodies from high-protein concentration solutions in a single step with a >90% purity. The dense magnetic core of these particles makes them five times more responsive to magnetic fields than commercial materials composed of polymer-(iron oxide) composites and a monolayer of phage could produce a 1000 fold higher antibody binding capacity. These new bionanomaterials appear to be well-suited to large-scale high-gradient magnetic fishing separation and promise to be cost effective as a result of the self-assembling and self-replicating properties of genetically engineered M13 bacteriophage. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Current progress of targetron technology: development, improvement and application in metabolic engineering.

PubMed

Liu, Ya-Jun; Zhang, Jie; Cui, Gu-Zhen; Cui, Qiu

2015-06-01

Targetrons are mobile group II introns that can recognize their DNA target sites by base-pairing RNA-DNA interactions with the aid of site-specific binding reverse transcriptases. Targetron technology stands out from recently developed gene targeting methods because of the flexibility, feasibility, and efficiency, and is particularly suitable for the genetic engineering of difficult microorganisms, including cellulolytic bacteria that are considered promising candidates for biomass conversion via consolidated bioprocessing. Along with the development of the thermotargetron method for thermophiles, targetron technology becomes increasingly important for the metabolic engineering of industrial microorganisms aiming at biofuel/chemical production. To summarize the current progress of targetron technology and provide new insights on the use of the technology, this paper reviews the retrohoming mechanisms of both mesophilic and thermophilic targetron methods based on various group II introns, investigates the improvement of targetron tools for high target efficiency and specificity, and discusses the current applications in the metabolic engineering for bacterial producers. Although there are still intellectual property and technical restrictions in targetron applications, we propose that targetron technology will contribute to both biochemistry research and the metabolic engineering for industrial productions. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reverse Core Engine with Thrust Reverser

NASA Technical Reports Server (NTRS)

Chandler, Jesse M. (Inventor); Suciu, Gabriel L. (Inventor)

2017-01-01

An engine system has a gas generator, a bi-fi wall surrounding at least a portion of the gas generator, a casing surrounding a fan, and the casing having first and second thrust reverser doors which in a deployed position abut each other and the bi-fi wall.

Antinociceptive Effect of Intrathecal Injection of Genetically Engineered Human Bone Marrow Stem Cells Expressing the Human Proenkephalin Gene in a Rat Model of Bone Cancer Pain.

PubMed

Sun, Yi; Tian, Yuke; Li, Haifeng; Zhang, Dengwen; Sun, Qiang

2017-01-01

Background . This study aimed to investigate the use of human bone marrow mesenchymal stem cells (hBMSCs) genetically engineered with the human proenkephalin (hPPE) gene to treat bone cancer pain (BCP) in a rat model. Methods . Primary cultured hBMSCs were passaged and modified with hPPE, and the cell suspensions (6 × 10 6 ) were then intrathecally injected into a rat model of BCP. Paw mechanical withdrawal threshold (PMWT) was measured before and after BCP. The effects of hPPE gene transfer on hBMSC bioactivity were analyzed in vitro and in vivo. Results . No changes were observed in the surface phenotypes and differentiation of hBMSCs after gene transfer. The hPPE-hBMSC group showed improved PMWT values on the ipsilateral side of rats with BCP from day 12 postoperatively, and the analgesic effect was reversed by naloxone. The levels of proinflammatory cytokines such as IL-1 β and IL-6 were ameliorated, and leucine-enkephalin (L-EK) secretion was augmented, in the hPPE-engineered hBMSC group. Conclusion . The intrathecal administration of BMSCs modified with the hPPE gene can effectively relieve pain caused by bone cancer in rats and might be a potentially therapeutic tool for cancer-related pain in humans.

Analysis of Ten Reverse Engineering Tools

NASA Astrophysics Data System (ADS)

Koskinen, Jussi; Lehmonen, Tero

Reverse engineering tools can be used in satisfying the information needs of software maintainers. Especially in case of maintaining large-scale legacy systems tool support is essential. Reverse engineering tools provide various kinds of capabilities to provide the needed information to the tool user. In this paper we analyze the provided capabilities in terms of four aspects: provided data structures, visualization mechanisms, information request specification mechanisms, and navigation features. We provide a compact analysis of ten representative reverse engineering tools for supporting C, C++ or Java: Eclipse Java Development Tools, Wind River Workbench (for C and C++), Understand (for C++), Imagix 4D, Creole, Javadoc, Javasrc, Source Navigator, Doxygen, and HyperSoft. The results of the study supplement the earlier findings in this important area.

Reverse Engineering Validation using a Benchmark Synthetic Gene Circuit in Human Cells

PubMed Central

Kang, Taek; White, Jacob T.; Xie, Zhen; Benenson, Yaakov; Sontag, Eduardo; Bleris, Leonidas

2013-01-01

Multi-component biological networks are often understood incompletely, in large part due to the lack of reliable and robust methodologies for network reverse engineering and characterization. As a consequence, developing automated and rigorously validated methodologies for unraveling the complexity of biomolecular networks in human cells remains a central challenge to life scientists and engineers. Today, when it comes to experimental and analytical requirements, there exists a great deal of diversity in reverse engineering methods, which renders the independent validation and comparison of their predictive capabilities difficult. In this work we introduce an experimental platform customized for the development and verification of reverse engineering and pathway characterization algorithms in mammalian cells. Specifically, we stably integrate a synthetic gene network in human kidney cells and use it as a benchmark for validating reverse engineering methodologies. The network, which is orthogonal to endogenous cellular signaling, contains a small set of regulatory interactions that can be used to quantify the reconstruction performance. By performing successive perturbations to each modular component of the network and comparing protein and RNA measurements, we study the conditions under which we can reliably reconstruct the causal relationships of the integrated synthetic network. PMID:23654266

Reverse engineering validation using a benchmark synthetic gene circuit in human cells.

PubMed

Kang, Taek; White, Jacob T; Xie, Zhen; Benenson, Yaakov; Sontag, Eduardo; Bleris, Leonidas

2013-05-17

Multicomponent biological networks are often understood incompletely, in large part due to the lack of reliable and robust methodologies for network reverse engineering and characterization. As a consequence, developing automated and rigorously validated methodologies for unraveling the complexity of biomolecular networks in human cells remains a central challenge to life scientists and engineers. Today, when it comes to experimental and analytical requirements, there exists a great deal of diversity in reverse engineering methods, which renders the independent validation and comparison of their predictive capabilities difficult. In this work we introduce an experimental platform customized for the development and verification of reverse engineering and pathway characterization algorithms in mammalian cells. Specifically, we stably integrate a synthetic gene network in human kidney cells and use it as a benchmark for validating reverse engineering methodologies. The network, which is orthogonal to endogenous cellular signaling, contains a small set of regulatory interactions that can be used to quantify the reconstruction performance. By performing successive perturbations to each modular component of the network and comparing protein and RNA measurements, we study the conditions under which we can reliably reconstruct the causal relationships of the integrated synthetic network.

78 FR 13286 - Sharing Certain Business Information Regarding the Introduction of Genetically Engineered...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-02-27

... Information Regarding the Introduction of Genetically Engineered Organisms With State and Tribal Government... proposing to amend our regulations regarding genetically engineered organisms regulated by the United States...). The regulations refer to such genetically engineered (GE) organisms and products as ``regulated...

Contact mechanics of reverse engineered distal humeral hemiarthroplasty implants.

PubMed

Willing, Ryan; King, Graham J W; Johnson, James A

2015-11-26

Erosion of articular cartilage is a concern following distal humeral hemiarthroplasty, because native cartilage surfaces are placed in contact with stiff metallic implant components, which causes decreases in contact area and increases in contact stresses. Recently, reverse engineered implants have been proposed which are intended to promote more natural contact mechanics by reproducing the native bone or cartilage shape. In this study, finite element modeling is used in order to calculate changes in cartilage contact areas and stresses following distal humeral hemiarthroplasty with commercially available and reverse engineered implant designs. At the ulna, decreases in contact area were -34±3% (p=0.002), -27±1% (p<0.001) and -14±2% (p=0.008) using commercially available, bone reverse engineered and cartilage reverse engineered designs, respectively. Peak contact stresses increased by 461±57% (p=0.008), 387±127% (p=0.229) and 165±16% (p=0.003). At the radius, decreases in contact area were -21±3% (p=0.013), -13±2% (p<0.006) and -6±1% (p=0.020), and peak contact stresses increased by 75±52% (p>0.999), 241±32% (p=0.010) and 61±10% (p=0.021). Between the three different implant designs, the cartilage reverse engineered design yielded the largest contact areas and lowest contact stresses, but was still unable to reproduce the contact mechanics of the native joint. These findings align with a growing body of evidence indicating that although reverse engineered hemiarthroplasty implants can provide small improvements in contact mechanics when compared with commercially available designs, further optimization of shape and material properties is required in order reproduce native joint contact mechanics. Copyright © 2015 Elsevier Ltd. All rights reserved.

Reverse Engineering Field Isolates of Myxoma Virus Demonstrates that Some Gene Disruptions or Losses of Function Do Not Explain Virulence Changes Observed in the Field

PubMed Central

Liu, June; Cattadori, Isabella M.; Sim, Derek G.; Eden, John-Sebastian; Read, Andrew F.

2017-01-01

ABSTRACT The coevolution of myxoma virus (MYXV) and wild European rabbits in Australia and Europe is a paradigm for the evolution of a pathogen in a new host species. Genomic analyses have identified the mutations that have characterized this evolutionary process, but defining causal mutations in the pathways from virulence to attenuation and back to virulence has not been possible. Using reverse genetics, we examined the roles of six selected mutations found in Australian field isolates of MYXV that fall in known or potential virulence genes. Several of these mutations occurred in genes previously identified as virulence genes in whole-gene knockout studies. Strikingly, no single or double mutation among the mutations tested had an appreciable impact on virulence. This suggests either that virulence evolution was defined by amino acid changes other than those analyzed here or that combinations of multiple mutations, possibly involving epistatic interactions or noncoding sequences, have been critical in the ongoing evolution of MYXV virulence. In sum, our results show that single-gene knockout studies of a progenitor virus can have little power to predict the impact of individual mutations seen in the field. The genetic determinants responsible for this canonical case of virulence evolution remain to be determined. IMPORTANCE The species jump of myxoma virus (MYXV) from the South American tapeti to the European rabbit populations of Australia and Europe is a canonical example of host-pathogen coevolution. Detailed molecular studies have identified multiple genes in MYXV that are critical for virulence, and genome sequencing has revealed the evolutionary history of MYXV in Australia and Europe. However, it has not been possible to categorically identify the key mutations responsible for the attenuation of or reversion to virulence during this evolutionary process. Here we use reverse genetics to examine the role of mutations in viruses isolated early and late in the Australian radiation of MYXV. Surprisingly, none of the candidate mutations that we identified as likely having roles in attenuation proved to be important for virulence. This indicates that considerable caution is warranted when interpreting the possible role of individual mutations during virulence evolution. PMID:28768866

The 727 airplane target thrust reverser static performance model test for refanned JT8D engines

NASA Technical Reports Server (NTRS)

Chow, C. T. P.; Atkey, E. N.

1974-01-01

The results of a scale model static performance test of target thrust reverser configurations for the Pratt and Whitney Aircraft JT8D-100 series engine are presented. The objective of the test was to select a series of suitable candidate reverser configurations for the subsequent airplane model wind tunnel ingestion and flight controls tests. Test results indicate that adequate reverse thrust performance with compatible engine airflow match is achievable for the selected configurations. Tapering of the lips results in loss of performance and only minimal flow directivity. Door pressure surveys were conducted on a selected number of lip and fence configurations to obtain data to support the design of the thrust reverser system.

14 CFR 25.1305 - Powerplant instruments.

Code of Federal Regulations, 2013 CFR

2013-01-01

... reverse pitch, for each reversing propeller. (c) For turbine engine-powered airplanes. In addition to the... required: (1) A gas temperature indicator for each engine. (2) A fuel flowmeter indicator for each engine... operated continuously but that is neither designed for continuous operation nor designed to prevent hazard...

14 CFR 25.1305 - Powerplant instruments.

Code of Federal Regulations, 2014 CFR

2014-01-01

... reverse pitch, for each reversing propeller. (c) For turbine engine-powered airplanes. In addition to the... required: (1) A gas temperature indicator for each engine. (2) A fuel flowmeter indicator for each engine... operated continuously but that is neither designed for continuous operation nor designed to prevent hazard...

14 CFR 25.1305 - Powerplant instruments.

Code of Federal Regulations, 2012 CFR

2012-01-01

... reverse pitch, for each reversing propeller. (c) For turbine engine-powered airplanes. In addition to the... required: (1) A gas temperature indicator for each engine. (2) A fuel flowmeter indicator for each engine... operated continuously but that is neither designed for continuous operation nor designed to prevent hazard...

14 CFR 25.1305 - Powerplant instruments.

Code of Federal Regulations, 2011 CFR

2011-01-01

... reverse pitch, for each reversing propeller. (c) For turbine engine-powered airplanes. In addition to the... required: (1) A gas temperature indicator for each engine. (2) A fuel flowmeter indicator for each engine... operated continuously but that is neither designed for continuous operation nor designed to prevent hazard...

14 CFR 25.1305 - Powerplant instruments.

Code of Federal Regulations, 2010 CFR

2010-01-01

... reverse pitch, for each reversing propeller. (c) For turbine engine-powered airplanes. In addition to the... required: (1) A gas temperature indicator for each engine. (2) A fuel flowmeter indicator for each engine... operated continuously but that is neither designed for continuous operation nor designed to prevent hazard...

Strains and Stressors: An Analysis of Touchscreen Learning in Genetically Diverse Mouse Strains

PubMed Central

Graybeal, Carolyn; Bachu, Munisa; Mozhui, Khyobeni; Saksida, Lisa M.; Bussey, Timothy J.; Sagalyn, Erica; Williams, Robert W.; Holmes, Andrew

2014-01-01

Touchscreen-based systems are growing in popularity as a tractable, translational approach for studying learning and cognition in rodents. However, while mouse strains are well known to differ in learning across various settings, performance variation between strains in touchscreen learning has not been well described. The selection of appropriate genetic strains and backgrounds is critical to the design of touchscreen-based studies and provides a basis for elucidating genetic factors moderating behavior. Here we provide a quantitative foundation for visual discrimination and reversal learning using touchscreen assays across a total of 35 genotypes. We found significant differences in operant performance and learning, including faster reversal learning in DBA/2J compared to C57BL/6J mice. We then assessed DBA/2J and C57BL/6J for differential sensitivity to an environmental insult by testing for alterations in reversal learning following exposure to repeated swim stress. Stress facilitated reversal learning (selectively during the late stage of reversal) in C57BL/6J, but did not affect learning in DBA/2J. To dissect genetic factors underlying these differences, we phenotyped a family of 27 BXD strains generated by crossing C57BL/6J and DBA/2J. There was marked variation in discrimination, reversal and extinction learning across the BXD strains, suggesting this task may be useful for identifying underlying genetic differences. Moreover, different measures of touchscreen learning were only modestly correlated in the BXD strains, indicating that these processes are comparatively independent at both genetic and phenotypic levels. Finally, we examined the behavioral structure of learning via principal component analysis of the current data, plus an archival dataset, totaling 765 mice. This revealed 5 independent factors suggestive of “reversal learning,” “motivation-related late reversal learning,” “discrimination learning,” “speed to respond,” and “motivation during discrimination.” Together, these findings provide a valuable reference to inform the choice of strains and genetic backgrounds in future studies using touchscreen-based tasks. PMID:24586288

Reverse engineering and identification in systems biology: strategies, perspectives and challenges.

PubMed

Villaverde, Alejandro F; Banga, Julio R

2014-02-06

The interplay of mathematical modelling with experiments is one of the central elements in systems biology. The aim of reverse engineering is to infer, analyse and understand, through this interplay, the functional and regulatory mechanisms of biological systems. Reverse engineering is not exclusive of systems biology and has been studied in different areas, such as inverse problem theory, machine learning, nonlinear physics, (bio)chemical kinetics, control theory and optimization, among others. However, it seems that many of these areas have been relatively closed to outsiders. In this contribution, we aim to compare and highlight the different perspectives and contributions from these fields, with emphasis on two key questions: (i) why are reverse engineering problems so hard to solve, and (ii) what methods are available for the particular problems arising from systems biology?

76 FR 8707 - Syngenta Seeds, Inc.; Determination of Nonregulated Status for Corn Genetically Engineered To...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-02-15

... for Corn Genetically Engineered To Produce an Enzyme That Facilitates Ethanol Production AGENCY... event 3272, which has been genetically engineered to produce a microbial enzyme that facilitates ethanol... transformation event 3272, which has been genetically engineered to produce a microbial enzyme that facilitates...

Liquid chromatographic determination of sennosides in Cassia angustifolia leaves.

PubMed

Srivastava, Alpuna; Pandey, Richa; Verma, Ram K; Gupta, Madan M

2006-01-01

A simple liquid chromatographic method was developed for the determination of sennosides B and A in leaves of Cassia angustifolia. These compounds were extracted from leaves with a mixture of methanol-water (70 + 30, v/v) after defatting with hexane. Analyte separation and quantitation were achieved by gradient reversed-phase liquid chromatography and UV absorbance at 270 nm using a photodiode array detector. The method involves the use of an RP-18 Lichrocart reversed-phase column (5 microm, 125 x 4.0 mm id) and a binary gradient mobile-phase profile. The various other aspects of analysis, namely, peak purity, similarity, recovery, repeatability, and robustness, were validated. Average recoveries of 98.5 and 98.6%, with a coefficient of variation of 0.8 and 0.3%, were obtained by spiking sample solution with 3 different concentration solutions of standards (60, 100, and 200 microg/mL). Detection limits were 10 microg/mL for sennoside B and 35 microg/mL for sennoside A, present in the sample solution. The quantitation limits were 28 and 100 microg/mL. The analytical method was applied to a large number of senna leaf samples. The new method provides a reliable tool for rapid screening of C. angustifolia samples in large numbers, which is needed in breeding/genetic engineering and genetic mapping experiments.

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

How to turn a genetic circuit into a synthetic tunable oscillator, or a bistable switch.

PubMed

Marucci, Lucia; Barton, David A W; Cantone, Irene; Ricci, Maria Aurelia; Cosma, Maria Pia; Santini, Stefania; di Bernardo, Diego; di Bernardo, Mario

2009-12-07

Systems and Synthetic Biology use computational models of biological pathways in order to study in silico the behaviour of biological pathways. Mathematical models allow to verify biological hypotheses and to predict new possible dynamical behaviours. Here we use the tools of non-linear analysis to understand how to change the dynamics of the genes composing a novel synthetic network recently constructed in the yeast Saccharomyces cerevisiae for In-vivo Reverse-engineering and Modelling Assessment (IRMA). Guided by previous theoretical results that make the dynamics of a biological network depend on its topological properties, through the use of simulation and continuation techniques, we found that the network can be easily turned into a robust and tunable synthetic oscillator or a bistable switch. Our results provide guidelines to properly re-engineering in vivo the network in order to tune its dynamics.

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.

Hamilton Standard Q-fan demonstrator dynamic pitch change test program, volume 1

NASA Technical Reports Server (NTRS)

Demers, W. J.; Nelson, D. J.; Wainauski, H. S.

1975-01-01

Tests of a full scale variable pitch fan engine to obtain data on the structural characteristics, response times, and fan/core engine compatibility during transient changes in blade angle, fan rpm, and engine power is reported. Steady state reverse thrust tests with a take off nozzle configuration were also conducted. The 1.4 meter diameter, 13 bladed controllable pitch fan was driven by a T55 L 11A engine with power and blade angle coordinated by a digital computer. The tests demonstrated an ability to change from full forward thrust to reverse thrust in less than one (1) second. Reverse thrust was effected through feather and through flat pitch; structural characteristics and engine/fan compatibility were within satisfactory limits.

Evaluation of a genetically modified foot-and-mouth disease virus vaccine candidate generated by reverse genetics

PubMed Central

2012-01-01

Background Foot-and-mouth disease (FMD) is the most economically important and highly contagious disease of cloven-hoofed animals worldwide. Control of the disease has been mainly based on large-scale vaccinations with whole-virus inactivated vaccines. In recent years, a series of outbreaks of type O FMD occurred in China (including Chinese Taipei, Chinese Hong Kong) posed a tremendous threat to Chinese animal husbandry. Its causative agent, type O FMDV, has evolved into three topotypes (East–South Asia (ME-SA), Southeast Asia (SEA), Cathay (CHY)) in these regions, which represents an important obstacle to disease control. The available FMD vaccine in China shows generally good protection against ME-SA and SEA topotype viruses infection, but affords insufficient protection against some variants of the CHY topotype. Therefore, the choice of a new vaccine strain is of fundamental importance. Results The present study describes the generation of a full-length infectious cDNA clone of FMDV vaccine strain and a genetically modified virus with some amino acid substitutions in antigenic sites 1, 3, and 4, based on the established infectious clone. The recombinant viruses had similar growth properties to the wild O/HN/CHA/93 virus. All swine immunized with inactivated vaccine prepared from the O/HN/CHA/93 were fully protected from challenge with the viruses of ME-SA and SEA topotypes and partially protected against challenge with the virus of CHY topotype at 28 days post-immunization. In contrast, the swine inoculated with the genetically modified vaccine were completely protected from the infection of viruses of the three topotypes. Conclusions Some amino acid substitutions in the FMDV vaccine strain genome did not have an effect on the ability of viral replication in vitro. The vaccine prepared from genetically modified FMDV by reverse genetics significantly improved the protective efficacy to the variant of the CHY topotype, compared with the wild O/HN/CHA/93 virus. Thus, the full-length cDNA clone of FMDV can be a useful tool to develop genetically engineered FMDV vaccine candidates to help control porcinophilic FMD epidemics in China. PMID:22591597

Slowly switching between environments facilitates reverse evolution in small populations.

PubMed

Tan, Longzhi; Gore, Jeff

2012-10-01

Natural populations must constantly adapt to ever-changing environmental conditions. A particularly interesting question is whether such adaptations can be reversed by returning the population to an ancestral environment. Such evolutionary reversals have been observed in both natural and laboratory populations. However, the factors that determine the reversibility of evolution are still under debate. The time scales of environmental change vary over a wide range, but little is known about how the rate of environmental change influences the reversibility of evolution. Here, we demonstrate computationally that slowly switching between environments increases the reversibility of evolution for small populations that are subject to only modest clonal interference. For small populations, slow switching reduces the mean number of mutations acquired in a new environment and also increases the probability of reverse evolution at each of these "genetic distances." As the population size increases, slow switching no longer reduces the genetic distance, thus decreasing the evolutionary reversibility. We confirm this effect using both a phenomenological model of clonal interference and also a Wright-Fisher stochastic simulation that incorporates genetic diversity. Our results suggest that the rate of environmental change is a key determinant of the reversibility of evolution, and provides testable hypotheses for experimental evolution. © 2012 The Author(s). Evolution© 2012 The Society for the Study of Evolution.

Reverse engineering and identification in systems biology: strategies, perspectives and challenges

PubMed Central

Villaverde, Alejandro F.; Banga, Julio R.

2014-01-01

The interplay of mathematical modelling with experiments is one of the central elements in systems biology. The aim of reverse engineering is to infer, analyse and understand, through this interplay, the functional and regulatory mechanisms of biological systems. Reverse engineering is not exclusive of systems biology and has been studied in different areas, such as inverse problem theory, machine learning, nonlinear physics, (bio)chemical kinetics, control theory and optimization, among others. However, it seems that many of these areas have been relatively closed to outsiders. In this contribution, we aim to compare and highlight the different perspectives and contributions from these fields, with emphasis on two key questions: (i) why are reverse engineering problems so hard to solve, and (ii) what methods are available for the particular problems arising from systems biology? PMID:24307566

76 FR 63278 - Bayer CropScience LP; Determination of Nonregulated Status for Cotton Genetically Engineered for...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-10-12

... for Cotton Genetically Engineered for Insect Resistance and Herbicide Tolerance AGENCY: Animal and... determination that a genetically engineered cotton developed by Bayer CropScience LP, designated as TwinLink TM cotton (events T304-40 and GHB119), which has been genetically engineered to be tolerant to the herbicide...

Seeking perfection: a Kantian look at human genetic engineering.

PubMed

Gunderson, Martin

2007-01-01

It is tempting to argue that Kantian moral philosophy justifies prohibiting both human germ-line genetic engineering and non-therapeutic genetic engineering because they fail to respect human dignity. There are, however, good reasons for resisting this temptation. In fact, Kant's moral philosophy provides reasons that support genetic engineering-even germ-line and non-therapeutic. This is true of Kant's imperfect duties to seek one's own perfection and the happiness of others. It is also true of the categorical imperative. Kant's moral philosophy does, however, provide limits to justifiable genetic engineering.

Static Performance of a Wing-Mounted Thrust Reverser Concept

NASA Technical Reports Server (NTRS)

Asbury, Scott C.; Yetter, Jeffrey A.

1998-01-01

An experimental investigation was conducted in the Jet-Exit Test Facility at NASA Langley Research Center to study the static aerodynamic performance of a wing-mounted thrust reverser concept applicable to subsonic transport aircraft. This innovative engine powered thrust reverser system is designed to utilize wing-mounted flow deflectors to produce aircraft deceleration forces. Testing was conducted using a 7.9%-scale exhaust system model with a fan-to-core bypass ratio of approximately 9.0, a supercritical left-hand wing section attached via a pylon, and wing-mounted flow deflectors attached to the wing section. Geometric variations of key design parameters investigated for the wing-mounted thrust reverser concept included flow deflector angle and chord length, deflector edge fences, and the yaw mount angle of the deflector system (normal to the engine centerline or parallel to the wing trailing edge). All tests were conducted with no external flow and high pressure air was used to simulate core and fan engine exhaust flows. Test results indicate that the wing-mounted thrust reverser concept can achieve overall thrust reverser effectiveness levels competitive with (parallel mount), or better than (normal mount) a conventional cascade thrust reverser system. By removing the thrust reverser system from the nacelle, the wing-mounted concept offers the nacelle designer more options for improving nacelle aero dynamics and propulsion-airframe integration, simplifying nacelle structural designs, reducing nacelle weight, and improving engine maintenance access.

The Influenza A Virus Genotype Determines the Antiviral Function of NF-κB

PubMed Central

Dam, Sharmistha; Kracht, Michael; Pleschka, Stephan

2016-01-01

ABSTRACT The role of NF-κB in influenza A virus (IAV) infection does not reveal a coherent picture, as pro- and also antiviral functions of this transcription factor have been described. To address this issue, we used clustered regularly interspaced short palindromic repeat with Cas9 (CRISPR-Cas9)-mediated genome engineering to generate murine MLE-15 cells lacking two essential components of the NF-κB pathway. Cells devoid of either the central NF-κB essential modulator (NEMO) scaffold protein and thus defective in IκB kinase (IKK) activation or cells not expressing the NF-κB DNA-binding and transactivation subunit p65 were tested for propagation of the SC35 virus, which has an avian host range, and its mouse-adapted variant, SC35M. While NF-κB was not relevant for replication of SC35M, the absence of NF-κB activity increased replication of the nonadapted SC35 virus. This antiviral effect of NF-κB was most prominent upon infection of cells with low virus titers as they usually occur during the initiation phase of IAV infection. The defect in NF-κB signaling resulted in diminished IAV-triggered phosphorylation of interferon regulatory factor 3 (IRF3) and expression of the antiviral beta interferon (IFN-β) gene. To identify the viral proteins responsible for NF-κB dependency, reassortant viruses were generated by reverse genetics. SC35 viruses containing the SC35M segment encoding neuraminidase (NA) were completely inert to the inhibitory effect of NF-κB, emphasizing the importance of the viral genotype for susceptibility to the antiviral functions of NF-κB. IMPORTANCE This study addresses two different issues. First, we investigated the role of the host cell transcription factor NF-κB in IAV replication by genetic manipulation of IAVs by reverse genetics combined with targeted genome engineering of host cells using CRISPR-Cas9. The analysis of these two highly defined genetic systems indicated that the IAV genotype can influence whether NF-κB displays an antiviral function and thus might in part explain incoherent results from the literature. Second, we found that perturbation of NF-κB function greatly improved the growth of a nonadapted IAV, suggesting that NF-κB may contribute to the maintenance of the host species barrier. PMID:27356900

Modularization of genetic elements promotes synthetic metabolic engineering.

PubMed

Qi, Hao; Li, Bing-Zhi; Zhang, Wen-Qian; Liu, Duo; Yuan, Ying-Jin

2015-11-15

In the context of emerging synthetic biology, metabolic engineering is moving to the next stage powered by new technologies. Systematical modularization of genetic elements makes it more convenient to engineer biological systems for chemical production or other desired purposes. In the past few years, progresses were made in engineering metabolic pathway using synthetic biology tools. Here, we spotlighted the topic of implementation of modularized genetic elements in metabolic engineering. First, we overviewed the principle developed for modularizing genetic elements and then discussed how the genetic modules advanced metabolic engineering studies. Next, we picked up some milestones of engineered metabolic pathway achieved in the past few years. Last, we discussed the rapid raised synthetic biology field of "building a genome" and the potential in metabolic engineering. Copyright © 2015 Elsevier Inc. All rights reserved.

Virus-based Photo-Responsive Nanowires Formed By Linking Site-Directed Mutagenesis and Chemical Reaction

NASA Astrophysics Data System (ADS)

Murugesan, Murali; Abbineni, Gopal; Nimmo, Susan L.; Cao, Binrui; Mao, Chuanbin

2013-05-01

Owing to the genetic flexibility and error-free bulk production, bio-nanostructures such as filamentous phage showed great potential in materials synthesis, however, their photo-responsive behaviour is neither explored nor unveiled. Here we show M13 phage genetically engineered with tyrosine residues precisely fused to the major coat protein is converted into a photo-responsive organic nanowire by a site-specific chemical reaction with an aromatic amine to form an azo dye structure on the surface. The resulting azo-M13-phage nanowire exhibits reversible photo-responsive properties due to the photo-switchable cis-trans isomerisation of the azo unit formed on the phage. This result shows that site-specific display of a peptide on bio-nanostructures through site-directed genetic mutagenesis can be translated into site-directed chemical reaction for developing advanced materials. The photo-responsive properties of the azo-M13-phage nanowires may open the door for the development of light controllable smart devices for use in non-linear optics, holography data storage, molecular antenna, and actuators.

Virus-based Photo-Responsive Nanowires Formed By Linking Site-Directed Mutagenesis and Chemical Reaction

PubMed Central

Murugesan, Murali; Abbineni, Gopal; Nimmo, Susan L.; Cao, Binrui; Mao, Chuanbin

2013-01-01

Owing to the genetic flexibility and error-free bulk production, bio-nanostructures such as filamentous phage showed great potential in materials synthesis, however, their photo-responsive behaviour is neither explored nor unveiled. Here we show M13 phage genetically engineered with tyrosine residues precisely fused to the major coat protein is converted into a photo-responsive organic nanowire by a site-specific chemical reaction with an aromatic amine to form an azo dye structure on the surface. The resulting azo-M13-phage nanowire exhibits reversible photo-responsive properties due to the photo-switchable cis-trans isomerisation of the azo unit formed on the phage. This result shows that site-specific display of a peptide on bio-nanostructures through site-directed genetic mutagenesis can be translated into site-directed chemical reaction for developing advanced materials. The photo-responsive properties of the azo-M13-phage nanowires may open the door for the development of light controllable smart devices for use in non-linear optics, holography data storage, molecular antenna, and actuators. PMID:23673356

Reverse Flow Engine Core Having a Ducted Fan with Integrated Secondary Flow Blades

NASA Technical Reports Server (NTRS)

Kisska, Michael K. (Inventor); Princen, Norman H. (Inventor); Kuehn, Mark S. (Inventor); Cosentino, Gary B. (Inventor)

2014-01-01

Secondary air flow is provided for a ducted fan having a reverse flow turbine engine core driving a fan blisk. The fan blisk incorporates a set of thrust fan blades extending from an outer hub and a set of integral secondary flow blades extending intermediate an inner hub and the outer hub. A nacelle provides an outer flow duct for the thrust fan blades and a secondary flow duct carries flow from the integral secondary flow blades as cooling air for components of the reverse flow turbine engine.

Application of reverse engineering in the medical industry.

NASA Astrophysics Data System (ADS)

Kaleev, A. A.; Kashapov, L. N.; Kashapov, N. F.; Kashapov, R. N.

2017-09-01

The purpose of this research is to develop on the basis of existing analogs new design of ophthalmologic microsurgical tweezers by using reverse engineering techniques. Virtual model was obtained by using a three-dimensional scanning system Solutionix Rexcan 450 MP. Geomagic Studio program was used to remove defects and inaccuracies of the obtained parametric model. A prototype of the finished model was made on the installation of laser stereolithography Projet 6000. Total time of the creation was 16 hours from the reverse engineering procedure to 3D-printing of the prototype.

A genetically-engineered von Willebrand disease type 2B mouse model displays defects in hemostasis and inflammation.

PubMed

Adam, Frédéric; Casari, Caterina; Prévost, Nicolas; Kauskot, Alexandre; Loubière, Cécile; Legendre, Paulette; Repérant, Christelle; Baruch, Dominique; Rosa, Jean-Philippe; Bryckaert, Marijke; de Groot, Philip G; Christophe, Olivier D; Lenting, Peter J; Denis, Cécile V

2016-05-23

von Willebrand disease (VWD)-type 2B is characterized by gain-of-function mutations in the von Willebrand factor (VWF) A1-domain, leading to increased affinity for its platelet-receptor, glycoprotein Ibα. We engineered the first knock-in (KI) murine model for VWD-type 2B by introducing the p.V1316M mutation in murine VWF. Homozygous KI-mice replicated human VWD-type 2B with macrothrombocytopenia (platelet counts reduced by 55%, platelet volume increased by 44%), circulating platelet-aggregates and a severe bleeding tendency. Also, vessel occlusion was deficient in the FeCl3-induced thrombosis model. Platelet aggregation induced by thrombin or collagen was defective for KI-mice at all doses. KI-mice manifested a loss of high molecular weight multimers and increased multimer degradation. In a model of VWF-string formation, the number of platelets/string and string-lifetime were surprisingly enhanced in KI-mice, suggesting that proteolysis of VWF/p.V1316M is differentially regulated in the circulation versus the endothelial surface. Furthermore, we observed increased leukocyte recruitment during an inflammatory response induced by the reverse passive Arthus reaction. This points to an active role of VWF/p.V1316M in the exfiltration of leukocytes under inflammatory conditions. In conclusion, our genetically-engineered VWD-type 2B mice represent an original model to study the consequences of spontaneous VWF-platelet interactions and the physiopathology of this human disease.

GRASP/Ada (Graphical Representations of Algorithms, Structures, and Processes for Ada): The development of a program analysis environment for Ada. Reverse engineering tools for Ada, task 1, phase 2

NASA Technical Reports Server (NTRS)

Cross, James H., II

1990-01-01

The study, formulation, and generation of structures for Ada (GRASP/Ada) are discussed in this second phase report of a three phase effort. Various graphical representations that can be extracted or generated from source code are described and categorized with focus on reverse engineering. The overall goal is to provide the foundation for a CASE (computer-aided software design) environment in which reverse engineering and forward engineering (development) are tightly coupled. Emphasis is on a subset of architectural diagrams that can be generated automatically from source code with the control structure diagram (CSD) included for completeness.

Genetic engineering possibilities for CELSS: A bibliography and summary of techniques

NASA Technical Reports Server (NTRS)

Johnson, E. J.

1982-01-01

A bibliography of the most useful techniques employed in genetic engineering of higher plants, bacteria associated with plants, and plant cell cultures is provided. A resume of state-of-the-art genetic engineering of plants and bacteria is presented. The potential application of plant bacterial genetic engineering to CELSS (Controlled Ecological Life Support System) program and future research needs are discussed.

Rewiring the severe acute respiratory syndrome coronavirus (SARS-CoV) transcription circuit: Engineering a recombination-resistant genome

NASA Astrophysics Data System (ADS)

Yount, Boyd; Roberts, Rhonda S.; Lindesmith, Lisa; Baric, Ralph S.

2006-08-01

Live virus vaccines provide significant protection against many detrimental human and animal diseases, but reversion to virulence by mutation and recombination has reduced appeal. Using severe acute respiratory syndrome coronavirus as a model, we engineered a different transcription regulatory circuit and isolated recombinant viruses. The transcription network allowed for efficient expression of the viral transcripts and proteins, and the recombinant viruses replicated to WT levels. Recombinant genomes were then constructed that contained mixtures of the WT and mutant regulatory circuits, reflecting recombinant viruses that might occur in nature. Although viable viruses could readily be isolated from WT and recombinant genomes containing homogeneous transcription circuits, chimeras that contained mixed regulatory networks were invariantly lethal, because viable chimeric viruses were not isolated. Mechanistically, mixed regulatory circuits promoted inefficient subgenomic transcription from inappropriate start sites, resulting in truncated ORFs and effectively minimize viral structural protein expression. Engineering regulatory transcription circuits of intercommunicating alleles successfully introduces genetic traps into a viral genome that are lethal in RNA recombinant progeny viruses. regulation | systems biology | vaccine design

A versatile genetic tool for post-translational control of gene expression in Drosophila melanogaster

PubMed Central

Sethi, Sachin

2017-01-01

Several techniques have been developed to manipulate gene expression temporally in intact neural circuits. However, the applicability of current tools developed for in vivo studies in Drosophila is limited by their incompatibility with existing GAL4 lines and side effects on physiology and behavior. To circumvent these limitations, we adopted a strategy to reversibly regulate protein degradation with a small molecule by using a destabilizing domain (DD). We show that this system is effective across different tissues and developmental stages. We further show that this system can be used to control in vivo gene expression levels with low background, large dynamic range, and in a reversible manner without detectable side effects on the lifespan or behavior of the animal. Additionally, we engineered tools for chemically controlling gene expression (GAL80-DD) and recombination (FLP-DD). We demonstrate the applicability of this technology in manipulating neuronal activity and for high-efficiency sparse labeling of neuronal populations. PMID:29140243

Optocontrol of glutamate receptor activity by single side-chain photoisomerization

PubMed Central

Klippenstein, Viktoria; Hoppmann, Christian; Ye, Shixin; Wang, Lei; Paoletti, Pierre

2017-01-01

Engineering light-sensitivity into proteins has wide ranging applications in molecular studies and neuroscience. Commonly used tethered photoswitchable ligands, however, require solvent-accessible protein labeling, face structural constrains, and are bulky. Here, we designed a set of optocontrollable NMDA receptors by directly incorporating single photoswitchable amino acids (PSAAs) providing genetic encodability, reversibility, and site tolerance. We identified several positions within the multi-domain receptor endowing robust photomodulation. PSAA photoisomerization at the GluN1 clamshell hinge is sufficient to control glycine sensitivity and activation efficacy. Strikingly, in the pore domain, flipping of a M3 residue within a conserved transmembrane cavity impacts both gating and permeation properties. Our study demonstrates the first detection of molecular rearrangements in real-time due to the reversible light-switching of single amino acid side-chains, adding a dynamic dimension to protein site-directed mutagenesis. This novel approach to interrogate neuronal protein function has general applicability in the fast expanding field of optopharmacology. DOI: http://dx.doi.org/10.7554/eLife.25808.001 PMID:28534738

Reverse and forward engineering of protein pattern formation.

PubMed

Kretschmer, Simon; Harrington, Leon; Schwille, Petra

2018-05-26

Living systems employ protein pattern formation to regulate important life processes in space and time. Although pattern-forming protein networks have been identified in various prokaryotes and eukaryotes, their systematic experimental characterization is challenging owing to the complex environment of living cells. In turn, cell-free systems are ideally suited for this goal, as they offer defined molecular environments that can be precisely controlled and manipulated. Towards revealing the molecular basis of protein pattern formation, we outline two complementary approaches: the biochemical reverse engineering of reconstituted networks and the de novo design, or forward engineering, of artificial self-organizing systems. We first illustrate the reverse engineering approach by the example of the Escherichia coli Min system, a model system for protein self-organization based on the reversible and energy-dependent interaction of the ATPase MinD and its activating protein MinE with a lipid membrane. By reconstituting MinE mutants impaired in ATPase stimulation, we demonstrate how large-scale Min protein patterns are modulated by MinE activity and concentration. We then provide a perspective on the de novo design of self-organizing protein networks. Tightly integrated reverse and forward engineering approaches will be key to understanding and engineering the intriguing phenomenon of protein pattern formation.This article is part of the theme issue 'Self-organization in cell biology'. © 2018 The Author(s).

77 FR 41366 - Syngenta Biotechnology, Inc.; Availability of Petition, Plant Pest Risk Assessment, and...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-13

... engineered organisms and products. We are soliciting comments on whether this genetically engineered corn is... pests. Such genetically engineered organisms and products are considered ``regulated articles.'' The... Assessment for Determination of Nonregulated Status of Corn Genetically Engineered for Insect Resistance...

Selected Readings in Genetic Engineering

ERIC Educational Resources Information Center

Mertens, Thomas R.; Robinson, Sandra K.

1973-01-01

Describes different sources of readings for understanding issues and concepts of genetic engineering. Broad categories of reading materials are: concerns about genetic engineering; its background; procedures; and social, ethical and legal issues. References are listed. (PS)

Dynamics of high-bypass-engine thrust reversal using a variable-pitch fan

NASA Technical Reports Server (NTRS)

Schaefer, J. W.; Sagerser, D. R.; Stakolich, E. G.

1977-01-01

The test program demonstrated that successful and rapid forward-to reverse-thrust transients can be performed without any significant engine operational limitations for fan blade pitch changes through either feather pitch or flat pitch. For through-feather-pitch operation with a flight inlet, fan stall problems were encountered, and a fan blade overshoot technique was used to establish reverse thrust.

Engineered Intrinsic Bioremediation of Ammonium Perchlorate in Groundwater

DTIC Science & Technology

2010-12-01

German Collection of Microorganisms and Cell Cultures) GA Genetic Algorithms GA-ANN Genetic Algorithm Artificial Neural Network GMO genetically...for in situ treatment of perchlorate in groundwater. This is accomplished without the addition of genetically engineered microorganisms ( GMOs ) to the...perchlorate, even in the presence of oxygen and without the addition of genetically engineered microorganisms ( GMOs ) to the environment. This approach

Alleviation of carbon catabolite repression in Enterobacter aerogenes for efficient utilization of sugarcane molasses for 2,3-butanediol production.

PubMed

Jung, Moo-Young; Jung, Hwi-Min; Lee, Jinwon; Oh, Min-Kyu

2015-01-01

Due to its cost-effectiveness and rich sugar composition, sugarcane molasses is considered to be a promising carbon source for biorefinery. However, the sugar mixture in sugarcane molasses is not consumed as efficiently as glucose in microbial fermentation due to complex interactions among their utilizing pathways, such as carbon catabolite repression (CCR). In this study, 2,3-butanediol-producing Enterobacter aerogenes was engineered to alleviate CCR and improve sugar utilization by modulating its carbon preference. The gene encoding catabolite repressor/activator (Cra) was deleted in the genome of E. aerogenes to increase the fructose consumption rate. However, the deletion mutation repressed sucrose utilization, resulting in the accumulation of sucrose in the fermentation medium. Cra regulation on expression of the scrAB operon involved in sucrose catabolism was verified by reverse transcription and real-time PCR, and the efficiency of sucrose utilization was restored by disrupting the scrR gene and overexpressing the scrAB operon. In addition, overexpression of the ptsG gene involved in glucose utilization enhanced the glucose preference among mixed sugars, which relieved glucose accumulation in fed-batch fermentation. In fed-batch fermentation using sugarcane molasses, the maximum titer of 2,3-butanediol production by the mutant reached 140.0 g/L at 54 h, which was by far the highest titer of 2,3-butanediol with E. aerogenes achieved through genetic engineering. We have developed genetically engineered E. aerogenes as a 2,3-butanediol producer that efficiently utilizes sugarcane molasses. The fermentation efficiency was dramatically improved by the alleviation of CCR and modulation of carbon preference. These results offer a metabolic engineering approach for achieving highly efficient utilization of mixed sugars for the biorefinery industry.

A gene network simulator to assess reverse engineering algorithms.

PubMed

Di Camillo, Barbara; Toffolo, Gianna; Cobelli, Claudio

2009-03-01

In the context of reverse engineering of biological networks, simulators are helpful to test and compare the accuracy of different reverse-engineering approaches in a variety of experimental conditions. A novel gene-network simulator is presented that resembles some of the main features of transcriptional regulatory networks related to topology, interaction among regulators of transcription, and expression dynamics. The simulator generates network topology according to the current knowledge of biological network organization, including scale-free distribution of the connectivity and clustering coefficient independent of the number of nodes in the network. It uses fuzzy logic to represent interactions among the regulators of each gene, integrated with differential equations to generate continuous data, comparable to real data for variety and dynamic complexity. Finally, the simulator accounts for saturation in the response to regulation and transcription activation thresholds and shows robustness to perturbations. It therefore provides a reliable and versatile test bed for reverse engineering algorithms applied to microarray data. Since the simulator describes regulatory interactions and expression dynamics as two distinct, although interconnected aspects of regulation, it can also be used to test reverse engineering approaches that use both microarray and protein-protein interaction data in the process of learning. A first software release is available at http://www.dei.unipd.it/~dicamill/software/netsim as an R programming language package.

Reverse engineering biomolecular systems using -omic data: challenges, progress and opportunities.

PubMed

Quo, Chang F; Kaddi, Chanchala; Phan, John H; Zollanvari, Amin; Xu, Mingqing; Wang, May D; Alterovitz, Gil

2012-07-01

Recent advances in high-throughput biotechnologies have led to the rapid growing research interest in reverse engineering of biomolecular systems (REBMS). 'Data-driven' approaches, i.e. data mining, can be used to extract patterns from large volumes of biochemical data at molecular-level resolution while 'design-driven' approaches, i.e. systems modeling, can be used to simulate emergent system properties. Consequently, both data- and design-driven approaches applied to -omic data may lead to novel insights in reverse engineering biological systems that could not be expected before using low-throughput platforms. However, there exist several challenges in this fast growing field of reverse engineering biomolecular systems: (i) to integrate heterogeneous biochemical data for data mining, (ii) to combine top-down and bottom-up approaches for systems modeling and (iii) to validate system models experimentally. In addition to reviewing progress made by the community and opportunities encountered in addressing these challenges, we explore the emerging field of synthetic biology, which is an exciting approach to validate and analyze theoretical system models directly through experimental synthesis, i.e. analysis-by-synthesis. The ultimate goal is to address the present and future challenges in reverse engineering biomolecular systems (REBMS) using integrated workflow of data mining, systems modeling and synthetic biology.

Commercialising genetically engineered animal biomedical products.

PubMed

Sullivan, Eddie J; Pommer, Jerry; Robl, James M

2008-01-01

Research over the past two decades has increased the quality and quantity of tools available to produce genetically engineered animals. The number of potentially viable biomedical products from genetically engineered animals is increasing. However, moving from cutting-edge research to development and commercialisation of a biomedical product that is useful and wanted by the public has significant challenges. Even early stage development of genetically engineered animal applications requires consideration of many steps, including quality assurance and quality control, risk management, gap analysis, founder animal establishment, cell banking, sourcing of animals and animal-derived material, animal facilities, product collection facilities and processing facilities. These steps are complicated and expensive. Biomedical applications of genetically engineered animals have had some recent successes and many applications are well into development. As researchers consider applications for their findings, having a realistic understanding of the steps involved in the development and commercialisation of a product, produced in genetically engineered animals, is useful in determining the risk of genetic modification to the animal nu. the potential public benefit of the application.

76 FR 78232 - Monsanto Co.; Determination of Nonregulated Status for Soybean Genetically Engineered To Have a...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-12-16

... peer review of safety tests, and health effects of genetically modified organisms and glyphosate. APHIS...] Monsanto Co.; Determination of Nonregulated Status for Soybean Genetically Engineered To Have a Modified... that there is reason to believe are plant pests. Such genetically engineered organisms and products are...

What Ideas Do Students Associate with "Biotechnology" and "Genetic Engineering"?

ERIC Educational Resources Information Center

Hill, Ruaraidh; Stanisstreet, Martin; Boyes, Edward

2000-01-01

Explores the ideas that students aged 16-19 associate with the terms 'biotechnology' and 'genetic engineering'. Indicates that some students see biotechnology as risky whereas genetic engineering was described as ethically wrong. (Author/ASK)

76 FR 39812 - Scotts Miracle-Gro Co.; Regulatory Status of Kentucky Bluegrass Genetically Engineered for...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-07-07

...] Scotts Miracle-Gro Co.; Regulatory Status of Kentucky Bluegrass Genetically Engineered for Herbicide... engineered for herbicide tolerance without the use of plant pest components, does not meet the definition of... has been genetically engineered for herbicide tolerance, does not meet the definition of a regulated...

Multiple-Color Optical Activation, Silencing, and Desynchronization of Neural Activity, with Single-Spike Temporal Resolution

PubMed Central

Han, Xue; Boyden, Edward S.

2007-01-01

The quest to determine how precise neural activity patterns mediate computation, behavior, and pathology would be greatly aided by a set of tools for reliably activating and inactivating genetically targeted neurons, in a temporally precise and rapidly reversible fashion. Having earlier adapted a light-activated cation channel, channelrhodopsin-2 (ChR2), for allowing neurons to be stimulated by blue light, we searched for a complementary tool that would enable optical neuronal inhibition, driven by light of a second color. Here we report that targeting the codon-optimized form of the light-driven chloride pump halorhodopsin from the archaebacterium Natronomas pharaonis (hereafter abbreviated Halo) to genetically-specified neurons enables them to be silenced reliably, and reversibly, by millisecond-timescale pulses of yellow light. We show that trains of yellow and blue light pulses can drive high-fidelity sequences of hyperpolarizations and depolarizations in neurons simultaneously expressing yellow light-driven Halo and blue light-driven ChR2, allowing for the first time manipulations of neural synchrony without perturbation of other parameters such as spiking rates. The Halo/ChR2 system thus constitutes a powerful toolbox for multichannel photoinhibition and photostimulation of virally or transgenically targeted neural circuits without need for exogenous chemicals, enabling systematic analysis and engineering of the brain, and quantitative bioengineering of excitable cells. PMID:17375185

Genetic engineering applied to agriculture has a long row to hoe.

PubMed

Miller, Henry I

2018-01-02

In spite of the lack of scientific justification for skepticism about crops modified with molecular techniques of genetic engineering, they have been the most scrutinized agricultural products in human history. The assumption that "genetically engineered" or "genetically modified" is a meaningful - and dangerous - classification has led to excessive and dilatory regulation. The modern molecular techniques are an extension, or refinement, of older, less precise, less predictable methods of genetic modification, but as long as today's activists and regulators remain convinced that so called "GMOs" represent a distinct and dangerous category of research and products, genetic engineering will fall short of its potential.

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

28. MESTA STEAM ENGINE, INSTALLED BY THE CORRIGAN, McKINNEY COMPANY ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

28. MESTA STEAM ENGINE, INSTALLED BY THE CORRIGAN, McKINNEY COMPANY IN 1916, STILL DRIVES THE 44-INCH REVERSING BLOOMING MILL. THE TWIN TANDAM, COMPOUND CONDENSING, REVERSING STEAM ENGINE HAS A RATED CAPACITY OF 35,000 H.P. IT WAS BUILT BY THE MESTA MACHINE COMPANY OF PITTSBURGH. - Corrigan, McKinney Steel Company, 3100 East Forty-fifth Street, Cleveland, Cuyahoga County, OH

Genetic Engineering

ERIC Educational Resources Information Center

Phillips, John

1973-01-01

Presents a review of genetic engineering, in which the genotypes of plants and animals (including human genotypes) may be manipulated for the benefit of the human species. Discusses associated problems and solutions and provides an extensive bibliography of literature relating to genetic engineering. (JR)

The Potential of Genetic Engineering in Agriculture to Affect Global Stability

DTIC Science & Technology

2013-04-17

manipulation in agriculture is thousands of years old, dating back to man’s first efforts of plant domestication. Over the last 200 years, and especially the...engineering.” In agriculture, genetic engineering describes the science of manipulating the genetic material (DNA) of plants by adding or taking...nature run its course. This paper does not delve into the science or even the raging safety debate over the use of genetic engineering in plants that

FLP-18 Functions through the G-Protein-Coupled Receptors NPR-1 and NPR-4 to Modulate Reversal Length in Caenorhabditis elegans

PubMed Central

Dahiya, Yogesh; Babu, Kavita

2018-01-01

Animal behavior is critically dependent on the activity of neuropeptides. Reversals, one of the most conspicuous behaviors in Caenorhabditis elegans, plays an important role in determining the navigation strategy of the animal. Our experiments on hermaphrodite C. elegans show the involvement of a neuropeptide FLP-18 in modulating reversal length in these hermaphrodites. We show that FLP-18 controls the reversal length by regulating the activity of AVA interneurons through the G-protein-coupled neuropeptide receptors, NPR-4 and NPR-1. We go on to show that the site of action of these receptors is the AVA interneuron for NPR-4 and the ASE sensory neurons for NPR-1. We further show that mutants in the neuropeptide, flp-18, and its receptors show increased reversal lengths. Consistent with the behavioral data, calcium levels in the AVA neuron of freely reversing C. elegans were significantly higher and persisted for longer durations in flp-18, npr-1, npr-4, and npr-1 npr-4 genetic backgrounds compared with wild-type control animals. Finally, we show that increasing FLP-18 levels through genetic and physiological manipulations causes shorter reversal lengths. Together, our analysis suggests that the FLP-18/NPR-1/NPR-4 signaling is a pivotal point in the regulation of reversal length under varied genetic and environmental conditions. PMID:29712787

Antibiotic Combinations That Enable One-Step, Targeted Mutagenesis of Chromosomal Genes.

PubMed

Lee, Wonsik; Do, Truc; Zhang, Ge; Kahne, Daniel; Meredith, Timothy C; Walker, Suzanne

2018-06-08

Targeted modification of bacterial chromosomes is necessary to understand new drug targets, investigate virulence factors, elucidate cell physiology, and validate results of -omics-based approaches. For some bacteria, reverse genetics remains a major bottleneck to progress in research. Here, we describe a compound-centric strategy that combines new negative selection markers with known positive selection markers to achieve simple, efficient one-step genome engineering of bacterial chromosomes. The method was inspired by the observation that certain nonessential metabolic pathways contain essential late steps, suggesting that antibiotics targeting a late step can be used to select for the absence of genes that control flux into the pathway. Guided by this hypothesis, we have identified antibiotic/counterselectable markers to accelerate reverse engineering of two increasingly antibiotic-resistant pathogens, Staphylococcus aureus and Acinetobacter baumannii. For S. aureus, we used wall teichoic acid biosynthesis inhibitors to select for the absence of tarO and for A. baumannii, we used colistin to select for the absence of lpxC. We have obtained desired gene deletions, gene fusions, and promoter swaps in a single plating step with perfect efficiency. Our method can also be adapted to generate markerless deletions of genes using FLP recombinase. The tools described here will accelerate research on two important pathogens, and the concept we outline can be readily adapted to any organism for which a suitable target pathway can be identified.

DECOMPOSTION OF GENETICALLY ENGINEERED TOBACCO UNDER FIELD CONDITIONS: PERSISTENCE OF THE PROTEINASE INHIBITOR I PRODUCT AND EFFECTS OF SOIL MICROBIAL RESPIRATION AND PROTOZOA, NEMATODE AND MICROARTHR

EPA Science Inventory

1. To evaluate the potential effects of genetically engineered (transgenic) plants on soil ecosystems, litterbags containing leaves of non-engineered (parental) and transgenic tobacco plants were buried in field plots. The transgenic tobacco plants were genetically engineered to ...

78 FR 44199 - Semiannual Regulatory Agenda, Spring 2013

Federal Register 2010, 2011, 2012, 2013, 2014

2013-07-23

..., interstate movement, and environmental release of certain genetically engineered organisms. This rule will... genetically engineered plants and certain other genetically engineered organisms. Timetable: Action Date FR... Citrus Canker; 0579-AC05 Compensation for Certified Citrus Nursery Stock. 17 Introduction of Organisms...

Analysis and documentation of QCSEE (Quiet Clean Short-haul Experimental Engine) over-the-wing exhaust system development

NASA Technical Reports Server (NTRS)

Ammer, R. C.; Kutney, J. T.

1977-01-01

A static scale model test program was conducted in the static test area of the NASA-Langley 9.14- by 18.29 m(30- by 60-ft) Full-Scale Wind Tunnel Facility to develop an over-the-wing (OTW) nozzle and reverser configuration for the Quiet Clean Short-Haul Experimental Engine (QCSEE). Three nozzles and one basic reverser configuration were tested over the QCSEE takeoff and approach power nozzle pressure ratio range between 1.1 and 1.3. The models were scaled to 8.53% of QCSEE engine size and tested behind two 13.97-cm (5.5-in.) diameter tip-turbine-driven fan simulators coupled in tandem. An OTW nozzle and reverser configuration was identified which satisfies the QCSEE experimental engine requirements in terms of nozzle cycle area variation capability and reverse thrust level, and provides good jet flow spreading over a wing upper surface for achievement of high propulsive lift performance.

Quantum engine efficiency bound beyond the second law of thermodynamics.

PubMed

Niedenzu, Wolfgang; Mukherjee, Victor; Ghosh, Arnab; Kofman, Abraham G; Kurizki, Gershon

2018-01-11

According to the second law, the efficiency of cyclic heat engines is limited by the Carnot bound that is attained by engines that operate between two thermal baths under the reversibility condition whereby the total entropy does not increase. Quantum engines operating between a thermal and a squeezed-thermal bath have been shown to surpass this bound. Yet, their maximum efficiency cannot be determined by the reversibility condition, which may yield an unachievable efficiency bound above unity. Here we identify the fraction of the exchanged energy between a quantum system and a bath that necessarily causes an entropy change and derive an inequality for this change. This inequality reveals an efficiency bound for quantum engines energised by a non-thermal bath. This bound does not imply reversibility, unless the two baths are thermal. It cannot be solely deduced from the laws of thermodynamics.

Numerical Prediction of the Influence of Thrust Reverser on Aeroengine's Aerodynamic Stability

NASA Astrophysics Data System (ADS)

Zhiqiang, Wang; Xigang, Shen; Jun, Hu; Xiang, Gao; Liping, Liu

2017-11-01

A numerical method was developed to predict the aerodynamic stability of a high bypass ratio turbofan engine, at the landing stage of a large transport aircraft, when the thrust reverser was deployed. 3D CFD simulation and 2D aeroengine aerodynamic stability analysis code were performed in this work, the former is to achieve distortion coefficient for the analysis of engine stability. The 3D CFD simulation was divided into two steps, the single engine calculation and the integrated aircraft and engine calculation. Results of the CFD simulation show that with the decreasing of relative wind Mach number, the engine inlet will suffer more severe flow distortion. The total pressure and total temperature distortion coefficients at the inlet of the engines were obtained from the results of the numerical simulation. Then an aeroengine aerodynamic stability analysis program was used to quantitatively analyze the aerodynamic stability of the high bypass ratio turbofan engine. The results of the stability analysis show that the engine can work stably, when the reverser flow is re-ingested. But the anti-distortion ability of the booster is weaker than that of the fan and high pressure compressor. It is a weak link of engine stability.

Wind tunnel test of model target thrust reversers for the Pratt and Whitney aircraft JT8D-100 series engines installed on a 727-200 airplane

NASA Technical Reports Server (NTRS)

Hambly, D.

1974-01-01

The results of a low speed wind tunnel test of 0.046 scale model target thrust reversers installed on a 727-200 model airplane are presented. The full airplane model was mounted on a force balance, except for the nacelles and thrust reversers, which were independently mounted and isolated from it. The installation had the capability of simulating the inlet airflows and of supplying the correct proportions of primary and secondary air to the nozzles. The objectives of the test were to assess the compatibility of the thrust reversers target door design with the engine and airplane. The following measurements were made: hot gas ingestion at the nacelle inlets; model lift, drag, and pitching moment; hot gas impingement on the airplane structure; and qualitative assessment of the rudder effectiveness. The major parameters controlling hot gas ingestion were found to be thrust reverser orientation, engine power setting, and the lip height of the bottom thrust reverser doors on the side nacelles. The thrust reversers tended to increase the model lift, decrease the drag, and decrease the pitching moment.

Reverse engineering systems models of regulation: discovery, prediction and mechanisms.

PubMed

Ashworth, Justin; Wurtmann, Elisabeth J; Baliga, Nitin S

2012-08-01

Biological systems can now be understood in comprehensive and quantitative detail using systems biology approaches. Putative genome-scale models can be built rapidly based upon biological inventories and strategic system-wide molecular measurements. Current models combine statistical associations, causative abstractions, and known molecular mechanisms to explain and predict quantitative and complex phenotypes. This top-down 'reverse engineering' approach generates useful organism-scale models despite noise and incompleteness in data and knowledge. Here we review and discuss the reverse engineering of biological systems using top-down data-driven approaches, in order to improve discovery, hypothesis generation, and the inference of biological properties. Copyright © 2011 Elsevier Ltd. All rights reserved.

Genetic engineering of industrial strains of Saccharomyces cerevisiae.

PubMed

Le Borgne, Sylvie

2012-01-01

Genetic engineering has been successfully applied to Saccharomyces cerevisiae laboratory strains for different purposes: extension of substrate range, improvement of productivity and yield, elimination of by-products, improvement of process performance and cellular properties, and extension of product range. The potential of genetically engineered yeasts for the massive production of biofuels as bioethanol and other nonfuel products from renewable resources as lignocellulosic biomass hydrolysates has been recognized. For such applications, robust industrial strains of S. cerevisiae have to be used. Here, some relevant genetic and genomic characteristics of industrial strains are discussed in relation to the problematic of the genetic engineering of such strains. General molecular tools applicable to the manipulation of S. cerevisiae industrial strains are presented and examples of genetically engineered industrial strains developed for the production of bioethanol from lignocellulosic biomass are given.

Reinventing the ames test as a quantitative lab that connects classical and molecular genetics.

PubMed

Goodson-Gregg, Nathan; De Stasio, Elizabeth A

2009-01-01

While many institutions use a version of the Ames test in the undergraduate genetics laboratory, students typically are not exposed to techniques or procedures beyond qualitative analysis of phenotypic reversion, thereby seriously limiting the scope of learning. We have extended the Ames test to include both quantitative analysis of reversion frequency and molecular analysis of revertant gene sequences. By giving students a role in designing their quantitative methods and analyses, students practice and apply quantitative skills. To help students connect classical and molecular genetic concepts and techniques, we report here procedures for characterizing the molecular lesions that confer a revertant phenotype. We suggest undertaking reversion of both missense and frameshift mutants to allow a more sophisticated molecular genetic analysis. These modifications and additions broaden the educational content of the traditional Ames test teaching laboratory, while simultaneously enhancing students' skills in experimental design, quantitative analysis, and data interpretation.

Genetic engineering of live attenuated influenza viruses.

PubMed

Jin, Hong; Chen, Zhongying; Liu, Jonathan; Kemble, George

2012-01-01

The first live attenuated influenza vaccine (LAIV) was licensed in the USA in 2003; it is a trivalent vaccine composed of two type A (H1N1 and H3N2) and one type B influenza virus each at 10(7) fluorescent focus units (FFU). Each influenza vaccine strain is a reassortant virus that contains the hemagglutinin (HA) and neuraminidase (NA) gene segments from a wild-type influenza virus and the six internal protein gene segments from a master donor virus (MDV) of either cold-adapted A/Ann Arbor/6/60 or B/Ann Arbor/1/66. MDV confers the cold-adapted, temperature-sensitive, and attenuation phenotypes to the vaccine strains. The reassortant vaccine seeds are currently produced by reverse genetics and amplified in specific pathogen-free (SPF) 9-11 days old embryonated chicken eggs for manufacture. In addition, MDCK cell culture manufacture processes have been developed to produce LAIV for research use and with modifications for clinical and/or commercial grade material production.

Stochastic models for inferring genetic regulation from microarray gene expression data.

PubMed

Tian, Tianhai

2010-03-01

Microarray expression profiles are inherently noisy and many different sources of variation exist in microarray experiments. It is still a significant challenge to develop stochastic models to realize noise in microarray expression profiles, which has profound influence on the reverse engineering of genetic regulation. Using the target genes of the tumour suppressor gene p53 as the test problem, we developed stochastic differential equation models and established the relationship between the noise strength of stochastic models and parameters of an error model for describing the distribution of the microarray measurements. Numerical results indicate that the simulated variance from stochastic models with a stochastic degradation process can be represented by a monomial in terms of the hybridization intensity and the order of the monomial depends on the type of stochastic process. The developed stochastic models with multiple stochastic processes generated simulations whose variance is consistent with the prediction of the error model. This work also established a general method to develop stochastic models from experimental information. 2009 Elsevier Ireland Ltd. All rights reserved.

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.

Genetically engineered nanocarriers for drug delivery.

PubMed

Shi, Pu; Gustafson, Joshua A; MacKay, J Andrew

2014-01-01

Cytotoxicity, low water solubility, rapid clearance from circulation, and off-target side-effects are common drawbacks of conventional small-molecule drugs. To overcome these shortcomings, many multifunctional nanocarriers have been proposed to enhance drug delivery. In concept, multifunctional nanoparticles might carry multiple agents, control release rate, biodegrade, and utilize target-mediated drug delivery; however, the design of these particles presents many challenges at the stage of pharmaceutical development. An emerging solution to improve control over these particles is to turn to genetic engineering. Genetically engineered nanocarriers are precisely controlled in size and structure and can provide specific control over sites for chemical attachment of drugs. Genetically engineered drug carriers that assemble nanostructures including nanoparticles and nanofibers can be polymeric or non-polymeric. This review summarizes the recent development of applications in drug and gene delivery utilizing nanostructures of polymeric genetically engineered drug carriers such as elastin-like polypeptides, silk-like polypeptides, and silk-elastin-like protein polymers, and non-polymeric genetically engineered drug carriers such as vault proteins and viral proteins.

Genetically engineered nanocarriers for drug delivery

PubMed Central

Shi, Pu; Gustafson, Joshua A; MacKay, J Andrew

2014-01-01

Cytotoxicity, low water solubility, rapid clearance from circulation, and off-target side-effects are common drawbacks of conventional small-molecule drugs. To overcome these shortcomings, many multifunctional nanocarriers have been proposed to enhance drug delivery. In concept, multifunctional nanoparticles might carry multiple agents, control release rate, biodegrade, and utilize target-mediated drug delivery; however, the design of these particles presents many challenges at the stage of pharmaceutical development. An emerging solution to improve control over these particles is to turn to genetic engineering. Genetically engineered nanocarriers are precisely controlled in size and structure and can provide specific control over sites for chemical attachment of drugs. Genetically engineered drug carriers that assemble nanostructures including nanoparticles and nanofibers can be polymeric or non-polymeric. This review summarizes the recent development of applications in drug and gene delivery utilizing nanostructures of polymeric genetically engineered drug carriers such as elastin-like polypeptides, silk-like polypeptides, and silk-elastin-like protein polymers, and non-polymeric genetically engineered drug carriers such as vault proteins and viral proteins. PMID:24741309

Cell-Based Genotoxicity Testing

NASA Astrophysics Data System (ADS)

Reifferscheid, Georg; Buchinger, Sebastian

Genotoxicity test systems that are based on bacteria display an important role in the detection and assessment of DNA damaging chemicals. They belong to the basic line of test systems due to their easy realization, rapidness, broad applicability, high sensitivity and good reproducibility. Since the development of the Salmonella microsomal mutagenicity assay by Ames and coworkers in the early 1970s, significant development in bacterial genotoxicity assays was achieved and is still a subject matter of research. The basic principle of the mutagenicity assay is a reversion of a growth inhibited bacterial strain, e.g., due to auxotrophy, back to a fast growing phenotype (regain of prototrophy). Deeper knowledge of the ­mutation events allows a mechanistic understanding of the induced DNA-damage by the utilization of base specific tester strains. Collections of such specific tester strains were extended by genetic engineering. Beside the reversion assays, test systems utilizing the bacterial SOS-response were invented. These methods are based on the fusion of various SOS-responsive promoters with a broad variety of reporter genes facilitating numerous methods of signal detection. A very important aspect of genotoxicity testing is the bioactivation of ­xenobiotics to DNA-damaging compounds. Most widely used is the extracellular metabolic activation by making use of rodent liver homogenates. Again, genetic engineering allows the construction of highly sophisticated bacterial tester strains with significantly enhanced sensitivity due to overexpression of enzymes that are involved in the metabolism of xenobiotics. This provides mechanistic insights into the toxification and detoxification pathways of xenobiotics and helps explaining the chemical nature of hazardous substances in unknown mixtures. In summary, beginning with "natural" tester strains the rational design of bacteria led to highly specific and sensitive tools for a rapid, reliable and cost effective ­genotoxicity testing that is of outstanding importance in the risk assessment of compounds (REACH) and in ecotoxicology.

Remote control of molecular motors using light-activated gearshifting

NASA Astrophysics Data System (ADS)

Bryant, Zev

2013-03-01

Engineering molecular motors with dynamically controllable properties will allow selective perturbation of mechanical processes in vivo and provide sophisticated components for directed nanoscale transport in vitro. We previously constructed myosin motors that respond to a change in [Ca++] by reversing their direction of motion along the polarized actin filament. To expand the potential applications of controllable molecular motors, we have now developed myosins that shift gears in response to blue light illumination. Light is a versatile control signal that can be readily modulated in time and space, and is generally orthogonal to cellular signaling. Using structure-guided protein engineering, we have incorporated LOV photoreceptor domains into the lever arms of chimeric myosins, resulting in motors that robustly speed up, slow down, or switch directions upon illumination. These genetically encoded motors should be directly deployable inside living cells. Our successful designs include constructs based on two different myosin classes, and we show that optical velocity control can be implemented in motors that move at microns/sec speeds, enabling practical biological and bioengineering applications.

Inventing the Invented for STEM Understanding

ERIC Educational Resources Information Center

Stansell, Alicia; Tyler-Wood, Tandra; Stansell, Christina

2016-01-01

The reverse engineering of simple inventions that were of historic significance is now possible in a classroom by using digital models provided by places like the Smithsonian. The digital models can facilitate the mastery of students' STEM learning by utilizing digital fabrication in maker spaces to provide an opportunity for reverse engineer and…

A FIELD STUDY WITH GENETICALLY ENGINEERED ALFALFA INOCULATED WITH RECOMBINANT SINORHIZOBIUM MELILOTI: EFFECTS ON THE SOIL ECOSYSTEM

EPA Science Inventory

The agricultural use of genetically engineered plants and microorganisms has become increasingly common. Because genetically engineered plants and microorganisms can produce compounds foreign to their environment, there is concern that they may become established outside of thei...

What's in a name: the Vermont Genetically Engineered Food Labeling Act

PubMed Central

McPherson, Malia J.

2014-01-01

On May 8, 2014, Vermont passed the Vermont Genetically Engineered Food Labeling Act (Act) requiring labels on certain genetically engineered foods. Once the bill takes effect July 1, 2016, all Vermont-retailed foods with more than 0.9% of their total weight in genetically modified ingredients must be labeled with language stating, “may be partially produced with genetic engineering.” As genetically engineered food are considered scientifically equivalent to their traditional counterparts and are not subject to federal labeling by the FDA, the Act presents several legal questions. Several of the legal questions have been raised in a recent lawsuit filed by the Grocery Manufactures Association that claims the Act violates the First Amendment, Supremacy Clause, and Commerce Clause. This paper will discuss why the Second Circuit could strike down the Act as unconstitutional as to each claim. PMID:27774175

76 FR 44891 - Monsanto Co.; Availability of Petition, Plant Pest Risk Assessment, and Environmental Assessment...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-07-27

... Determination of Nonregulated Status for Corn Genetically Engineered for Drought Tolerance AGENCY: Animal and... nonregulated status for corn designated as MON 87460, which has been genetically engineered for drought... nonregulated status for corn designated as MON 87460, which has been genetically engineered for drought...

78 FR 66892 - BASF Plant Science LP; Availability of Plant Pest Risk Assessment and Environmental Assessment...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-11-07

... Determination of Nonregulated Status of Soybean Genetically Engineered for Herbicide Resistance AGENCY: Animal... genetically engineered for resistance to herbicides in the imidazolinone family. We are soliciting comments on... genetically engineered for resistance to herbicides in the imidazolinone family. The petition states that this...

76 FR 5780 - Determination of Regulated Status of Alfalfa Genetically Engineered for Tolerance to the...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-02-02

...] Determination of Regulated Status of Alfalfa Genetically Engineered for Tolerance to the Herbicide Glyphosate... for tolerance to the herbicide glyphosate based on APHIS' final environmental impact statement. FOR... regulated status of alfalfa genetically engineered for tolerance to the herbicide glyphosate based on an...

Genetic Engineering of Plants. Agricultural Research Opportunities and Policy Concerns.

ERIC Educational Resources Information Center

Roberts, Leslie

Plant scientists and science policymakers from government, private companies, and universities met at a convocation on the genetic engineering of plants. During the convocation, researchers described some of the ways genetic engineering may be used to address agricultural problems. Policymakers delineated and debated changes in research funding…

76 FR 37771 - Monsanto Co.; Availability of Petition, Plant Pest Risk Assessment, and Environmental Assessment...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-06-28

... Determination of Nonregulated Status for Soybean Genetically Engineered To Have a Modified Fatty Acid Profile... soybean designated as MON 87705, which has been genetically engineered to have a modified fatty acid... our regulations concerning the introduction of certain genetically engineered organisms and products...

Non-genetic engineering of cells for drug delivery and cell-based therapy.

PubMed

Wang, Qun; Cheng, Hao; Peng, Haisheng; Zhou, Hao; Li, Peter Y; Langer, Robert

2015-08-30

Cell-based therapy is a promising modality to address many unmet medical needs. In addition to genetic engineering, material-based, biochemical, and physical science-based approaches have emerged as novel approaches to modify cells. Non-genetic engineering of cells has been applied in delivering therapeutics to tissues, homing of cells to the bone marrow or inflammatory tissues, cancer imaging, immunotherapy, and remotely controlling cellular functions. This new strategy has unique advantages in disease therapy and is complementary to existing gene-based cell engineering approaches. A better understanding of cellular systems and different engineering methods will allow us to better exploit engineered cells in biomedicine. Here, we review non-genetic cell engineering techniques and applications of engineered cells, discuss the pros and cons of different methods, and provide our perspectives on future research directions. Copyright © 2014 Elsevier B.V. All rights reserved.

The Influenza A Virus Genotype Determines the Antiviral Function of NF-κB.

PubMed

Dam, Sharmistha; Kracht, Michael; Pleschka, Stephan; Schmitz, M Lienhard

2016-09-01

The role of NF-κB in influenza A virus (IAV) infection does not reveal a coherent picture, as pro- and also antiviral functions of this transcription factor have been described. To address this issue, we used clustered regularly interspaced short palindromic repeat with Cas9 (CRISPR-Cas9)-mediated genome engineering to generate murine MLE-15 cells lacking two essential components of the NF-κB pathway. Cells devoid of either the central NF-κB essential modulator (NEMO) scaffold protein and thus defective in IκB kinase (IKK) activation or cells not expressing the NF-κB DNA-binding and transactivation subunit p65 were tested for propagation of the SC35 virus, which has an avian host range, and its mouse-adapted variant, SC35M. While NF-κB was not relevant for replication of SC35M, the absence of NF-κB activity increased replication of the nonadapted SC35 virus. This antiviral effect of NF-κB was most prominent upon infection of cells with low virus titers as they usually occur during the initiation phase of IAV infection. The defect in NF-κB signaling resulted in diminished IAV-triggered phosphorylation of interferon regulatory factor 3 (IRF3) and expression of the antiviral beta interferon (IFN-β) gene. To identify the viral proteins responsible for NF-κB dependency, reassortant viruses were generated by reverse genetics. SC35 viruses containing the SC35M segment encoding neuraminidase (NA) were completely inert to the inhibitory effect of NF-κB, emphasizing the importance of the viral genotype for susceptibility to the antiviral functions of NF-κB. This study addresses two different issues. First, we investigated the role of the host cell transcription factor NF-κB in IAV replication by genetic manipulation of IAVs by reverse genetics combined with targeted genome engineering of host cells using CRISPR-Cas9. The analysis of these two highly defined genetic systems indicated that the IAV genotype can influence whether NF-κB displays an antiviral function and thus might in part explain incoherent results from the literature. Second, we found that perturbation of NF-κB function greatly improved the growth of a nonadapted IAV, suggesting that NF-κB may contribute to the maintenance of the host species barrier. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Genetically Modified Food: Knowledge and Attitude of Teachers and Students

NASA Astrophysics Data System (ADS)

Mohapatra, Animesh K.; Priyadarshini, Deepika; Biswas, Antara

2010-10-01

The concepts behind the technology of genetic modification of organisms and its applications are complex. A diverse range of opinions, public concern and considerable media interest accompanies the subject. This study explores the knowledge and attitudes of science teachers and senior secondary biology students about the application of a rapidly expanding technology, genetic engineering, to food production. The results indicated significant difference in understanding of concepts related with genetically engineered food stuffs between teachers and students. The most common ideas about genetically modified food were that cross bred plants and genetically modified plants are not same, GM organisms are produced by inserting a foreign gene into a plant or animal and are high yielding. More teachers thought that genetically engineered food stuffs were unsafe for the environment. Both teachers and students showed number of misconceptions, for example, the pesticidal proteins produced by GM organisms have indirect effects through bioaccumulation, induces production of allergic proteins, genetic engineering is production of new genes, GM plants are leaky sieves and that transgenes are more likely to introgress into wild species than mutated species. In general, more students saw benefits while teachers were cautious about the advantages of genetically engineered food stuffs.

Computer-aided dental prostheses construction using reverse engineering.

PubMed

Solaberrieta, E; Minguez, R; Barrenetxea, L; Sierra, E; Etxaniz, O

2014-01-01

The implementation of computer-aided design/computer-aided manufacturing (CAD/CAM) systems with virtual articulators, which take into account the kinematics, constitutes a breakthrough in the construction of customised dental prostheses. This paper presents a multidisciplinary protocol involving CAM techniques to produce dental prostheses. This protocol includes a step-by-step procedure using innovative reverse engineering technologies to transform completely virtual design processes into customised prostheses. A special emphasis is placed on a novel method that permits a virtual location of the models. The complete workflow includes the optical scanning of the patient, the use of reverse engineering software and, if necessary, the use of rapid prototyping to produce CAD temporary prostheses.

Sex reversal triggers the rapid transition from genetic to temperature-dependent sex.

PubMed

Holleley, Clare E; O'Meally, Denis; Sarre, Stephen D; Marshall Graves, Jennifer A; Ezaz, Tariq; Matsubara, Kazumi; Azad, Bhumika; Zhang, Xiuwen; Georges, Arthur

2015-07-02

Sex determination in animals is amazingly plastic. Vertebrates display contrasting strategies ranging from complete genetic control of sex (genotypic sex determination) to environmentally determined sex (for example, temperature-dependent sex determination). Phylogenetic analyses suggest frequent evolutionary transitions between genotypic and temperature-dependent sex determination in environmentally sensitive lineages, including reptiles. These transitions are thought to involve a genotypic system becoming sensitive to temperature, with sex determined by gene-environment interactions. Most mechanistic models of transitions invoke a role for sex reversal. Sex reversal has not yet been demonstrated in nature for any amniote, although it occurs in fish and rarely in amphibians. Here we make the first report of reptile sex reversal in the wild, in the Australian bearded dragon (Pogona vitticeps), and use sex-reversed animals to experimentally induce a rapid transition from genotypic to temperature-dependent sex determination. Controlled mating of normal males to sex-reversed females produces viable and fertile offspring whose phenotypic sex is determined solely by temperature (temperature-dependent sex determination). The W sex chromosome is eliminated from this lineage in the first generation. The instantaneous creation of a lineage of ZZ temperature-sensitive animals reveals a novel, climate-induced pathway for the rapid transition between genetic and temperature-dependent sex determination, and adds to concern about adaptation to rapid global climate change.

Computational metabolic engineering strategies for growth-coupled biofuel production by Synechocystis.

PubMed

Shabestary, Kiyan; Hudson, Elton P

2016-12-01

Chemical and fuel production by photosynthetic cyanobacteria is a promising technology but to date has not reached competitive rates and titers. Genome-scale metabolic modeling can reveal limitations in cyanobacteria metabolism and guide genetic engineering strategies to increase chemical production. Here, we used constraint-based modeling and optimization algorithms on a genome-scale model of Synechocystis PCC6803 to find ways to improve productivity of fermentative, fatty-acid, and terpene-derived fuels. OptGene and MOMA were used to find heuristics for knockout strategies that could increase biofuel productivity. OptKnock was used to find a set of knockouts that led to coupling between biofuel and growth. Our results show that high productivity of fermentation or reversed beta-oxidation derived alcohols such as 1-butanol requires elimination of NADH sinks, while terpenes and fatty-acid based fuels require creating imbalances in intracellular ATP and NADPH production and consumption. The FBA-predicted productivities of these fuels are at least 10-fold higher than those reported so far in the literature. We also discuss the physiological and practical feasibility of implementing these knockouts. This work gives insight into how cyanobacteria could be engineered to reach competitive biofuel productivities.

Murine genetically engineered and human xenograft models of chronic lymphocytic leukemia.

PubMed

Chen, Shih-Shih; Chiorazzi, Nicholas

2014-07-01

Chronic lymphocytic leukemia (CLL) is a genetically complex disease, with multiple factors having an impact on onset, progression, and response to therapy. Genetic differences/abnormalities have been found in hematopoietic stem cells from patients, as well as in B lymphocytes of individuals with monoclonal B-cell lymphocytosis who may develop the disease. Furthermore, after the onset of CLL, additional genetic alterations occur over time, often causing disease worsening and altering patient outcomes. Therefore, being able to genetically engineer mouse models that mimic CLL or at least certain aspects of the disease will help us understand disease mechanisms and improve treatments. This notwithstanding, because neither the genetic aberrations responsible for leukemogenesis and progression nor the promoting factors that support these are likely identical in character or influences for all patients, genetically engineered mouse models will only completely mimic CLL when all of these factors are precisely defined. In addition, multiple genetically engineered models may be required because of the heterogeneity in susceptibility genes among patients that can have an effect on genetic and environmental characteristics influencing disease development and outcome. For these reasons, we review the major murine genetically engineered and human xenograft models in use at the present time, aiming to report the advantages and disadvantages of each. Copyright © 2014 Elsevier Inc. All rights reserved.

Quiet Clean Short-Haul Experimental Engine (QCSEE) acoustic and aerodynamic tests on a scale model over-the-wing thrust reverser and forward thrust nozzle

NASA Technical Reports Server (NTRS)

Stimpert, D. L.

1978-01-01

An acoustic and aerodynamic test program was conducted on a 1/6.25 scale model of the Quiet, Clean, Short-Haul Experimental Engine (QCSEE) forward thrust over-the-wing (OTW) nozzle and OTW thrust reverser. In reverse thrust, the effect of reverser geometry was studied by parametric variations in blocker spacing, blocker height, lip angle, and lip length. Forward thrust nozzle tests determined the jet noise levels of the cruise and takeoff nozzles, the effect of opening side doors to achieve takeoff thrust, and scrubbing noise of the cruise and takeoff jet on a simulated wing surface. Velocity profiles are presented for both forward and reverse thrust nozzles. An estimate of the reverse thrust was made utilizing the measured centerline turning angle.

26 CFR 1.861-18 - Classification of transactions involving computer programs.

Code of Federal Regulations, 2011 CFR

2011-04-01

... on a single disk for a one-time payment with restrictions on transfer and reverse engineering, which... license. The license is stated to be perpetual. Under the license no reverse engineering, decompilation... fee, on a World Wide Web home page on the Internet. P, the Country Z resident, in return for payment...

26 CFR 1.861-18 - Classification of transactions involving computer programs.

Code of Federal Regulations, 2010 CFR

2010-04-01

... on a single disk for a one-time payment with restrictions on transfer and reverse engineering, which... license. The license is stated to be perpetual. Under the license no reverse engineering, decompilation... fee, on a World Wide Web home page on the Internet. P, the Country Z resident, in return for payment...

Recognition vs Reverse Engineering in Boolean Concepts Learning

ERIC Educational Resources Information Center

Shafat, Gabriel; Levin, Ilya

2012-01-01

This paper deals with two types of logical problems--recognition problems and reverse engineering problems, and with the interrelations between these types of problems. The recognition problems are modeled in the form of a visual representation of various objects in a common pattern, with a composition of represented objects in the pattern.…

Reverse Engineering Course at Philadelphia University in Jordan

ERIC Educational Resources Information Center

Younis, M. Bani; Tutunji, T.

2012-01-01

Reverse engineering (RE) is the process of testing and analysing a system or a device in order to identify, understand and document its functionality. RE is an efficient tool in industrial benchmarking where competitors' products are dissected and evaluated for performance and costs. RE can play an important role in the re-configuration and…

Teach CAD and Measuring Skills through Reverse Engineering

ERIC Educational Resources Information Center

Board, Keith

2012-01-01

This article describes a reverse engineering activity that gives students hands-on, minds-on experience with measuring tools, machine parts, and CAD. The author developed this activity to give students an abundance of practical experience with measuring tools. Equally important, it provides a good interface between the virtual world of CAD 3D…

A parallel implementation of the network identification by multiple regression (NIR) algorithm to reverse-engineer regulatory gene networks.

PubMed

Gregoretti, Francesco; Belcastro, Vincenzo; di Bernardo, Diego; Oliva, Gennaro

2010-04-21

The reverse engineering of gene regulatory networks using gene expression profile data has become crucial to gain novel biological knowledge. Large amounts of data that need to be analyzed are currently being produced due to advances in microarray technologies. Using current reverse engineering algorithms to analyze large data sets can be very computational-intensive. These emerging computational requirements can be met using parallel computing techniques. It has been shown that the Network Identification by multiple Regression (NIR) algorithm performs better than the other ready-to-use reverse engineering software. However it cannot be used with large networks with thousands of nodes--as is the case in biological networks--due to the high time and space complexity. In this work we overcome this limitation by designing and developing a parallel version of the NIR algorithm. The new implementation of the algorithm reaches a very good accuracy even for large gene networks, improving our understanding of the gene regulatory networks that is crucial for a wide range of biomedical applications.

A Predictive Approach to Network Reverse-Engineering

NASA Astrophysics Data System (ADS)

Wiggins, Chris

2005-03-01

A central challenge of systems biology is the ``reverse engineering" of transcriptional networks: inferring which genes exert regulatory control over which other genes. Attempting such inference at the genomic scale has only recently become feasible, via data-intensive biological innovations such as DNA microrrays (``DNA chips") and the sequencing of whole genomes. In this talk we present a predictive approach to network reverse-engineering, in which we integrate DNA chip data and sequence data to build a model of the transcriptional network of the yeast S. cerevisiae capable of predicting the response of genes in unseen experiments. The technique can also be used to extract ``motifs,'' sequence elements which act as binding sites for regulatory proteins. We validate by a number of approaches and present comparison of theoretical prediction vs. experimental data, along with biological interpretations of the resulting model. En route, we will illustrate some basic notions in statistical learning theory (fitting vs. over-fitting; cross- validation; assessing statistical significance), highlighting ways in which physicists can make a unique contribution in data- driven approaches to reverse engineering.

Silk-elastin-like protein biomaterials for the controlled delivery of therapeutics.

PubMed

Huang, Wenwen; Rollett, Alexandra; Kaplan, David L

2015-05-01

Genetically engineered biomaterials are useful for controlled delivery owing to their rational design, tunable structure-function, biocompatibility, degradability and target specificity. Silk-elastin-like proteins (SELPs), a family of genetically engineered recombinant protein polymers, possess these properties. Additionally, given the benefits of combining semi-crystalline silk-blocks and elastomeric elastin-blocks, SELPs possess multi-stimuli-responsive properties and tunability, thereby becoming promising candidates for targeted cancer therapeutics delivery and controlled gene release. An overview of SELP biomaterials for drug delivery and gene release is provided. Biosynthetic strategies used for SELP production, fundamental physicochemical properties and self-assembly mechanisms are discussed. The review focuses on sequence-structure-function relationships, stimuli-responsive features and current and potential drug delivery applications. The tunable material properties allow SELPs to be pursued as promising biomaterials for nanocarriers and injectable drug release systems. Current applications of SELPs have focused on thermally-triggered biomaterial formats for the delivery of therapeutics, based on local hyperthermia in tumors or infections. Other prominent controlled release applications of SELPs as injectable hydrogels for gene release have also been pursued. Further biomedical applications that utilize other stimuli to trigger the reversible material responses of SELPs for targeted delivery, including pH, ionic strength, redox, enzymatic stimuli and electric field, are in progress. Exploiting these additional stimuli-responsive features will provide a broader range of functional biomaterials for controlled therapeutics release and tissue regeneration.

U.S. Adults with Agricultural Experience Report More Genetic Engineering Familiarity than Those Without

ERIC Educational Resources Information Center

Stofer, Kathryn A.; Schiebel, Tracee M.

2017-01-01

Researchers and pollsters still debate the acceptance of genetic engineering technology among U.S. adults, and continue to assess their knowledge as part of this research. While decision-making may not rely entirely on knowledge, querying opinions and perceptions rely on public understanding of genetic engineering terms. Experience with…

Can Man Control His Biological Evolution? A Symposium on Genetic Engineering. Genetic Engineering

ERIC Educational Resources Information Center

Ramsey, Paul

1972-01-01

Presented are issues related to genetic engineering. Increased knowledge of techniques to manipulate genes are apt to create confusion about moral values in relation to unborn babies and other living organisms on earth. Human beings may use this knowledge to disturb the balance maintained by nature. (PS)

Reverse Transcription Quantitative Polymerase Chain Reaction for Detection of and Differentiation Between RNA and DNA of HIV-1-Based Lentiviral Vectors.

PubMed

Pavlovic, Melanie; Koehler, Nina; Anton, Martina; Dinkelmeier, Anna; Haase, Maren; Stellberger, Thorsten; Busch, Ulrich; Baiker, Armin E

2017-08-01

The purpose of the described method is the detection of and differentiation between RNA and DNA of human immunodeficiency virus (HIV)-derived lentiviral vectors (LV) in cell culture supernatants and swab samples. For the analytical surveillance of genetic engineering, operations methods for the detection of the HIV-1-based LV generations are required. Furthermore, for research issues, it is important to prove the absence of LV particles for downgrading experimental settings in terms of the biosafety level. Here, a quantitative polymerase chain reaction method targeting the long terminal repeat U5 subunit and the start sequence of the packaging signal ψ is described. Numerous controls are included in order to monitor the technical procedure.

Lingual Haematoma due to Tenecteplase in a Patient with Acute Myocardial Infarction

PubMed Central

Bal, Muhlis; Salturk, Ziya; Ateş, Ahmet Hakan; Yağcı, Serkan; Coşkun Bal, Gökçen

2013-01-01

The use of intravenous thrombolytic agents has revolutionised the treatment of acute myocardial infarction. However, the improvement in mortality rate achieved with these drugs is tempered by the risk of serious bleeding complications, including intracranial haemorrhage. Tenecteplase is a genetically engineered mutant tissue plasminogen activator. Haemorrhagic complications of tissue plasminogen activator (tPA) are well known. Compared to other tPAs, tenecteplase use leads to lower rates of bleeding complications. Here, we report a case of unusual site of spontaneous bleeding, intralingual haematoma during tenecteplase therapy following acute myocardial infarction, which caused significant upper airway obstruction and required tracheotomy to maintain the patient's airway. Clinical dilemmas related to securing the airway or reversing the effects of tissue plasminogen activator are discussed. PMID:23862086

A Reverse-Translational Approach to Bipolar Disorder: Rodent and human studies in the Behavioral Pattern Monitor

PubMed Central

Young, Jared W.; Minassian, Arpi; Paulus, Martin P.; Geyer, Mark A.; Perry, William

2007-01-01

Mania is the defining feature of Bipolar Disorder (BD). There has been limited progress in understanding the neurobiological underpinnings of BD mania and developing novel therapeutics, in part due to a paucity of relevant animal models with translational potential. Hyperactivity is a cardinal symptom of mania, traditionally measured in humans using observer-rated scales. Multivariate assessment of unconditioned locomotor behavior using the rat Behavioral Pattern Monitor (BPM) developed in our laboratory has shown that hyperactivity includes complex multifaceted behaviors. The BPM has been used to demonstrate differential effects of drugs on locomotor activity and exploratory behavior in rats. Studies of genetically engineered mice in a mouse BPM have confirmed its utility as a cross-species tool. In a “reverse-translational” approach to this work, we developed the human BPM to characterize motor activity in BD patients. Increased activity, object interactions, and altered locomotor patterns provide multidimensional phenotypes to model in the rodent BPM. This unique approach to modeling BD provides an opportunity to identify the neurobiology underlying BD mania and test novel antimanic agents. PMID:17706782

UAV Mission Optimization through Hybrid-Electric Propulsion

NASA Astrophysics Data System (ADS)

Blackwelder, Philip Scott

Hybrid-electric powertrain leverages the superior range of petrol based systems with the quiet and emission free benefits of electric propulsion. The major caveat to hybrid-electric powertrain in an airplane is that it is inherently heavier than conventional petroleum powertrain due mostly to the low energy density of battery technology. The first goal of this research is to develop mission planning code to match powertrain components for a small-scale unmanned aerial vehicle (UAV) to complete a standard surveillance mission within a set of user input parameters. The second goal is to promote low acoustic profile loitering through mid-flight engine starting. The two means by which midmission engine starting will be addressed is through reverse thrust from the propeller and a servo actuated gear to couple and decouple the engine and motor. The mission planning code calculates the power required to complete a mission and assists the user in sourcing powertrain components including the propeller, motor, battery, motor controller, engine and fuel. Reverse thrust engine starting involves characterizing an off the shelf variable pitch propeller and using its torque coefficient to calculate the advance ratio required to provide sufficient torque and speed to start an engine. Geared engine starting works like the starter in a conventional automobile. A servo actuated gear will couple the motor to the engine to start it and decouple once the engine has started. Reverse thrust engine starting was unsuccessful due to limitations of available off the shelf variable pitch propellers. However, reverse thrust engine starting could be realized through a custom larger diameter propeller. Geared engine starting was a success, though the system was unable to run fully as intended. Due to counter-clockwise crank rotation of the engine and the right-hand threads on the crankshaft, cranking the engine resulted in the nut securing the engine starter gear to back off as the engine cranked. A second nut was added to secure the starter gear but at the expense of removing the engine drive pulley. Removing the engine pulley meant that the starter gear must remain engaged to transmit torque to the propeller shaft as opposed to the engine pulley. This issue can be resolved using different hardware, however changing the mounting hardware would require additional modifications to the associated component which time would not permit. Though battery technology still proves to be the main constraint of electrified powertrain, careful design and mission planning can help minimize the weight penalties incurred. The mission planning code complements previous research by comparing the weight penalties of a blended climb versus an engine only climb and selecting the lightest option. Though reverse thrust engine starting proved unsuccessful, the success of geared engine starting now allows the engine to be shut off during loiter reducing both acoustic profile and fuel consumption during loiter.

Genetic Engineering of Alfalfa (Medicago sativa L.).

PubMed

Wang, Dan; Khurshid, Muhammad; Sun, Zhan Min; Tang, Yi Xiong; Zhou, Mei Liang; Wu, Yan Min

2016-01-01

Alfalfa is excellent perennial legume forage for its extensive ecological adaptability, high nutrition value, palatability and biological nitrogen fixation. It plays a very important role in the agriculture, animal husbandry and ecological construction. It is cultivated in all continents. With the development of modern plant breeding and genetic engineering techniques, a large amount of work has been carried out on alfalfa. Here we summarize the recent research advances in genetic engineering of alfalfa breeding, including transformation, quality improvement, stress resistance and as a bioreactor. The review article can enables us to understand the research method, direction and achievements of genetic engineering technology of Alfalfa.

Synthesis and Evolution of a Threose Nucleic Acid Aptamer Bearing 7-Deaza-7-Substituted Guanosine Residues.

PubMed

Mei, Hui; Liao, Jen-Yu; Jimenez, Randi M; Wang, Yajun; Bala, Saikat; McCloskey, Cailen; Switzer, Christopher; Chaput, John C

2018-05-02

In vitro selection experiments carried out on artificial genetic polymers require robust and faithful methods for copying genetic information back and forth between DNA and xeno-nucleic acids (XNA). Previously, we have shown that Kod-RI, an engineered polymerase developed to transcribe DNA templates into threose nucleic acid (TNA), can function with high fidelity in the absence of manganese ions. However, the transcriptional efficiency of this enzyme diminishes greatly when individual templates are replaced with libraries of DNA sequences, indicating that manganese ions are still required for in vitro selection. Unfortunately, the presence of manganese ions in the transcription mixture leads to the misincorporation of tGTP nucleotides opposite dG residues in the templating strand, which are detected as G-to-C transversions when the TNA is reverse transcribed back into DNA. Here we report the synthesis and fidelity of TNA replication using 7-deaza-7-modified guanosine base analogues in the DNA template and incoming TNA nucleoside triphosphate. Our findings reveal that tGTP misincorporation occurs via a Hoogsteen base pair in which the incoming tGTP residue adopts a syn conformation with respect to the sugar. Substitution of tGTP for 7-deaza-7-phenyl tGTP enabled the synthesis of TNA polymers with >99% overall fidelity. A TNA library containing the 7-deaza-7-phenyl guanine analogue was used to evolve a biologically stable TNA aptamer that binds to HIV reverse transcriptase with low nanomolar affinity.

Sex Reversal in Birds.

PubMed

Major, Andrew T; Smith, Craig A

2016-01-01

Sexual differentiation in birds is controlled genetically as in mammals, although the sex chromosomes are different. Males have a ZZ sex chromosome constitution, while females are ZW. Gene(s) on the sex chromosomes must initiate gonadal sex differentiation during embryonic life, inducing paired testes in ZZ individuals and unilateral ovaries in ZW individuals. The traditional view of avian sexual differentiation aligns with that expounded for other vertebrates; upon sexual differentiation, the gonads secrete sex steroid hormones that masculinise or feminise the rest of the body. However, recent studies on naturally occurring or experimentally induced avian sex reversal suggest a significant role for direct genetic factors, in addition to sex hormones, in regulating sexual differentiation of the soma in birds. This review will provide an overview of sex determination in birds and both naturally and experimentally induced sex reversal, with emphasis on the key role of oestrogen. We then consider how recent studies on sex reversal and gynandromorphic birds (half male:half female) are shaping our understanding of sexual differentiation in avians and in vertebrates more broadly. Current evidence shows that sexual differentiation in birds is a mix of direct genetic and hormonal mechanisms. Perturbation of either of these components may lead to sex reversal. © 2016 S. Karger AG, Basel.

Efficient and Robust Paramyxoviridae Reverse Genetics Systems

PubMed Central

Beaty, Shannon M.; Won, Sohui T.; Hong, Patrick; Lyons, Michael; Vigant, Frederic; Freiberg, Alexander N.; tenOever, Benjamin R.; Duprex, W. Paul

2017-01-01

ABSTRACT The notoriously low efficiency of Paramyxoviridae reverse genetics systems has posed a limiting barrier to the study of viruses in this family. Previous approaches to reverse genetics have utilized a wide variety of techniques to overcome the technical hurdles. Although robustness (i.e., the number of attempts that result in successful rescue) has been improved in some systems with the use of stable cell lines, the efficiency of rescue (i.e., the proportion of transfected cells that yield at least one successful rescue event) has remained low. We have substantially increased rescue efficiency for representative viruses from all five major Paramyxoviridae genera (from ~1 in 106-107 to ~1 in 102-103 transfected cells) by the addition of a self-cleaving hammerhead ribozyme (Hh-Rbz) sequence immediately preceding the start of the recombinant viral antigenome and the use of a codon-optimized T7 polymerase (T7opt) gene to drive paramyxovirus rescue. Here, we report a strategy for robust, reliable, and high-efficiency rescue of paramyxovirus reverse genetics systems, featuring several major improvements: (i) a vaccinia virus-free method, (ii) freedom to use any transfectable cell type for viral rescue, (iii) a single-step transfection protocol, and (iv) use of the optimal T7 promoter sequence for high transcription levels from the antigenomic plasmid without incorporation of nontemplated G residues. The robustness of our T7opt-HhRbz system also allows for greater latitude in the ratios of transfected accessory plasmids used that result in successful rescue. Thus, our system may facilitate the rescue and interrogation of the increasing number of emerging paramyxoviruses. IMPORTANCE The ability to manipulate the genome of paramyxoviruses and evaluate the effects of these changes at the phenotypic level is a powerful tool for the investigation of specific aspects of the viral life cycle and viral pathogenesis. However, reverse genetics systems for paramyxoviruses are notoriously inefficient, when successful. The ability to efficiently and robustly rescue paramyxovirus reverse genetics systems can be used to answer basic questions about the biology of paramyxoviruses, as well as to facilitate the considerable translational efforts being devoted to developing live attenuated paramyxovirus vaccine vectors. PMID:28405630

Moral Fantasy in Genetic Engineering.

ERIC Educational Resources Information Center

Boone, C. Keith

1984-01-01

Discusses the main ethical issues generated by the new genetics and suggests ways to think about them. Concerns include "playing God," violation of the natural order of the universe, and abuse of genetic technology. Critical distinctions for making difficult decisions about genetic engineering issues are noted. (DH)

Genetic Engineering Strategies for Enhanced Biodiesel Production.

PubMed

Hegde, Krishnamoorthy; Chandra, Niharika; Sarma, Saurabh Jyoti; Brar, Satinder Kaur; Veeranki, Venkata Dasu

2015-07-01

The focus on biodiesel research has shown a tremendous growth over the last few years. Several microbial and plant sources are being explored for the sustainable biodiesel production to replace the petroleum diesel. Conventional methods of biodiesel production have several limitations related to yield and quality, which led to development of new engineering strategies to improve the biodiesel production in plants, and microorganisms. Substantial progress in utilizing algae, yeast, and Escherichia coli for the renewable production of biodiesel feedstock via genetic engineering of fatty acid metabolic pathways has been reported in the past few years. However, in most of the cases, the successful commercialization of such engineering strategies for sustainable biodiesel production is yet to be seen. This paper systematically presents the drawbacks in the conventional methods for biodiesel production and an exhaustive review on the present status of research in genetic engineering strategies for production of biodiesel in plants, and microorganisms. Further, we summarize the technical challenges need to be tackled to make genetic engineering technology economically sustainable. Finally, the need and prospects of genetic engineering technology for the sustainable biodiesel production and the recommendations for the future research are discussed.

77 FR 41350 - Monsanto Co.; Determination of Nonregulated Status of Soybean Genetically Engineered To Produce...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-13

... MON 87769, which has been genetically engineered to produce stearidonic acid, an omega-3 fatty acid... 87769, which has been genetically engineered to produce stearidonic acid, an omega-3 fatty acid not... NEPA (40 CFR parts 1500-1508), (3) USDA regulations implementing NEPA (7 CFR part 1b), and (4) APHIS...

A field release of genetically engineered gypsy moth (Lymantria dispar L.) Nuclear Polyhedrosis Virus (LdNPV)

Treesearch

Vincent D' Amico; Joseph S. Elkinton; John D. Podgwaite; James M. Slavicek; Michael L. McManus; John P. Burand

1999-01-01

The gypsy moth (Lymantria dispar L.) nuclear polyhedrosis virus was genetically engineered for nonpersistence by removal of the gene coding for polyhedrin production and stabilized using a coocclusion process. A β-galactosidase marker gene was inserted into the genetically engineered virus (LdGEV) so that infected larvae could be tested for...

78 FR 13303 - Stine Seed Farm, Inc.; Availability of Plant Pest Risk Assessment, Environmental Assessment, and...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-02-27

... reason to believe are plant pests. Such genetically engineered organisms (GE) and products are considered... genetically engineered organisms. Paragraph (e) of Sec. 340.6 provides that APHIS will publish a notice in the... Preliminary Decision for an Extension of a Determination of Nonregulated Status of Corn Genetically Engineered...

Interactions between Multiple Genetic Determinants in the 5′ UTR and VP1 Capsid Control Pathogenesis of Chronic Post-Viral Myopathy caused by Coxsackievirus B1

PubMed Central

Sandager, Maribeth M.; Nugent, Jaime L.; Schulz, Wade L.; Messner, Ronald P.; Tam, Patricia E.

2008-01-01

Mice infected with coxsackievirus B1 Tucson (CVB1T) develop chronic, post-viral myopathy (PVM) with clinical manifestations of hind limb muscle weakness and myositis. The objective of the current study was to establish the genetic basis of myopathogenicity in CVB1T. Using a reverse genetics approach, full attenuation of PVM could only be achieved by simultaneously mutating four sites located at C706U in the 5′ untranslated region (5′ UTR) and at Y87F, V136A, and T276A in the VP1 capsid. Engineering these four myopathic determinants into an amyopathic CVB1T variant restored the ability to cause PVM. Moreover, these same four determinants controlled PVM expression in a second strain of mice, indicating that the underlying mechanism is operational in mice of different genetic backgrounds. Modeling studies predict that C706U alters both local and long-range pairing in the 5′ UTR, and that VP1 determinants are located on the capsid surface. However, these differences did not affect viral titers, temperature stability, pH stability, or the antibody response to virus. These studies demonstrate that PVM develops from a complex interplay between viral determinants in the 5′ UTR and VP1 capsid and have uncovered intriguing similarities between genetic determinants that cause PVM and those involved in pathogenesis of other enteroviruses. PMID:18029287

Reversible Quantum Brownian Heat Engines for Electrons

NASA Astrophysics Data System (ADS)

Humphrey, T. E.; Newbury, R.; Taylor, R. P.; Linke, H.

2002-08-01

Brownian heat engines use local temperature gradients in asymmetric potentials to move particles against an external force. The energy efficiency of such machines is generally limited by irreversible heat flow carried by particles that make contact with different heat baths. Here we show that, by using a suitably chosen energy filter, electrons can be transferred reversibly between reservoirs that have different temperatures and electrochemical potentials. We apply this result to propose heat engines based on mesoscopic semiconductor ratchets, which can quasistatically operate arbitrarily close to Carnot efficiency.

Reversible quantum heat engines for electrons

NASA Astrophysics Data System (ADS)

Linke, Heiner; Humphrey, Tammy E.; Newbury, Richard; Taylor, Richard P.

2002-03-01

Brownian heat engines use local temperature gradients in asymmetric potentials to move particles against an external force. The energy efficiency of such machines is generally limited by irreversible heat flow carried by particles that make contact with different heat baths. Here we show that, by using a suitably chosen energy filter, electrons can be transferred reversibly between reservoirs that have different temperatures and electrochemical potentials. We apply this result to propose heat engines based on quantum ratchets, which can quasistatically operate at Carnot efficiency.

Human Genetic Engineering: A Survey of Student Value Stances

ERIC Educational Resources Information Center

Wilson, Sara McCormack; And Others

1975-01-01

Assesses the values of high school and college students relative to human genetic engineering and recommends that biology educators explore instructional strategies merging human genetic information with value clarification techniques. (LS)

REAR DETAIL OF RIGHT ENGINE AND WING. THRUST REVERSER REMAINS ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

REAR DETAIL OF RIGHT ENGINE AND WING. THRUST REVERSER REMAINS OPEN. MECHANICS JONI BAINE (R) AND BILL THEODORE(L) OPEN FLAP CARRIAGE ACCESS WITH AN IMPACT GUN. THEY WILL CHECK TRANSMISSION FLUID AND OIL THE JACK SCREW. AT FAR LEFT UTILITY MECHANICS BEGIN BODY POLISHING. - Greater Buffalo International Airport, Maintenance Hangar, Buffalo, Erie County, NY

The Use of Reverse Engineering to Analyse Student Computer Programs.

ERIC Educational Resources Information Center

Vanneste, Philip; And Others

1996-01-01

Discusses how the reverse engineering approach can generate feedback on computer programs without the user having any prior knowledge of what the program was designed to do. This approach uses the cognitive model of programming knowledge to interpret both context independent and dependent errors in the same words and concepts as human programmers.…

An Application of Reverse Engineering to Automatic Item Generation: A Proof of Concept Using Automatically Generated Figures

ERIC Educational Resources Information Center

Lorié, William A.

2013-01-01

A reverse engineering approach to automatic item generation (AIG) was applied to a figure-based publicly released test item from the Organisation for Economic Cooperation and Development (OECD) Programme for International Student Assessment (PISA) mathematical literacy cognitive instrument as part of a proof of concept. The author created an item…

Genome-Wide Association Mapping Combined with Reverse Genetics Identifies New Effectors of Low Water Potential-Induced Proline Accumulation in Arabidopsis1[W][OPEN

PubMed Central

Verslues, Paul E.; Lasky, Jesse R.; Juenger, Thomas E.; Liu, Tzu-Wen; Kumar, M. Nagaraj

2014-01-01

Arabidopsis (Arabidopsis thaliana) exhibits natural genetic variation in drought response, including varying levels of proline (Pro) accumulation under low water potential. As Pro accumulation is potentially important for stress tolerance and cellular redox control, we conducted a genome-wide association (GWAS) study of low water potential-induced Pro accumulation using a panel of natural accessions and publicly available single-nucleotide polymorphism (SNP) data sets. Candidate genomic regions were prioritized for subsequent study using metrics considering both the strength and spatial clustering of the association signal. These analyses found many candidate regions likely containing gene(s) influencing Pro accumulation. Reverse genetic analysis of several candidates identified new Pro effector genes, including thioredoxins and several genes encoding Universal Stress Protein A domain proteins. These new Pro effector genes further link Pro accumulation to cellular redox and energy status. Additional new Pro effector genes found include the mitochondrial protease LON1, ribosomal protein RPL24A, protein phosphatase 2A subunit A3, a MADS box protein, and a nucleoside triphosphate hydrolase. Several of these new Pro effector genes were from regions with multiple SNPs, each having moderate association with Pro accumulation. This pattern supports the use of summary approaches that incorporate clusters of SNP associations in addition to consideration of individual SNP probability values. Further GWAS-guided reverse genetics promises to find additional effectors of Pro accumulation. The combination of GWAS and reverse genetics to efficiently identify new effector genes may be especially applicable for traits difficult to analyze by other genetic screening methods. PMID:24218491

Over-the-wing model thrust reverser noise tests

NASA Technical Reports Server (NTRS)

Goodykoontz, J.; Gutierrez, O.

1977-01-01

Static acoustic tests were conducted on a 1/12 scale model over-the-wing target type thrust reverser. The model configuration simulates a design that is applicable to the over-the-wing short-haul advanced technology engine. Aerodynamic screening tests of a variety of reverser designs identified configurations that satisfied a reverse thrust requirement of 35 percent of forward thrust at a nozzle pressure ratio of 1.29. The variations in the reverser configuration included, blocker door angle, blocker door lip angle and shape, and side skirt shape. Acoustic data are presented and compared for the various configurations. The model data scaled to a single full size engine show that peak free field perceived noise (PN) levels at a 152.4 meter sideline distance range from 98 to 104 PNdb.

Reverse-engineering the Arabidopsis thaliana transcriptional network under changing environmental conditions

PubMed Central

Carrera, Javier; Rodrigo, Guillermo; Jaramillo, Alfonso; Elena, Santiago F

2009-01-01

Background Understanding the molecular mechanisms plants have evolved to adapt their biological activities to a constantly changing environment is an intriguing question and one that requires a systems biology approach. Here we present a network analysis of genome-wide expression data combined with reverse-engineering network modeling to dissect the transcriptional control of Arabidopsis thaliana. The regulatory network is inferred by using an assembly of microarray data containing steady-state RNA expression levels from several growth conditions, developmental stages, biotic and abiotic stresses, and a variety of mutant genotypes. Results We show that the A. thaliana regulatory network has the characteristic properties of hierarchical networks. We successfully applied our quantitative network model to predict the full transcriptome of the plant for a set of microarray experiments not included in the training dataset. We also used our model to analyze the robustness in expression levels conferred by network motifs such as the coherent feed-forward loop. In addition, the meta-analysis presented here has allowed us to identify regulatory and robust genetic structures. Conclusions These data suggest that A. thaliana has evolved high connectivity in terms of transcriptional regulation among cellular functions involved in response and adaptation to changing environments, while gene networks constitutively expressed or less related to stress response are characterized by a lower connectivity. Taken together, these findings suggest conserved regulatory strategies that have been selected during the evolutionary history of this eukaryote. PMID:19754933

Inherited XX sex reversal originating from wild medaka populations.

PubMed

Shinomiya, A; Otake, H; Hamaguchi, S; Sakaizumi, M

2010-11-01

The teleost fish, medaka (Oryzias latipes), has an XX/XY sex-determining mechanism. A Y-linked DM domain gene, DMY, has been isolated by positional cloning as the sex-determining gene in this species. Previously, we conducted a field survey of genotypic sex and found that approximately 1% of wild medaka are sex-reversed (XX males and XY females). Here, we performed genetic analyses of nine spontaneous XX sex-reversed males to elucidate its genetic basis. In all cases, the F(1) progeny were all females, whereas XX males reappeared in the backcross (BC) progeny, suggesting that XX sex reversal is a recessive trait. Although the incidences of sex reversal in the BC progeny were mostly low, 40% were males derived from one XX male. We performed linkage analysis using 55 BC males and located a single major factor, sda-1 (sex-determining autosomal factor-1), controlling sex reversal in an autosomal linkage group. Thus, genes involved in the sex-determining pathway can be isolated from spontaneous mutants in wild populations.

Genetic Engineering Workshop Report, 2010

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

Allen, J; Slezak, T

2010-11-03

The Lawrence Livermore National Laboratory (LLNL) Bioinformatics group has recently taken on a role in DTRA's Transformation Medical Technologies (TMT) program. The high-level goal of TMT is to accelerate the development of broad-spectrum countermeasures. To achieve this goal, there is a need to assess the genetic engineering (GE) approaches, potential application as well as detection and mitigation strategies. LLNL was tasked to coordinate a workshop to determine the scope of investments that DTRA should make to stay current with the rapid advances in genetic engineering technologies, so that accidental or malicious uses of GE technologies could be adequately detected andmore » characterized. Attachment A is an earlier report produced by LLNL for TMT that provides some relevant background on Genetic Engineering detection. A workshop was held on September 23-24, 2010 in Springfield, Virginia. It was attended by a total of 55 people (see Attachment B). Twenty four (44%) of the attendees were academic researchers involved in GE or bioinformatics technology, 6 (11%) were from DTRA or the TMT program management, 7 (13%) were current TMT performers (including Jonathan Allen and Tom Slezak of LLNL who hosted the workshop), 11 (20%) were from other Federal agencies, and 7 (13%) were from industries that are involved in genetic engineering. Several attendees could be placed in multiple categories. There were 26 attendees (47%) who were from out of the DC area and received travel assistance through Invitational Travel Orders (ITOs). We note that this workshop could not have been as successful without the ability to invite experts from outside of the Beltway region. This workshop was an unclassified discussion of the science behind current genetic engineering capabilities. US citizenship was not required for attendance. While this may have limited some discussions concerning risk, we felt that it was more important for this first workshop to focus on the scientific state of the art. We also consciously chose to not dwell on matters of policy (for example, screening of commercial gene or oligo synthesis orders), as multiple other forums for policy discussion have taken place in recent years. We acknowledge that other workshops on topics relevant to genetic engineering should be held, some of which may need to take place at higher classification levels. The workshop moderators would like to acknowledge the enthusiastic participation of the attendees in the discussions. Special thanks are given to Sofi Ibrahim, for his extensive assistance on helping this report reach its final form. The genetic engineering workshop brought together a diverse mix of genetic engineering pioneers and experts, Federal agency representatives concerned with abuses of genetic engineering, TMT performers, bioinformatics experts, and representatives from industry involved with large-scale genetic engineering and synthetic biology. Several talks established the current range of genetic engineering capabilities and the relative difficulties of identifying and characterizing the results of their use. Extensive discussions established a number of recommendations to DTRA of how to direct future research investments so that any mis-use of genetic engineering techniques can be promptly identified and characterized.« less

Genetic Engineering: A Matter that Requires Further Refinement in Spanish Secondary School Textbooks

ERIC Educational Resources Information Center

Martinez-Gracia, M. V.; Gil-Quylez, M. J.; Osada, J.

2003-01-01

Genetic engineering is now an integral part of many high school textbooks but little work has been done to assess whether it is being properly addressed. A checklist with 19 items was used to analyze how genetic engineering is presented in biology textbooks commonly used in Spanish high schools, including the content, its relationship with…

Genetically engineered orange petunias on the market.

PubMed

Bashandy, Hany; Teeri, Teemu H

2017-08-01

Unauthorized genetically engineered orange petunias were found on the market. Genetic engineering of petunia was shown to lead to novel flower color some 20 years ago. Here we show that petunia lines with orange flowers, generated for scientific purposes, apparently found their way to petunia breeding programmes, intentionally or unintentionally. Today they are widely available, but have not been registered for commerce.

The Effect of Case Teaching on Meaningful and Retentive Learning When Studying Genetic Engineering

ERIC Educational Resources Information Center

Güccük, Ahmet; Köksal, Mustafa Serdar

2017-01-01

The purpose of this study is to investigate the effects of case teaching on how students learn about genetic engineering, in terms of meaningful learning and retention of learning. The study was designed as quasi-experimental research including 63 8th graders (28 boys and 35 girls). To collect data, genetic engineering achievement tests were…

German politics of genetic engineering and its deconstruction.

PubMed

Gottweis, H

1995-05-01

Policy-making, as exemplified by biotechnology policy, can be understood as an attempt to manage a field of discursivity, to construct regularity in a dispersed multitude of combinable elements. Following this perspective of politics as a textual process, the paper interprets the politicization of genetic engineering in Germany as a defence of the political as a regime of heterogeneity, as a field of 'dissensus' rather than 'consensus', and a rejection of the idea that the framing of technological transformation is an autonomous process. From its beginning in the early 1970s, genetic engineering was symbolically entrenched as a key technology of the future, and as an integral element of the German politics of modernization. Attempts by new social movements and the Green Party to displace the egalitarian imaginary of democratic discourse into the politics of genetic engineering were construed by the political élites as an attack on the political order of post-World War II Germany. The 1990 Genetic Engineering Law attempted a closure of this controversy. But it is precisely the homogenizing idiom of this 'settlement' which continues to nourish the social movements and their radical challenge to the definitions and codings of the politics of genetic engineering.

The use of genetically-engineered animals in science: perspectives of Canadian Animal Care Committee members.

PubMed

Ormandy, Elisabeth H; Dale, Julie; Griffin, Gilly

2013-05-01

The genetic engineering of animals for their use in science challenges the implementation of refinement and reduction in several areas, including the invasiveness of the procedures involved, unanticipated welfare concerns, and the numbers of animals required. Additionally, the creation of genetically-engineered animals raises problems with the Canadian system of reporting animal numbers per Category of Invasiveness, as well as raising issues of whether ethical limits can, or should, be placed on genetic engineering. A workshop was held with the aim of bringing together Canadian animal care committee members to discuss these issues, to reflect on progress that has been made in addressing them, and to propose ways of overcoming any challenges. Although previous literature has made recommendations with regard to refinement and reduction when creating new genetically-engineered animals, the perception of the workshop participants was that some key opportunities are being missed. The participants identified the main roadblocks to the implementation of refinement and reduction alternatives as confidentiality, cost and competition. If the scientific community is to make progress concerning the implementation of refinement and reduction, particularly in the creation and use of genetically-engineered animals, addressing these roadblocks needs to be a priority. 2013 FRAME.

Genetic engineering including superseding microinjection: new ways to make GM pigs.

PubMed

Galli, Cesare; Perota, Andrea; Brunetti, Dario; Lagutina, Irina; Lazzari, Giovanna; Lucchini, Franco

2010-01-01

Techniques for genetic engineering of swine are providing genetically modified animals of importance for the field of xenotransplantation, animal models for human diseases and for a variety of research applications. Many of these modifications have been directed toward avoiding naturally existing cellular and antibody responses to species-specific antigens. A number of techniques are today available to engineering the genome of mammals, these range from the well established less efficient method of DNA microinjection into the zygote, the use of viral vectors, to the more recent use of somatic cell nuclear transfer. The use of enzymatic engineering that are being developed now will refine the precision of the genetic modification combined with the use of new vectors like transposons. The use of somatic cell nuclear transfer is currently the most efficient way to generate genetically modified pigs. The development of enzymatic engineering with zinc-finger nucleases, recombinases and transposons will revolutionize the field. Nevertheless, genetic engineering in large domesticated animals will remain a challenging task. Recent improvements in several fields of cell and molecular biology offer new promises and opportunities toward an easier, cost-effective and efficient generation of transgenic pigs. © 2010 John Wiley & Sons A/S.

Genetically Engineered Cyanobacteria

NASA Technical Reports Server (NTRS)

Zhou, Ruanbao (Inventor); Gibbons, William (Inventor)

2015-01-01

The disclosed embodiments provide cyanobacteria spp. that have been genetically engineered to have increased production of carbon-based products of interest. These genetically engineered hosts efficiently convert carbon dioxide and light into carbon-based products of interest such as long chained hydrocarbons. Several constructs containing polynucleotides encoding enzymes active in the metabolic pathways of cyanobacteria are disclosed. In many instances, the cyanobacteria strains have been further genetically modified to optimize production of the carbon-based products of interest. The optimization includes both up-regulation and down-regulation of particular genes.

Genetic engineering of Ganoderma lucidum for the efficient production of ganoderic acids.

PubMed

Xu, Jun-Wei; Zhong, Jian-Jiang

2015-01-01

Ganoderma lucidum is a well-known traditional medicinal mushroom that produces ganoderic acids with numerous interesting bioactivities. Genetic engineering is an efficient approach to improve ganoderic acid biosynthesis. However, reliable genetic transformation methods and appropriate genetic manipulation strategies remain underdeveloped and thus should be enhanced. We previously established a homologous genetic transformation method for G. lucidum; we also applied the established method to perform the deregulated overexpression of a homologous 3-hydroxy-3-methyl-glutaryl coenzyme A reductase gene in G. lucidum. Engineered strains accumulated more ganoderic acids than wild-type strains. In this report, the genetic transformation systems of G. lucidum are described; current trends are also presented to improve ganoderic acid production through the genetic manipulation of G. lucidum.

Lassa Virus Reverse Genetics.

PubMed

Martínez-Sobrido, Luis; Paessler, Slobodan; de la Torre, Juan Carlos

2017-01-01

The Old World (OW) arenavirus Lassa (LASV ) is estimated to infect several hundred thousand people yearly in West Africa, resulting in high numbers of Lassa fever (LF), a viral hemorrhagic fever (HF) disease associated with high morbidity and mortality. To date, no licensed vaccines are available to LASV infections, and anti-LASV drug therapy is limited to an off-label use of ribavirin (Rib) that is only partially effective. The development of reverse genetics has provided investigators with a novel and powerful approach for the investigation of the molecular, cell biology, and pathogenesis of LASV. The use of cell-based LASV minigenome (MG) systems has allowed examining the cis- and trans-acting factors involved in genome replication and gene transcription and the identification of novel drugable LASV targets. Likewise, it is now feasible to rescue infectious recombinant (r)LASV entirely from cloned cDNAs containing predetermined mutations in their genomes to investigate virus-host interactions and mechanisms of pathogenesis, as well as to facilitate screens to identify antiviral drugs against LASV and the implementation of novel strategies to develop live-attenuated vaccines (LAV). In this chapter we will summarize the state-of-the-art experimental procedures for implementation of LASV reverse genetics. In addition, we will briefly discuss some significant translational research developments that have been made possible upon the development of LASV reverse genetics.

Reverse genetics in high throughput: rapid generation of complete negative strand RNA virus cDNA clones and recombinant viruses thereof.

PubMed

Nolden, T; Pfaff, F; Nemitz, S; Freuling, C M; Höper, D; Müller, T; Finke, Stefan

2016-04-05

Reverse genetics approaches are indispensable tools for proof of concepts in virus replication and pathogenesis. For negative strand RNA viruses (NSVs) the limited number of infectious cDNA clones represents a bottleneck as clones are often generated from cell culture adapted or attenuated viruses, with limited potential for pathogenesis research. We developed a system in which cDNA copies of complete NSV genomes were directly cloned into reverse genetics vectors by linear-to-linear RedE/T recombination. Rapid cloning of multiple rabies virus (RABV) full length genomes and identification of clones identical to field virus consensus sequence confirmed the approache's reliability. Recombinant viruses were recovered from field virus cDNA clones. Similar growth kinetics of parental and recombinant viruses, preservation of field virus characters in cell type specific replication and virulence in the mouse model were confirmed. Reduced titers after reporter gene insertion indicated that the low level of field virus replication is affected by gene insertions. The flexibility of the strategy was demonstrated by cloning multiple copies of an orthobunyavirus L genome segment. This important step in reverse genetics technology development opens novel avenues for the analysis of virus variability combined with phenotypical characterization of recombinant viruses at a clonal level.

Development of a PCR-Based Reverse Genetics System for an Attenuated Duck Tembusu Virus Strain

PubMed Central

Wu, Xiaogang; Shi, Ying; Yan, Dawei; Li, Xuesong; Yan, Pixi; Gao, Xuyuan; Zhang, Yuee; Yu, Lei; Ren, Chaochao; Li, Guoxin; Yan, Liping; Teng, Qiaoyang; Li, Zejun

2016-01-01

The infectious disease caused by the duck Tembusu virus (DTMUV) has resulted in massive economic losses to the Chinese duck industry in China since 2010. Research on the molecular basis of DTMUV pathogenicity has been hampered by the lack of a reliable reverse genetics system for this virus. Here we developed a PCR-based reverse genetics system with high fidelity for the attenuated DTMUV strain FX2010-180P. The rescued virus was characterized by using both indirect immunofluorescence assays (IFA) and whole genome sequencing. The rescued virus (rFX2010-180P) grew to similar titers as compared with the wild-type virus in DF-1 cells, and had similar replication and immunogenicity properties in ducks. To determine whether exogenous proteins could be expressed from DTMUV, both an internal ribosomal entry site (IRES) and the enhanced green fluorescent protein (eGFP) gene were introduced between the NS5 gene and the 3' non-coding sequence of FX2010-180P. A recombinant DTMUV expressing eGFP was rescued, but eGFP expression was unstable after 4 passages in DF-1 cells due to a deletion of 1,294 nucleotides. The establishment of a reliable reverse genetics system for FX2010-180P provides a foundation for future studies of DTMUV. PMID:27248497

Development of a PCR-Based Reverse Genetics System for an Attenuated Duck Tembusu Virus Strain.

PubMed

Wu, Xiaogang; Shi, Ying; Yan, Dawei; Li, Xuesong; Yan, Pixi; Gao, Xuyuan; Zhang, Yuee; Yu, Lei; Ren, Chaochao; Li, Guoxin; Yan, Liping; Teng, Qiaoyang; Li, Zejun

2016-01-01

The infectious disease caused by the duck Tembusu virus (DTMUV) has resulted in massive economic losses to the Chinese duck industry in China since 2010. Research on the molecular basis of DTMUV pathogenicity has been hampered by the lack of a reliable reverse genetics system for this virus. Here we developed a PCR-based reverse genetics system with high fidelity for the attenuated DTMUV strain FX2010-180P. The rescued virus was characterized by using both indirect immunofluorescence assays (IFA) and whole genome sequencing. The rescued virus (rFX2010-180P) grew to similar titers as compared with the wild-type virus in DF-1 cells, and had similar replication and immunogenicity properties in ducks. To determine whether exogenous proteins could be expressed from DTMUV, both an internal ribosomal entry site (IRES) and the enhanced green fluorescent protein (eGFP) gene were introduced between the NS5 gene and the 3' non-coding sequence of FX2010-180P. A recombinant DTMUV expressing eGFP was rescued, but eGFP expression was unstable after 4 passages in DF-1 cells due to a deletion of 1,294 nucleotides. The establishment of a reliable reverse genetics system for FX2010-180P provides a foundation for future studies of DTMUV.

Genetic screens for mutations affecting development of Xenopus tropicalis.

PubMed

Goda, Tadahiro; Abu-Daya, Anita; Carruthers, Samantha; Clark, Matthew D; Stemple, Derek L; Zimmerman, Lyle B

2006-06-01

We present here the results of forward and reverse genetic screens for chemically-induced mutations in Xenopus tropicalis. In our forward genetic screen, we have uncovered 77 candidate phenotypes in diverse organogenesis and differentiation processes. Using a gynogenetic screen design, which minimizes time and husbandry space expenditures, we find that if a phenotype is detected in the gynogenetic F2 of a given F1 female twice, it is highly likely to be a heritable abnormality (29/29 cases). We have also demonstrated the feasibility of reverse genetic approaches for obtaining carriers of mutations in specific genes, and have directly determined an induced mutation rate by sequencing specific exons from a mutagenized population. The Xenopus system, with its well-understood embryology, fate map, and gain-of-function approaches, can now be coupled with efficient loss-of-function genetic strategies for vertebrate functional genomics and developmental genetics.

Evaluating a common semi-mechanistic mathematical model of gene-regulatory networks

PubMed Central

2015-01-01

Modeling and simulation of gene-regulatory networks (GRNs) has become an important aspect of modern systems biology investigations into mechanisms underlying gene regulation. A key challenge in this area is the automated inference (reverse-engineering) of dynamic, mechanistic GRN models from gene expression time-course data. Common mathematical formalisms for representing such models capture two aspects simultaneously within a single parameter: (1) Whether or not a gene is regulated, and if so, the type of regulator (activator or repressor), and (2) the strength of influence of the regulator (if any) on the target or effector gene. To accommodate both roles, "generous" boundaries or limits for possible values of this parameter are commonly allowed in the reverse-engineering process. This approach has several important drawbacks. First, in the absence of good guidelines, there is no consensus on what limits are reasonable. Second, because the limits may vary greatly among different reverse-engineering experiments, the concrete values obtained for the models may differ considerably, and thus it is difficult to compare models. Third, if high values are chosen as limits, the search space of the model inference process becomes very large, adding unnecessary computational load to the already complex reverse-engineering process. In this study, we demonstrate that restricting the limits to the [−1, +1] interval is sufficient to represent the essential features of GRN systems and offers a reduction of the search space without loss of quality in the resulting models. To show this, we have carried out reverse-engineering studies on data generated from artificial and experimentally determined from real GRN systems. PMID:26356485

Reverse engineering time discrete finite dynamical systems: a feasible undertaking?

PubMed

Delgado-Eckert, Edgar

2009-01-01

With the advent of high-throughput profiling methods, interest in reverse engineering the structure and dynamics of biochemical networks is high. Recently an algorithm for reverse engineering of biochemical networks was developed by Laubenbacher and Stigler. It is a top-down approach using time discrete dynamical systems. One of its key steps includes the choice of a term order, a technicality imposed by the use of Gröbner-bases calculations. The aim of this paper is to identify minimal requirements on data sets to be used with this algorithm and to characterize optimal data sets. We found minimal requirements on a data set based on how many terms the functions to be reverse engineered display. Furthermore, we identified optimal data sets, which we characterized using a geometric property called "general position". Moreover, we developed a constructive method to generate optimal data sets, provided a codimensional condition is fulfilled. In addition, we present a generalization of their algorithm that does not depend on the choice of a term order. For this method we derived a formula for the probability of finding the correct model, provided the data set used is optimal. We analyzed the asymptotic behavior of the probability formula for a growing number of variables n (i.e. interacting chemicals). Unfortunately, this formula converges to zero as fast as , where and . Therefore, even if an optimal data set is used and the restrictions in using term orders are overcome, the reverse engineering problem remains unfeasible, unless prodigious amounts of data are available. Such large data sets are experimentally impossible to generate with today's technologies.

Cranioplasty prosthesis manufacturing based on reverse engineering technology

PubMed Central

Chrzan, Robert; Urbanik, Andrzej; Karbowski, Krzysztof; Moskała, Marek; Polak, Jarosław; Pyrich, Marek

2012-01-01

Summary Background Most patients with large focal skull bone loss after craniectomy are referred for cranioplasty. Reverse engineering is a technology which creates a computer-aided design (CAD) model of a real structure. Rapid prototyping is a technology which produces physical objects from virtual CAD models. The aim of this study was to assess the clinical usefulness of these technologies in cranioplasty prosthesis manufacturing. Material/Methods CT was performed on 19 patients with focal skull bone loss after craniectomy, using a dedicated protocol. A material model of skull deficit was produced using computer numerical control (CNC) milling, and individually pre-operatively adjusted polypropylene-polyester prosthesis was prepared. In a control group of 20 patients a prosthesis was manually adjusted to each patient by a neurosurgeon during surgery, without using CT-based reverse engineering/rapid prototyping. In each case, the prosthesis was implanted into the patient. The mean operating times in both groups were compared. Results In the group of patients with reverse engineering/rapid prototyping-based cranioplasty, the mean operating time was shorter (120.3 min) compared to that in the control group (136.5 min). The neurosurgeons found the new technology particularly useful in more complicated bone deficits with different curvatures in various planes. Conclusions Reverse engineering and rapid prototyping may reduce the time needed for cranioplasty neurosurgery and improve the prosthesis fitting. Such technologies may utilize data obtained by commonly used spiral CT scanners. The manufacturing of individually adjusted prostheses should be commonly used in patients planned for cranioplasty with synthetic material. PMID:22207125

In vitro assessment of the contact mechanics of reverse-engineered distal humeral hemiarthroplasty prostheses.

PubMed

Willing, Ryan; Lapner, Michael; King, Graham J W; Johnson, James A

2014-11-01

Distal humeral hemiarthroplasty alters cartilage contact mechanics, which may predispose to osteoarthritis. Current prostheses do not replicate the native anatomy, and therefore contribute to these changes. We hypothesized that prostheses reverse-engineered from the native bone shape would provide similar contact patterns as the native articulation. Reverse-engineered hemiarthroplasty prostheses were manufactured for five cadaveric elbows based on CT images of the distal humerus. Passive flexion trials with constant muscle forces were performed with the native articulation intact while bone motions were recorded using a motion tracking system. Motion trials were then repeated after the distal humerus was replaced with a corresponding reverse-engineered prosthesis. Contact areas and patterns were reconstructed using computer models created from CT scan images combined with the motion tracker data. The total contact areas, as well as the contact area within smaller sub-regions of the ulna and radius, were analyzed for changes resulting from hemiarthroplasty using repeated-measures ANOVAs. Contact area at the ulna and radius decreased on average 42% (SD 19%, P=.008) and 41% (SD 42%, P=.096), respectively. Contact area decreases were not uniform throughout the different sub-regions, suggesting that contact patterns were also altered. Reverse-engineered prostheses did not reproduce the same contact pattern as the native joints, possibly because the thickness of the distal humerus cartilage layer was neglected when generating the prosthesis shapes or as a consequence of the increased stiffness of the metallic implants. Alternative design strategies and materials for hemiarthroplasty should be considered in future work. Copyright © 2014 Elsevier Ltd. All rights reserved.

Reversible and irreversible heat engine and refrigerator cycles

NASA Astrophysics Data System (ADS)

Leff, Harvey S.

2018-05-01

Although no reversible thermodynamic cycles exist in nature, nearly all cycles covered in textbooks are reversible. This is a review, clarification, and extension of results and concepts for quasistatic, reversible and irreversible processes and cycles, intended primarily for teachers and students. Distinctions between the latter process types are explained, with emphasis on clockwise (CW) and counterclockwise (CCW) cycles. Specific examples of each are examined, including Carnot, Kelvin and Stirling cycles. For the Stirling cycle, potentially useful task-specific efficiency measures are proposed and illustrated. Whether a cycle behaves as a traditional refrigerator or heat engine can depend on whether it is reversible or irreversible. Reversible and irreversible-quasistatic CW cycles both satisfy Carnot's inequality for thermal efficiency, η ≤ η C a r n o t . Irreversible CCW cycles with two reservoirs satisfy the coefficient of performance inequality K ≤ K C a r n o t . However, an arbitrary reversible cycle satisfies K ≥ K C a r n o t when compared with a reversible Carnot cycle operating between its maximum and minimum temperatures, a potentially counterintuitive result.

The ATPG Attack for Reverse Engineering of Combinational Hybrid Custom-Programmable Circuits

DTIC Science & Technology

2017-03-23

The ATPG Attack for Reverse Engineering of Combinational Hybrid Custom- Programmable Circuits Raza Shafiq Hamid Mahmoodi Houman Homayoun Hassan... programmable circuits. While functionality of programmable cells are only known to trusted parties, effective techniques for activation and propagation...of the cells are introduced. The ATPG attack carefully studies dependency of programmable cells to develop their (partial) truth tables. Results

Genetically Engineered Immunotherapy for Advanced Cancer

Cancer.gov

In this trial, doctors will collect T lymphocytes from patients with advanced mesothelin-expressing cancer and genetically engineer them to recognize mesothelin. The gene-engineered cells will be multiplied and infused into the patient to fight the cancer

High Yields of 2,3-Butanediol and Mannitol in Lactococcus lactis through Engineering of NAD+ Cofactor Recycling ▿ †

PubMed Central

Gaspar, Paula; Neves, Ana Rute; Gasson, Michael J.; Shearman, Claire A.; Santos, Helena

2011-01-01

Manipulation of NADH-dependent steps, and particularly disruption of the las-located lactate dehydrogenase (ldh) gene in Lactococcus lactis, is common to engineering strategies envisaging the accumulation of reduced end products other than lactate. Reverse transcription-PCR experiments revealed that three out of the four genes assigned to lactate dehydrogenase in the genome of L. lactis, i.e., the ldh, ldhB, and ldhX genes, were expressed in the parental strain MG1363. Given that genetic redundancy is often a major cause of metabolic instability in engineered strains, we set out to develop a genetically stable lactococcal host tuned for the production of reduced compounds. Therefore, the ldhB and ldhX genes were sequentially deleted in L. lactis FI10089, a strain with a deletion of the ldh gene. The single, double, and triple mutants, FI10089, FI10089ΔldhB, and FI10089ΔldhBΔldhX, showed similar growth profiles and displayed mixed-acid fermentation, ethanol being the main reduced end product. Hence, the alcohol dehydrogenase-encoding gene, the adhE gene, was inactivated in FI10089, but the resulting strain reverted to homolactic fermentation due to induction of the ldhB gene. The three lactate dehydrogenase-deficient mutants were selected as a background for the production of mannitol and 2,3-butanediol. Pathways for the biosynthesis of these compounds were overexpressed under the control of a nisin promoter, and the constructs were analyzed with respect to growth parameters and product yields under anaerobiosis. Glucose was efficiently channeled to mannitol (maximal yield, 42%) or to 2,3-butanediol (maximal yield, 67%). The theoretical yield for 2,3-butanediol was achieved. We show that FI10089ΔldhB is a valuable basis for engineering strategies aiming at the production of reduced compounds. PMID:21841021

Reverse engineering physical models employing a sensor integration between 3D stereo detection and contact digitization

NASA Astrophysics Data System (ADS)

Chen, Liang-Chia; Lin, Grier C. I.

1997-12-01

A vision-drive automatic digitization process for free-form surface reconstruction has been developed, with a coordinate measurement machine (CMM) equipped with a touch-triggered probe and a CCD camera, in reverse engineering physical models. The process integrates 3D stereo detection, data filtering, Delaunay triangulation, adaptive surface digitization into a single process of surface reconstruction. By using this innovative approach, surface reconstruction can be implemented automatically and accurately. Least-squares B- spline surface models with the controlled accuracy of digitization can be generated for further application in product design and manufacturing processes. One industrial application indicates that this approach is feasible, and the processing time required in reverse engineering process can be significantly reduced up to more than 85%.

Mobile Timekeeping Application Built on Reverse-Engineered JPL Infrastructure

NASA Technical Reports Server (NTRS)

Witoff, Robert J.

2013-01-01

Every year, non-exempt employees cumulatively waste over one man-year tracking their time and using the timekeeping Web page to save those times. This app eliminates this waste. The innovation is a native iPhone app. Libraries were built around a reverse- engineered JPL API. It represents a punch-in/punch-out paradigm for timekeeping. It is accessible natively via iPhones, and features ease of access. Any non-exempt employee can natively punch in and out, as well as save and view their JPL timecard. This app is built on custom libraries created by reverse-engineering the standard timekeeping application. Communication is through custom libraries that re-route traffic through BrowserRAS (remote access service). This has value at any center where employees track their time.

Reverse Genetics of Newcastle Disease Virus.

PubMed

Cardenas-Garcia, Stivalis; Afonso, Claudio L

2017-01-01

Reverse genetics allows for the generation of recombinant viruses or vectors used in functional studies, vaccine development, and gene therapy. This technique enables genetic manipulation and cloning of viral genomes, gene mutation through site-directed mutagenesis, along with gene insertion or deletion, among other studies. An in vitro infection-based system including the highly attenuated vaccinia virus Ankara strain expressing the T7 RNA polymerase from bacteriophage T7, with co-transfection of three helper plasmids and a full-length cDNA plasmid, was successfully developed to rescue genetically modified Newcastle disease viruses in 1999. In this chapter, the materials and the methods involved in rescuing Newcastle disease virus (NDV) from cDNA, utilizing site-directed mutagenesis and gene replacement techniques, are described in detail.

Genetic engineering of Ganoderma lucidum for the efficient production of ganoderic acids

PubMed Central

Xu, Jun-Wei; Zhong, Jian-Jiang

2015-01-01

Ganoderma lucidum is a well-known traditional medicinal mushroom that produces ganoderic acids with numerous interesting bioactivities. Genetic engineering is an efficient approach to improve ganoderic acid biosynthesis. However, reliable genetic transformation methods and appropriate genetic manipulation strategies remain underdeveloped and thus should be enhanced. We previously established a homologous genetic transformation method for G. lucidum; we also applied the established method to perform the deregulated overexpression of a homologous 3-hydroxy-3-methyl-glutaryl coenzyme A reductase gene in G. lucidum. Engineered strains accumulated more ganoderic acids than wild-type strains. In this report, the genetic transformation systems of G. lucidum are described; current trends are also presented to improve ganoderic acid production through the genetic manipulation of G. lucidum. PMID:26588475

Sensitivity of Female Inbreds of Cucumis sativus to Sex Reversion by Gibberellin.

PubMed

Shifriss, O; George, W L

1964-03-27

Two female inbred cucumbers were developed by substituting gene Acr for acr in the genetic backgrounds of the monoecious races Marketer and Tokyo, which exhibit weak and strong male tendency respectively. Marketer females are resistant and Tokyo females are sensitive to sex reversion in response to treatments with gibberellin A(3). Resistance and sensitivity of this type appear to depend upon the genetic system which controls sex tendency.

Reverse Genetics for Newcastle Disease Virus as a Vaccine Vector.

PubMed

Kim, Shin-Hee; Samal, Siba K

2018-02-22

Newcastle disease virus (NDV) is an economically important pathogen in the poultry industry worldwide. Recovery of infectious NDV from cDNA using reverse genetics has made it possible to manipulate the genome of NDV. This has greatly contributed to our understanding of the molecular biology and pathogenesis of NDV. Furthermore, NDV has modular genome and accommodates insertion of a foreign gene as a transcriptional unit, thus enabling NDV as a vaccine vector against diseases of humans and animals. Avirulent NDV strains (e.g., LaSota and B1) have been commonly used as vaccine vectors. In this protocol, we have described reverse genetics of NDV to be used as a vaccine vector by exemplifying the recovery of NDV vectored avian influenza virus vaccine. Specifically, cloning and recovery of NDV expressing the hemagglutinin protein of highly pathogenic influenza virus were explained. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

Population genetic structure and direct observations reveal sex-reversed patterns of dispersal in a cooperative bird.

PubMed

Harrison, Xavier A; York, Jennifer E; Young, Andrew J

2014-12-01

Sex-biased dispersal is pervasive and has diverse evolutionary implications, but the fundamental drivers of dispersal sex biases remain unresolved. This is due in part to limited diversity within taxonomic groups in the direction of dispersal sex biases, which leaves hypothesis testing critically dependent upon identifying rare reversals of taxonomic norms. Here, we use a combination of observational and genetic data to demonstrate a rare reversal of the avian sex bias in dispersal in the cooperatively breeding white-browed sparrow weaver (Plocepasser mahali). Direct observations revealed that (i) natal philopatry was rare, with both sexes typically dispersing locally to breed, and (ii), unusually for birds, males bred at significantly greater distances from their natal group than females. Population genetic analyses confirmed these patterns, as (i) corrected Assignment index (AIc), FST tests and isolation-by-distance metrics were all indicative of longer dispersal distances among males than females, and (ii) spatial autocorrelation analysis indicated stronger within-group genetic structure among females than males. Examining the spatial scale of extra-group mating highlighted that the resulting 'sperm dispersal' could have acted in concert with individual dispersal to generate these genetic patterns, but gamete dispersal alone cannot account entirely for the sex differences in genetic structure observed. That leading hypotheses for the evolution of dispersal sex biases cannot readily account for these sex-reversed patterns of dispersal in white-browed sparrow weavers highlights the continued need for attention to alternative explanations for this enigmatic phenomenon. We highlight the potential importance of sex differences in the distances over which dispersal opportunities can be detected. © 2014 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.

Targeted mutagenesis of dengue virus type 2 replicon RNA by yeast in vivo recombination.

PubMed

Manzano, Mark; Padmanabhan, Radhakrishnan

2014-01-01

The use of cDNA infectious clones or subgenomic replicons is indispensable in studying flavivirus biology. Mutating nucleotides or amino acid residues gives important clues to their function in the viral life cycle. However, a major challenge to the establishment of a reverse genetics system for flaviviruses is the instability of their nucleotide sequences in Escherichia coli. Thus, direct cloning using conventional restriction enzyme-based procedures usually leads to unwanted rearrangements of the construct. In this chapter, we discuss a cloning strategy that bypasses traditional cloning procedures. We take advantage of the observations from previous studies that (1) unstable sequences in bacteria can be cloned in eukaryotic systems and (2) Saccharomyces cerevisiae has a well-studied genetics system to introduce sequences using homologous recombination. We describe a protocol to perform targeted mutagenesis in a subgenomic dengue virus 2 replicon. Our method makes use of homologous recombination in yeast using a linearized replicon and a PCR product containing the desired mutation. Constructs derived from this method can be propagated in E. coli with improved stability. Thus, yeast in vivo recombination provides an excellent strategy to genetically engineer flavivirus infectious clones or replicons because this system is compatible with inherently unstable sequences of flaviviruses and is not restricted by the limitations of traditional cloning procedures.

Genetically engineered mouse models of melanoma.

PubMed

Pérez-Guijarro, Eva; Day, Chi-Ping; Merlino, Glenn; Zaidi, M Raza

2017-06-01

Melanoma is a complex disease that exhibits highly heterogeneous etiological, histopathological, and genetic features, as well as therapeutic responses. Genetically engineered mouse (GEM) models provide powerful tools to unravel the molecular mechanisms critical for melanoma development and drug resistance. Here, we expound briefly the basis of the mouse modeling design, the available technology for genetic engineering, and the aspects influencing the use of GEMs to model melanoma. Furthermore, we describe in detail the currently available GEM models of melanoma. Cancer 2017;123:2089-103. © 2017 American Cancer Society. © 2017 American Cancer Society.

Testing the Role of p21 Activated Kinases in Schwannoma Formation Using a Novel Genetically Engineered Murine Model that Closely Phenocopies Human NF2 Disease

DTIC Science & Technology

2017-06-01

Kinases in Schwannoma Formation Using a Novel Genetically Engineered Murine Model that Closely Phenocopies Human NF2 Disease The views, opinions and...Role of p21 Activated Kinases in Schwannoma Formation Using a Novel Genetically Engineered Murine Model that Closely Phenocopies Human NF2 Disease Form...NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. The major goal of this research project was to genetically and pharmacologically test the requirement of PAK

Building a Genome Engineering Toolbox in Non-Model Prokaryotic Microbes

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

Eckert, Carrie A; Freed, Emily; Smolinski, Sharon

The realization of a sustainable bioeconomy requires our ability to understand and engineer complex design principles for the development of platform organisms capable of efficient conversion of cheap and sustainable feedstocks (e.g. sunlight, CO2, non-food biomass) to biofuels and bioproducts at sufficient titers and costs. For model microbes such as E. coli, advances in DNA reading and writing technologies are driving adoption of new paradigms for engineering biological systems. Unfortunately, microbes with properties of interest for the utilization of cheap and renewable feedstocks such as photosynthesis, autotrophic growth, and cellulose degradation have very few, if any, genetic tools for metabolicmore » engineering. Therefore, it is important to begin to develop 'design rules' for building a genetic toolbox for novel microbes. Here, we present an overview of our current understanding of these rules for the genetic manipulation of prokaryotic microbes and available genetic tools to expand our ability to genetically engineer non-model systems.« less

Reproductive cloning, genetic engineering and the autonomy of the child: the moral agent and the open future.

PubMed

Mameli, M

2007-02-01

Some authors have argued that the human use of reproductive cloning and genetic engineering should be prohibited because these biotechnologies would undermine the autonomy of the resulting child. In this paper, two versions of this view are discussed. According to the first version, the autonomy of cloned and genetically engineered people would be undermined because knowledge of the method by which these people have been conceived would make them unable to assume full responsibility for their actions. According to the second version, these biotechnologies would undermine autonomy by violating these people's right to an open future. There is no evidence to show that people conceived through cloning and genetic engineering would inevitably or even in general be unable to assume responsibility for their actions; there is also no evidence for the claim that cloning and genetic engineering would inevitably or even in general rob the child of the possibility to choose from a sufficiently large array of life plans.

Reproductive cloning, genetic engineering and the autonomy of the child: the moral agent and the open future

PubMed Central

Mameli, M

2007-01-01

Some authors have argued that the human use of reproductive cloning and genetic engineering should be prohibited because these biotechnologies would undermine the autonomy of the resulting child. In this paper, two versions of this view are discussed. According to the first version, the autonomy of cloned and genetically engineered people would be undermined because knowledge of the method by which these people have been conceived would make them unable to assume full responsibility for their actions. According to the second version, these biotechnologies would undermine autonomy by violating these people's right to an open future. There is no evidence to show that people conceived through cloning and genetic engineering would inevitably or even in general be unable to assume responsibility for their actions; there is also no evidence for the claim that cloning and genetic engineering would inevitably or even in general rob the child of the possibility to choose from a sufficiently large array of life plans. PMID:17264194

Current Progress of Genetically Engineered Pig Models for Biomedical Research

PubMed Central

Gün, Gökhan

2014-01-01

Abstract The first transgenic pigs were generated for agricultural purposes about three decades ago. Since then, the micromanipulation techniques of pig oocytes and embryos expanded from pronuclear injection of foreign DNA to somatic cell nuclear transfer, intracytoplasmic sperm injection-mediated gene transfer, lentiviral transduction, and cytoplasmic injection. Mechanistically, the passive transgenesis approach based on random integration of foreign DNA was developed to active genetic engineering techniques based on the transient activity of ectopic enzymes, such as transposases, recombinases, and programmable nucleases. Whole-genome sequencing and annotation of advanced genome maps of the pig complemented these developments. The full implementation of these tools promises to immensely increase the efficiency and, in parallel, to reduce the costs for the generation of genetically engineered pigs. Today, the major application of genetically engineered pigs is found in the field of biomedical disease modeling. It is anticipated that genetically engineered pigs will increasingly be used in biomedical research, since this model shows several similarities to humans with regard to physiology, metabolism, genome organization, pathology, and aging. PMID:25469311

Building a genome engineering toolbox in nonmodel prokaryotic microbes.

PubMed

Freed, Emily; Fenster, Jacob; Smolinski, Sharon L; Walker, Julie; Henard, Calvin A; Gill, Ryan; Eckert, Carrie A

2018-05-11

The realization of a sustainable bioeconomy requires our ability to understand and engineer complex design principles for the development of platform organisms capable of efficient conversion of cheap and sustainable feedstocks (e.g., sunlight, CO 2 , and nonfood biomass) into biofuels and bioproducts at sufficient titers and costs. For model microbes, such as Escherichia coli, advances in DNA reading and writing technologies are driving the adoption of new paradigms for engineering biological systems. Unfortunately, microbes with properties of interest for the utilization of cheap and renewable feedstocks, such as photosynthesis, autotrophic growth, and cellulose degradation, have very few, if any, genetic tools for metabolic engineering. Therefore, it is important to develop "design rules" for building a genetic toolbox for novel microbes. Here, we present an overview of our current understanding of these rules for the genetic manipulation of prokaryotic microbes and the available genetic tools to expand our ability to genetically engineer nonmodel systems. © 2018 Wiley Periodicals, Inc.

Lactococcus lactis As a Versatile Vehicle for Tolerogenic Immunotherapy

PubMed Central

Cook, Dana P.; Gysemans, Conny; Mathieu, Chantal

2018-01-01

Genetically modified Lactococcus lactis bacteria have been engineered as a tool to deliver bioactive proteins to mucosal tissues as a means to exert both local and systemic effects. They have an excellent safety profile, the result of years of human consumption in the food industry, as well as a lack of toxicity and immunogenicity. Also, containment strategies have been developed to promote further application as clinical protein-based therapeutics. Here, we review technological advancements made to enhanced the potential of L. lactis as live biofactories and discuss some examples of tolerogenic immunotherapies mediated by mucosal drug delivery via L. lactis. Additionally, we highlight their use to induce mucosal tolerance by targeted autoantigen delivery to the intestine as an approach to reverse autoimmune type 1 diabetes. PMID:29387056

Metabolic Engineering of Oleaginous Yeasts for Production of Fuels and Chemicals.

PubMed

Shi, Shuobo; Zhao, Huimin

2017-01-01

Oleaginous yeasts have been increasingly explored for production of chemicals and fuels via metabolic engineering. Particularly, there is a growing interest in using oleaginous yeasts for the synthesis of lipid-related products due to their high lipogenesis capability, robustness, and ability to utilize a variety of substrates. Most of the metabolic engineering studies in oleaginous yeasts focused on Yarrowia that already has plenty of genetic engineering tools. However, recent advances in systems biology and synthetic biology have provided new strategies and tools to engineer those oleaginous yeasts that have naturally high lipid accumulation but lack genetic tools, such as Rhodosporidium , Trichosporon , and Lipomyces . This review highlights recent accomplishments in metabolic engineering of oleaginous yeasts and recent advances in the development of genetic engineering tools in oleaginous yeasts within the last 3 years.

[Progress of research on genetic engineering antibody and its application in prevention and control of parasitic diseases].

PubMed

Yao, Yuan; Yu, Chuan-xin

2013-08-01

Antibody has extensive application prospects in the biomedical field. The inherent disadvantages of traditional polyclonal antibody and monoclonal antibody limit their application values. The humanized and fragmented antibody remodeling has given a rise to a series of genetic engineered antibody variant. This paper reviews the progress of research on genetic engineering antibody and its application in prevention and control of parasitic diseases.

Field performance of a genetically engineered strain of pink bollworm.

PubMed

Simmons, Gregory S; McKemey, Andrew R; Morrison, Neil I; O'Connell, Sinead; Tabashnik, Bruce E; Claus, John; Fu, Guoliang; Tang, Guolei; Sledge, Mickey; Walker, Adam S; Phillips, Caroline E; Miller, Ernie D; Rose, Robert I; Staten, Robert T; Donnelly, Christl A; Alphey, Luke

2011-01-01

Pest insects harm crops, livestock and human health, either directly or by acting as vectors of disease. The Sterile Insect Technique (SIT)--mass-release of sterile insects to mate with, and thereby control, their wild counterparts--has been used successfully for decades to control several pest species, including pink bollworm, a lepidopteran pest of cotton. Although it has been suggested that genetic engineering of pest insects provides potential improvements, there is uncertainty regarding its impact on their field performance. Discrimination between released and wild moths caught in monitoring traps is essential for estimating wild population levels. To address concerns about the reliability of current marking methods, we developed a genetically engineered strain of pink bollworm with a heritable fluorescent marker, to improve discrimination of sterile from wild moths. Here, we report the results of field trials showing that this engineered strain performed well under field conditions. Our data show that attributes critical to SIT in the field--ability to find a mate and to initiate copulation, as well as dispersal and persistence in the release area--were comparable between the genetically engineered strain and a standard strain. To our knowledge, these represent the first open-field experiments with a genetically engineered insect. The results described here provide encouragement for the genetic control of insect pests.

Energy Efficient Engine program advanced turbofan nacelle definition study

NASA Technical Reports Server (NTRS)

Howe, David C.; Wynosky, T. A.

1985-01-01

Advanced, low drag, nacelle configurations were defined for some of the more promising propulsion systems identified in the earlier Benefit/Cost Study, to assess the benefits associated with these advanced technology nacelles and formulate programs for developing these nacelles and low volume thrust reversers/spoilers to a state of technology readiness in the early 1990's. The study results established the design feasibility of advanced technology, slim line nacelles applicable to advanced technology, high bypass ratio turbofan engines. Design feasibility was also established for two low volume thrust reverse/spoiler concepts that meet or exceed the required effectiveness for these engines. These nacelle and thrust reverse/spoiler designs were shown to be applicable in engines with takeoff thrust sizes ranging from 24,000 to 60,000 pounds. The reduced weight, drag, and cost of the advanced technology nacelle installations relative to current technology nacelles offer a mission fuel burn savings ranging from 3.0 to 4.5 percent and direct operating cost plus interest improvements from 1.6 to 2.2 percent.

Using Genetically Engineered Animal Models in the Postgenomic Era to Understand Gene Function in Alcoholism

PubMed Central

Reilly, Matthew T.; Harris, R. Adron; Noronha, Antonio

2012-01-01

Over the last 50 years, researchers have made substantial progress in identifying genetic variations that underlie the complex phenotype of alcoholism. Not much is known, however, about how this genetic variation translates into altered biological function. Genetic animal models recapitulating specific characteristics of the human condition have helped elucidate gene function and the genetic basis of disease. In particular, major advances have come from the ability to manipulate genes through a variety of genetic technologies that provide an unprecedented capacity to determine gene function in the living organism and in alcohol-related behaviors. Even newer genetic-engineering technologies have given researchers the ability to control when and where a specific gene or mutation is activated or deleted, allowing investigators to narrow the role of the gene’s function to circumscribed neural pathways and across development. These technologies are important for all areas of neuroscience, and several public and private initiatives are making a new generation of genetic-engineering tools available to the scientific community at large. Finally, high-throughput “next-generation sequencing” technologies are set to rapidly increase knowledge of the genome, epigenome, and transcriptome, which, combined with genetically engineered mouse mutants, will enhance insight into biological function. All of these resources will provide deeper insight into the genetic basis of alcoholism. PMID:23134044

Using genetically engineered animal models in the postgenomic era to understand gene function in alcoholism.

PubMed

Reilly, Matthew T; Harris, R Adron; Noronha, Antonio

2012-01-01

Over the last 50 years, researchers have made substantial progress in identifying genetic variations that underlie the complex phenotype of alcoholism. Not much is known, however, about how this genetic variation translates into altered biological function. Genetic animal models recapitulating specific characteristics of the human condition have helped elucidate gene function and the genetic basis of disease. In particular, major advances have come from the ability to manipulate genes through a variety of genetic technologies that provide an unprecedented capacity to determine gene function in the living organism and in alcohol-related behaviors. Even newer genetic-engineering technologies have given researchers the ability to control when and where a specific gene or mutation is activated or deleted, allowing investigators to narrow the role of the gene's function to circumscribed neural pathways and across development. These technologies are important for all areas of neuroscience, and several public and private initiatives are making a new generation of genetic-engineering tools available to the scientific community at large. Finally, high-throughput "next-generation sequencing" technologies are set to rapidly increase knowledge of the genome, epigenome, and transcriptome, which, combined with genetically engineered mouse mutants, will enhance insight into biological function. All of these resources will provide deeper insight into the genetic basis of alcoholism.

CDKN2B loss promotes progression from benign melanocytic nevus to melanoma

PubMed Central

McNeal, Andrew S.; Liu, Kevin; Nakhate, Vihang; Natale, Christopher A.; Duperret, Elizabeth K.; Capell, Brian C.; Dentchev, Tzvete; Berger, Shelley L.; Herlyn, Meenhard; Seykora, John T.; Ridky, Todd W.

2015-01-01

Deletion of the entire CDKN2B-CDKN2A gene cluster is among the most common genetic events in cancer. The tumor-promoting effects are generally attributed to loss of CDKN2A-encoded p16 and p14ARF tumor suppressors. The degree to which the associated CDKN2B-encoded p15 loss contributes to human tumorigenesis is unclear. Here we show that CDKN2B is highly upregulated in benign melanocytic nevi, contributes to maintaining nevus melanocytes in a growth-arrested premalignant state, and is commonly lost in melanoma. Using primary melanocytes isolated directly from freshly excised human nevi naturally expressing the common BRAF(V600E) activating mutation, nevi progressing to melanoma, and normal melanocytes engineered to inducibly express BRAF(V600E), we show that BRAF activation results in reversible, TGFβ-dependent, p15 induction that halts proliferation. Further, we engineer human skin grafts containing nevus-derived melanocytes to establish a new, architecturally faithful, in vivo melanoma model, and demonstrate that p15 loss promotes the transition from benign nevus to melanoma. PMID:26183406

Photoactivable antibody binding protein: site-selective and covalent coupling of antibody.

PubMed

Jung, Yongwon; Lee, Jeong Min; Kim, Jung-won; Yoon, Jeongwon; Cho, Hyunmin; Chung, Bong Hyun

2009-02-01

Here we report new photoactivable antibody binding proteins, which site-selectively capture antibodies and form covalent conjugates with captured antibodies upon irradiation. The proteins allow the site-selective tagging and/or immobilization of antibodies with a highly preferred orientation and omit the need for prior antibody modifications. The minimal Fc-binding domain of protein G, a widely used antibody binding protein, was genetically and chemically engineered to contain a site-specific photo cross-linker, benzophenone. In addition, the domain was further mutated to have an enhanced Fc-targeting ability. This small engineered protein was successfully cross-linked only to the Fc region of the antibody without any nonspecific reactivity. SPR analysis indicated that antibodies can be site-selectively biotinylated through the present photoactivable protein. Furthermore, the system enabled light-induced covalent immobilization of antibodies directly on various solid surfaces, such as those of glass slides, gold chips, and small particles. Antibody coupling via photoactivable antibody binding proteins overcomes several limitations of conventional approaches, such as random chemical reactions or reversible protein binding, and offers a versatile tool for the field of immunosensors.

Anti-proliferative Effect of Engineered Neural Stem Cells Expressing Cytosine Deaminase and Interferon-β against Lymph Node–Derived Metastatic Colorectal Adenocarcinoma in Cellular and Xenograft Mouse Models

PubMed Central

Park, Geon-Tae; Kim, Seung U.; Choi, Kyung-Chul

2017-01-01

Purpose Genetically engineered stem cells may be advantageous for gene therapy against various human cancers due to their inherent tumor-tropic properties. In this study, genetically engineered human neural stem cells (HB1.F3) expressing Escherichia coli cytosine deaminase (CD) (HB1.F3.CD) and human interferon-β (IFN-β) (HB1.F3.CD.IFN-β) were employed against lymph node–derived metastatic colorectal adenocarcinoma. Materials and Methods CD can convert a prodrug, 5-fluorocytosine (5-FC), to active 5-fluorouracil, which inhibits tumor growth through the inhibition of DNA synthesis,while IFN-β also strongly inhibits tumor growth by inducing the apoptotic process. In reverse transcription polymerase chain reaction analysis, we confirmed that HB1.F3.CD cells expressed the CD gene and HB1.F3.CD.IFN-β cells expressed both CD and IFN-β genes. Results In results of a modified trans-well migration assay, HB1.F3.CD and HB1.F3.CD.IFN-β cells selectively migrated toward SW-620, human lymph node–derived metastatic colorectal adenocarcinoma cells. The viability of SW-620 cells was significantly reduced when co-cultured with HB1.F3.CD or HB1.F3.CD.IFN-β cells in the presence of 5-FC. In addition, it was found that the tumor-tropic properties of these engineered human neural stem cells (hNSCs) were attributed to chemoattractant molecules including stromal cell-derived factor 1, c-Kit, urokinase receptor, urokinase-type plasminogen activator, and C-C chemokine receptor type 2 secreted by SW-620 cells. In a xenograft mouse model, treatment with hNSC resulted in significantly inhibited growth of the tumor mass without virulent effects on the animals. Conclusion The current results indicate that engineered hNSCs and a prodrug treatment inhibited the growth of SW-620 cells. Therefore, hNSC therapy may be a clinically effective tool for the treatment of lymph node metastatic colorectal cancer. PMID:27188205

The results of a low-speed wind tunnel test to investigate the effects of the Refan JT8D engine target thrust reverser on the stability and control characteristics of the Boeing 727-200 airplane

NASA Technical Reports Server (NTRS)

Kupcis, E. A.

1974-01-01

The effects of the Refan JT8D side engine target thrust reverser on the stability and control characteristics of the Boeing 727-200 airplane were investigated using the Boeing-Vertol 20 x 20 ft Low-Speed Wind Tunnel. A powered model of the 727-200 was tested in groud effect in the landing configuration. The Refan target reverser configuration was evaluated relative to the basic production 727 airplane with its clamshell-deflector door thrust reverser design. The Refan configuration had slightly improved directional control characteristics relative to the basic airplane. Clocking the Refan thrust reversers 20 degrees outboard to direct the reverser flow away from the vertical tail, had little effect on directional control. However, clocking them 20 degrees inboard resulted in a complete loss of rudder effectiveness for speeds greater than 90 knots. Variations in Refan reverser lip/fence geometry had a minor effect on directional control.

Genetically modified "obligate" anaerobic Salmonella typhimurium as a therapeutic strategy for neuroblastoma.

PubMed

Guo, Zhu-Ling; Yu, Bin; Ning, Bo-Tao; Chan, Shing; Lin, Qiu-Bin; Li, James Chun-Bong; Huang, Jian-Dong; Chan, Godfrey Chi-Fung

2015-08-19

Neuroblastoma currently has poor prognosis, therefore we proposed a new strategy by targeting neuroblastoma with genetically engineered anaerobic Salmonella (Sal-YB1). Nude and nonobese diabetic-severe combined immunodeficiency (NOD-SCID) orthotopic mouse models were used, and Sal-YB1 was administered via tail vein. The therapeutic effectiveness, bio-safety, and mechanisms were studied. No mice died of therapy-related complications. Tumor size reduction was 70 and 30% in nude and NOD-SCID mice, respectively. No Salmonella was detected in the urine; 75% mice had positive stool culture if diaminopimelic acid was added, but all turned negative subsequently. Tumor tissues had more Sal-YB1 infiltration, necrosis, and shrinkage in Sal-YB1-treated mice. Significantly higher expression of TLR4, TNF-stimulated gene 6 protein (TSG6), and cleaved caspase 1, 3, 8, and 9 was found in the tumor masses of the Sal-YB1-treated group with a decrease of interleukin 1 receptor-associated kinase (IRAK) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα). There was a high release of TNFα both in human macrophages and mouse tumor tissues with Sal-YB1 treatment. The antitumor effect of the supernatant derived from macrophages treated with Sal-YB1 could be reversed with TNFα and pan-caspase inhibitors. This new approach in targeting neuroblastoma by bio-engineered Salmonella with the assistance of macrophages indirectly may have a clinical therapeutic impact in the future.

Forward and reverse mutagenesis in C. elegans

PubMed Central

Kutscher, Lena M.; Shaham, Shai

2014-01-01

Mutagenesis drives natural selection. In the lab, mutations allow gene function to be deciphered. C. elegans is highly amendable to functional genetics because of its short generation time, ease of use, and wealth of available gene-alteration techniques. Here we provide an overview of historical and contemporary methods for mutagenesis in C. elegans, and discuss principles and strategies for forward (genome-wide mutagenesis) and reverse (target-selected and gene-specific mutagenesis) genetic studies in this animal. PMID:24449699

An assessment of heavy ion irradiation mutagenesis for reverse genetics in wheat (Triticum aestivum L.).

PubMed

Fitzgerald, Timothy L; Powell, Jonathan J; Stiller, Jiri; Weese, Terri L; Abe, Tomoko; Zhao, Guangyao; Jia, Jizeng; McIntyre, C Lynne; Li, Zhongyi; Manners, John M; Kazan, Kemal

2015-01-01

Reverse genetic techniques harnessing mutational approaches are powerful tools that can provide substantial insight into gene function in plants. However, as compared to diploid species, reverse genetic analyses in polyploid plants such as bread wheat can present substantial challenges associated with high levels of sequence and functional similarity amongst homoeologous loci. We previously developed a high-throughput method to identify deletions of genes within a physically mutagenized wheat population. Here we describe our efforts to combine multiple homoeologous deletions of three candidate disease susceptibility genes (TaWRKY11, TaPFT1 and TaPLDß1). We were able to produce lines featuring homozygous deletions at two of the three homoeoloci for all genes, but this was dependent on the individual mutants used in crossing. Intriguingly, despite extensive efforts, viable lines possessing homozygous deletions at all three homoeoloci could not be produced for any of the candidate genes. To investigate deletion size as a possible reason for this phenomenon, we developed an amplicon sequencing approach based on synteny to Brachypodium distachyon to assess the size of the deletions removing one candidate gene (TaPFT1) in our mutants. These analyses revealed that genomic deletions removing the locus are relatively large, resulting in the loss of multiple additional genes. The implications of this work for the use of heavy ion mutagenesis for reverse genetic analyses in wheat are discussed.

An Assessment of Heavy Ion Irradiation Mutagenesis for Reverse Genetics in Wheat (Triticum aestivum L.)

PubMed Central

Fitzgerald, Timothy L.; Powell, Jonathan J.; Stiller, Jiri; Weese, Terri L.; Abe, Tomoko; Zhao, Guangyao; Jia, Jizeng; McIntyre, C. Lynne; Li, Zhongyi; Manners, John M.; Kazan, Kemal

2015-01-01

Reverse genetic techniques harnessing mutational approaches are powerful tools that can provide substantial insight into gene function in plants. However, as compared to diploid species, reverse genetic analyses in polyploid plants such as bread wheat can present substantial challenges associated with high levels of sequence and functional similarity amongst homoeologous loci. We previously developed a high-throughput method to identify deletions of genes within a physically mutagenized wheat population. Here we describe our efforts to combine multiple homoeologous deletions of three candidate disease susceptibility genes (TaWRKY11, TaPFT1 and TaPLDß1). We were able to produce lines featuring homozygous deletions at two of the three homoeoloci for all genes, but this was dependent on the individual mutants used in crossing. Intriguingly, despite extensive efforts, viable lines possessing homozygous deletions at all three homoeoloci could not be produced for any of the candidate genes. To investigate deletion size as a possible reason for this phenomenon, we developed an amplicon sequencing approach based on synteny to Brachypodium distachyon to assess the size of the deletions removing one candidate gene (TaPFT1) in our mutants. These analyses revealed that genomic deletions removing the locus are relatively large, resulting in the loss of multiple additional genes. The implications of this work for the use of heavy ion mutagenesis for reverse genetic analyses in wheat are discussed. PMID:25719507

Recovery of infectious classical swine fever virus (CSFV) from full-length genomic cDNA clones by a swine kidney cell line expressing bacteriophage T7 RNA polymerase.

PubMed

van Gennip, H G; van Rijn, P A; Widjojoatmodjo, M N; Moormann, R J

1999-03-01

A new method for the recovery of infectious classical swine fever virus (CSFV) from full-length genomic cDNA clones of the C-strain was developed. Classical reverse genetics is based on transfection of in vitro transcribed RNA to target cells to recover RNA viruses. However, the specific infectivity of such in vitro transcribed RNA in swine kidney cells is usually low. To improve reverse genetics for CSFV, a stable swine kidney cell line was established that expresses cytoplasmic bacteriophage T7 RNA polymerase (SK6.T7). A 200-fold increased virus titre was obtained from SK6.T7 cells transfected with linearized full-length cDNA compared to in vitro transcribed RNA, whereas transfection of circular full-length cDNA resulted in 20-fold increased virus titres. Viruses generated on the SK6.T7 cells are indistinguishable from the viruses generated by the classical reverse genetic procedures. These results show the improved recovery of infectious CSFV directly from full-length cDNAs. Furthermore, the reverse genetic procedures are simplified to a faster, one step protocol. We conclude that the SK6.T7 cell line will be a valuable tool for recovering mutant CSFV and will contribute to future pestivirus research.

Controlling transgene expression in subcutaneous implants using a skin lotion containing the apple metabolite phloretin.

PubMed

Gitzinger, Marc; Kemmer, Christian; El-Baba, Marie Daoud; Weber, Wilfried; Fussenegger, Martin

2009-06-30

Adjustable control of therapeutic transgenes in engineered cell implants after transdermal and topical delivery of nontoxic trigger molecules would increase convenience, patient compliance, and elimination of hepatic first-pass effect in future therapies. Pseudomonas putida DOT-T1E has evolved the flavonoid-triggered TtgR operon, which controls expression of a multisubstrate-specific efflux pump (TtgABC) to resist plant-derived defense metabolites in its rhizosphere habitat. Taking advantage of the TtgR operon, we have engineered a hybrid P. putida-mammalian genetic unit responsive to phloretin. This flavonoid is contained in apples, and, as such, or as dietary supplement, regularly consumed by humans. The engineered mammalian phloretin-adjustable control element (PEACE) enabled adjustable and reversible transgene expression in different mammalian cell lines and primary cells. Due to the short half-life of phloretin in culture, PEACE could also be used to program expression of difficult-to-produce protein therapeutics during standard bioreactor operation. When formulated in skin lotions and applied to the skin of mice harboring transgenic cell implants, phloretin was able to fine-tune target genes and adjust heterologous protein levels in the bloodstream of treated mice. PEACE-controlled target gene expression could foster advances in biopharmaceutical manufacturing as well as gene- and cell-based therapies.

Controlling transgene expression in subcutaneous implants using a skin lotion containing the apple metabolite phloretin

PubMed Central

Gitzinger, Marc; Kemmer, Christian; El-Baba, Marie Daoud; Weber, Wilfried; Fussenegger, Martin

2009-01-01

Adjustable control of therapeutic transgenes in engineered cell implants after transdermal and topical delivery of nontoxic trigger molecules would increase convenience, patient compliance, and elimination of hepatic first-pass effect in future therapies. Pseudomonas putida DOT-T1E has evolved the flavonoid-triggered TtgR operon, which controls expression of a multisubstrate-specific efflux pump (TtgABC) to resist plant-derived defense metabolites in its rhizosphere habitat. Taking advantage of the TtgR operon, we have engineered a hybrid P. putida–mammalian genetic unit responsive to phloretin. This flavonoid is contained in apples, and, as such, or as dietary supplement, regularly consumed by humans. The engineered mammalian phloretin-adjustable control element (PEACE) enabled adjustable and reversible transgene expression in different mammalian cell lines and primary cells. Due to the short half-life of phloretin in culture, PEACE could also be used to program expression of difficult-to-produce protein therapeutics during standard bioreactor operation. When formulated in skin lotions and applied to the skin of mice harboring transgenic cell implants, phloretin was able to fine-tune target genes and adjust heterologous protein levels in the bloodstream of treated mice. PEACE-controlled target gene expression could foster advances in biopharmaceutical manufacturing as well as gene- and cell-based therapies. PMID:19549857

Virus resistant plums through genetic engineering - from lab to market

USDA-ARS?s Scientific Manuscript database

Genetic engineering (GE) has the potential to revolutionize the genetic improvement of fruit trees and other specialty crops, to provide greater flexibility and speed in responding to changes in climate, production systems and market demands, and to maintain the competitiveness of American agricultu...

A Survey of Statistical Models for Reverse Engineering Gene Regulatory Networks

PubMed Central

Huang, Yufei; Tienda-Luna, Isabel M.; Wang, Yufeng

2009-01-01

Statistical models for reverse engineering gene regulatory networks are surveyed in this article. To provide readers with a system-level view of the modeling issues in this research, a graphical modeling framework is proposed. This framework serves as the scaffolding on which the review of different models can be systematically assembled. Based on the framework, we review many existing models for many aspects of gene regulation; the pros and cons of each model are discussed. In addition, network inference algorithms are also surveyed under the graphical modeling framework by the categories of point solutions and probabilistic solutions and the connections and differences among the algorithms are provided. This survey has the potential to elucidate the development and future of reverse engineering GRNs and bring statistical signal processing closer to the core of this research. PMID:20046885

A Genetically Engineered Mouse Model of Neuroblastoma Driven by Mutated ALK and MYCN

DTIC Science & Technology

2014-09-01

AWARD NUMBER: W81XWH-13-1-0220 TITLE: A Genetically Engineered Mouse Model of Neuroblastoma ...CONTRACT NUMBER A Genetically Engineered Mouse Model of Neuroblastoma Driven by Mutated ALK and MYCN 5b. GRANT NUMBER W81XWH-13-1-0220 5c...common ALK mutations in neuroblastoma , F1174L and R1275Q. We have determined that in tumors cells expressing mutated ALK, different downstream

Genetically Engineered Humanized Mouse Models for Preclinical Antibody Studies

PubMed Central

Proetzel, Gabriele; Wiles, Michael V.; Roopenian, Derry C.

2015-01-01

The use of genetic engineering has vastly improved our capabilities to create animal models relevant in preclinical research. With the recent advances in gene-editing technologies, it is now possible to very rapidly create highly tunable mouse models as needs arise. Here, we provide an overview of genetic engineering methods, as well as the development of humanized neonatal Fc receptor (FcRn) models and their use for monoclonal antibody in vivo studies. PMID:24150980

Developing Novel Therapeutic Approaches in Small Cell Lung Carcinoma Using Genetically Engineered Mouse Models and Human Circulating Tumor Cells

DTIC Science & Technology

2014-10-01

AD_________________ Award Number: W81XWH-13-1-0325 TITLE: Developing Novel Therapeutic Approaches in Small Cell Lung Carcinoma Using ...Genetically Engineered Mouse Models and Human Circulating Tumor Cells PRINCIPAL INVESTIGATOR: Jeffrey Engelman MD PhD CONTRACTING ORGANIZATION ...Novel Therapeutic Approaches in Small Cell Lung 5a. CONTRACT NUMBER W81XWH-13-1-0325 Carcinoma Using Genetically Engineered Mouse Models and 5b

Genetic engineering: a matter that requires further refinement in Spanish secondary school textbooks

NASA Astrophysics Data System (ADS)

Martínez-Gracia, M. V.; Gil-Quýlez, M. J.

2003-09-01

Genetic engineering is now an integral part of many high school textbooks but little work has been done to assess whether it is being properly addressed. A checklist with 19 items was used to analyze how genetic engineering is presented in biology textbooks commonly used in Spanish high schools, including the content, its relationship with fundamental genetic principles, and how it aims to improve the genetic literacy of students. The results show that genetic engineering was normally introduced without a clear reference to the universal genetic code, protein expression or the genetic material shared by all species. In most cases it was poorly defined, without a clear explanation of all the relevant processes involved. Some procedures (such as vectors) were explained in detail without considering previous student knowledge or skills. Some books emphasized applications such as the human genome project without describing DNA sequencing. All books included possible repercussions, but in most cases only fashionable topics such as human cloning. There was an excess of information that was not always well founded and hence was unsuitable to provide a meaningful understanding of DNA technology required for citizens in the twenty-first century.

Genetic diversity in natural populations of a soil bacterium across a landscape gradient

PubMed Central

McArthur, J. Vaun; Kovacic, David A.; Smith, Michael H.

1988-01-01

Genetic diversity in natural populations of the bacterium Pseudomonas cepacia was surveyed in 10 enzymes from 70 clones isolated along a landscape gradient. Estimates of genetic diversity, ranging from 0.54 to 0.70, were higher than any previously reported values of which we are aware and were positively correlated with habitat variability. Patterns of bacterial genetic diversity were correlated with habitat variability. Findings indicate that the source of strains used in genetic engineering will greatly affect the outcome of planned releases in variable environments. Selection of generalist strains may confer a large advantage to engineered populations, while selection of laboratory strains may result in quick elimination of the engineered strains. PMID:16594009

Metabolic Engineering of Oleaginous Yeasts for Production of Fuels and Chemicals

PubMed Central

Shi, Shuobo; Zhao, Huimin

2017-01-01

Oleaginous yeasts have been increasingly explored for production of chemicals and fuels via metabolic engineering. Particularly, there is a growing interest in using oleaginous yeasts for the synthesis of lipid-related products due to their high lipogenesis capability, robustness, and ability to utilize a variety of substrates. Most of the metabolic engineering studies in oleaginous yeasts focused on Yarrowia that already has plenty of genetic engineering tools. However, recent advances in systems biology and synthetic biology have provided new strategies and tools to engineer those oleaginous yeasts that have naturally high lipid accumulation but lack genetic tools, such as Rhodosporidium, Trichosporon, and Lipomyces. This review highlights recent accomplishments in metabolic engineering of oleaginous yeasts and recent advances in the development of genetic engineering tools in oleaginous yeasts within the last 3 years. PMID:29167664

Genetically engineering milk.

PubMed

Whitelaw, C Bruce A; Joshi, Akshay; Kumar, Satish; Lillico, Simon G; Proudfoot, Chris

2016-02-01

It has been thirty years since the first genetically engineered animal with altered milk composition was reported. During the intervening years, the world population has increased from 5bn to 7bn people. An increasing demand for protein in the human diet has followed this population expansion, putting huge stress on the food supply chain. Many solutions to the grand challenge of food security for all have been proposed and are currently under investigation and study. Amongst these, genetics still has an important role to play, aiming to continually enable the selection of livestock with enhanced traits. Part of the geneticist's tool box is the technology of genetic engineering. In this Invited Review, we indicate that this technology has come a long way, we focus on the genetic engineering of dairy animals and we argue that the new strategies for precision breeding demand proper evaluation as to how they could contribute to the essential increases in agricultural productivity our society must achieve.

Reverse engineering of integrated circuits

DOEpatents

Chisholm, Gregory H.; Eckmann, Steven T.; Lain, Christopher M.; Veroff, Robert L.

2003-01-01

Software and a method therein to analyze circuits. The software comprises several tools, each of which perform particular functions in the Reverse Engineering process. The analyst, through a standard interface, directs each tool to the portion of the task to which it is most well suited, rendering previously intractable problems solvable. The tools are generally used iteratively to produce a successively more abstract picture of a circuit, about which incomplete a priori knowledge exists.

Engineering Values into Genetic Engineering: A Proposed Analytic Framework for Scientific Social Responsibility

PubMed Central

Cho, Mildred K.

2016-01-01

Recent experiments have been used to “edit” genomes of various plant, animal and other species, including humans, with unprecedented precision. Furthermore, editing Cas9 endonuclease gene with a gene encoding the desired guide RNA into an organism, adjacent to an altered gene, could create a “gene drive” that could spread a trait through an entire population of organisms. These experiments represent advances along a spectrum of technological abilities that genetic engineers have been working on since the advent of recombinant DNA techniques. The scientific and bioethics communities have built substantial literatures about the ethical and policy implications of genetic engineering, especially in the age of bioterrorism. However, recent CRISPr/Cas experiments have triggered a rehashing of previous policy discussions, suggesting that the scientific community requires guidance on how to think about social responsibility. We propose a framework to enable analysis of social responsibility, using two examples of genetic engineering experiments. PMID:26632356

Engineering Values Into Genetic Engineering: A Proposed Analytic Framework for Scientific Social Responsibility.

PubMed

Sankar, Pamela L; Cho, Mildred K

2015-01-01

Recent experiments have been used to "edit" genomes of various plant, animal and other species, including humans, with unprecedented precision. Furthermore, editing the Cas9 endonuclease gene with a gene encoding the desired guide RNA into an organism, adjacent to an altered gene, could create a "gene drive" that could spread a trait through an entire population of organisms. These experiments represent advances along a spectrum of technological abilities that genetic engineers have been working on since the advent of recombinant DNA techniques. The scientific and bioethics communities have built substantial literatures about the ethical and policy implications of genetic engineering, especially in the age of bioterrorism. However, recent CRISPr/Cas experiments have triggered a rehashing of previous policy discussions, suggesting that the scientific community requires guidance on how to think about social responsibility. We propose a framework to enable analysis of social responsibility, using two examples of genetic engineering experiments.

The art and design of genetic screens: maize

USDA-ARS?s Scientific Manuscript database

Maize (Zea mays) is an excellent model for basic research. Genetic screens have informed our understanding of developmental processes, meiosis, epigenetics and biochemical pathways--not only in maize but also in other cereal crops. We discuss the forward and reverse genetic screens that are possible...

Quantum Stirling heat engine and refrigerator with single and coupled spin systems

NASA Astrophysics Data System (ADS)

Huang, Xiao-Li; Niu, Xin-Ya; Xiu, Xiao-Ming; Yi, Xue-Xi

2014-02-01

We study the reversible quantum Stirling cycle with a single spin or two coupled spins as the working substance. With the single spin as the working substance, we find that under certain conditions the reversed cycle of a heat engine is NOT a refrigerator, this feature holds true for a Stirling heat engine with an ion trapped in a shallow potential as its working substance. The efficiency of quantum Stirling heat engine can be higher than the efficiency of the Carnot engine, but the performance coefficient of the quantum Stirling refrigerator is always lower than its classical counterpart. With two coupled spins as the working substance, we find that a heat engine can turn to a refrigerator due to the increasing of the coupling constant, this can be explained by the properties of the isothermal line in the magnetic field-entropy plane.

The Effects of Meiosis/Genetics Integration and Instructional Sequence on College Biology Student Achievement in Genetics.

ERIC Educational Resources Information Center

Browning, Mark

The purpose of the research was to manipulate two aspects of genetics instruction in order to measure their effects on college, introductory biology students' achievement in genetics. One instructional sequence that was used dealt first with monohybrid autosomal inheritance patterns, then sex-linkage. The alternate sequence was the reverse.…

Genetic Engineering: The Modification of Man

ERIC Educational Resources Information Center

Sinsheimer, Robert L.

1970-01-01

Describes somatic and genetic manipulations of individual genotypes, using diabetes control as an example of the first mode that is potentially realizable be derepression or viral transduction of genes. Advocates the use of genetic engineering of the second mode to remove man from his biological limitations, but offers maxims to ensure the…

Current achievements and future directions in genetic engineering of european plum (Prunus domestica L.)

USDA-ARS?s Scientific Manuscript database

In most woody fruit species, transformation and regeneration are difficult. However, European plum (Prunus domestica) has been shown to be amenable to genetic improvement technologies from classical hybridization, to genetic engineering, to rapid cycle crop breeding (‘FasTrack’ breeding). Since th...

Chapter VIII. Contributions of propagation techniques and genetic modification to breeding - genetic engineering for disease resistance

USDA-ARS?s Scientific Manuscript database

Genetic engineering offers an opportunity to develop flower bulb crops with resistance to fungal, viral, and bacterial pathogens. Several of the flower bulb crops, Lilium spp., Gladiolus, Zantedeschia, Muscari, Hyacinthus, Narcissus, Ornithogalum, Iris, and Alstroemeria, have been transformed with t...

Reverse genetics: Its origins and prospects

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

Berg, P.

1991-04-01

The nucleotide sequence of a gene and its flanking segments alone will not tell us how its expression is regulated during development and differentiation, or in response to environmental changes. To comprehend the physiological significance of the molecular details requires biological analysis. Recombinant DNA techniques provide a powerful experimental approach. A strategy termed reverse genetics' utilizes the analysis of the activities of mutant and normal genes and experimentally constructed mutants to explore the relationship between gene structure and function thereby helping elucidate the relationship between genotype and phenotype.

77 FR 41356 - Monsanto Co.; Availability of Petition for Determination of Nonregulated Status of Soybean...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-13

... Engineered for Herbicide Tolerance AGENCY: Animal and Plant Health Inspection Service, USDA. ACTION: Notice... soybean designated as MON 87708, which has been genetically engineered for tolerance to the herbicide... MON 87708, which has been genetically engineered for tolerance to the herbicide dicamba, stating that...

77 FR 41353 - GENECTIVE SA; Availability of Petition for Determination of Nonregulated Status of Maize...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-13

... Engineered for Herbicide Tolerance AGENCY: Animal and Plant Health Inspection Service, USDA. ACTION: Notice... VCO-[Oslash]1981-5, which has been genetically engineered for tolerance to the herbicide glyphosate...- [Oslash]1981-5, which has been genetically engineered for tolerance to the herbicide glyphosate, stating...

Perspectives for genetic engineering for the phytoremediation of arsenic-contaminated environments: from imagination to reality?

PubMed

Zhu, Yong-Guan; Rosen, Barry P

2009-04-01

Phytoremediation to clean up arsenic-contaminated environments has been widely hailed as environmentally friendly and cost effective, and genetic engineering is believed to improve the efficiency and versatility of phytoremediation. Successful genetic engineering requires the thorough understanding of the mechanisms involved in arsenic tolerance and accumulation by natural plant species. Key mechanisms include arsenate reduction, arsenic sequestration in vacuoles of root or shoot, arsenic loading to the xylem, and volatilization through the leaves. Key advances include the identification of arsenic (As) translocation from root to shoot in the As hyperaccumulator, Pteris vittata, and the characterization of related key genes from hyperaccumulator and nonaccumulators. In this paper we have proposed three pathways for genetic engineering: arsenic sequestration in the root, hyperaccumulation of arsenic in aboveground tissues, and phytovolatilization.

A Hybrid Neural Network-Genetic Algorithm Technique for Aircraft Engine Performance Diagnostics

NASA Technical Reports Server (NTRS)

Kobayashi, Takahisa; Simon, Donald L.

2001-01-01

In this paper, a model-based diagnostic method, which utilizes Neural Networks and Genetic Algorithms, is investigated. Neural networks are applied to estimate the engine internal health, and Genetic Algorithms are applied for sensor bias detection and estimation. This hybrid approach takes advantage of the nonlinear estimation capability provided by neural networks while improving the robustness to measurement uncertainty through the application of Genetic Algorithms. The hybrid diagnostic technique also has the ability to rank multiple potential solutions for a given set of anomalous sensor measurements in order to reduce false alarms and missed detections. The performance of the hybrid diagnostic technique is evaluated through some case studies derived from a turbofan engine simulation. The results show this approach is promising for reliable diagnostics of aircraft engines.

Genetic tool development and systemic regulation in biosynthetic technology.

PubMed

Dai, Zhongxue; Zhang, Shangjie; Yang, Qiao; Zhang, Wenming; Qian, Xiujuan; Dong, Weiliang; Jiang, Min; Xin, Fengxue

2018-01-01

With the increased development in research, innovation, and policy interest in recent years, biosynthetic technology has developed rapidly, which combines engineering, electronics, computer science, mathematics, and other disciplines based on classical genetic engineering and metabolic engineering. It gives a wider perspective and a deeper level to perceive the nature of life via cell mechanism, regulatory networks, or biological evolution. Currently, synthetic biology has made great breakthrough in energy, chemical industry, and medicine industries, particularly in the programmable genetic control at multiple levels of regulation to perform designed goals. In this review, the most advanced and comprehensive developments achieved in biosynthetic technology were represented, including genetic engineering as well as synthetic genomics. In addition, the superiority together with the limitations of the current genome-editing tools were summarized.

Genome engineering in cattle: recent technological advancements.

PubMed

Wang, Zhongde

2015-02-01

Great strides in technological advancements have been made in the past decade in cattle genome engineering. First, the success of cloning cattle by somatic cell nuclear transfer (SCNT) or chromatin transfer (CT) is a significant advancement that has made obsolete the need for using embryonic stem (ES) cells to conduct cell-mediated genome engineering, whereby site-specific genetic modifications can be conducted in bovine somatic cells via DNA homologous recombination (HR) and whereby genetically engineered cattle can subsequently be produced by animal cloning from the genetically modified cells. With this approach, a chosen bovine genomic locus can be precisely modified in somatic cells, such as to knock out (KO) or knock in (KI) a gene via HR, a gene-targeting strategy that had almost exclusively been used in mouse ES cells. Furthermore, by the creative application of embryonic cloning to rejuvenate somatic cells, cattle genome can be sequentially modified in the same line of somatic cells and complex genetic modifications have been achieved in cattle. Very recently, the development of designer nucleases-such as zinc finger nucleases (ZFNs) and transcription activator-like effector nuclease (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-has enabled highly efficient and more facile genome engineering in cattle. Most notably, by employing such designer nucleases, genomes can be engineered at single-nucleotide precision; this process is now often referred to as genome or gene editing. The above achievements are a drastic departure from the traditional methods of creating genetically modified cattle, where foreign DNAs are randomly integrated into the animal genome, most often along with the integrations of bacterial or viral DNAs. Here, I review the most recent technological developments in cattle genome engineering by highlighting some of the major achievements in creating genetically engineered cattle for agricultural and biomedical applications.

Engineering Bacterial Thiosulfate and Tetrathionate Sensors for Detecting Gut Inflammation

DTIC Science & Technology

2017-04-03

Article Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation Kristina N-M Daeffler1 , Jeffrey D Galley2, Ravi U...interest in using genetically engineered sensor bacteria to study gut microbiota pathways, and diagnose or treat associated diseases. Here, we...understood. Genetically engineered sensor bacteria have untapped potential as tools for analyzing gut pathways. Bacteria have evolved sensors of a large

Using a Formal Approach for Reverse Engineering and Design Recovery to Support Software Reuse

NASA Technical Reports Server (NTRS)

Gannod, Gerald C.

2002-01-01

This document describes 3rd year accomplishments and summarizes overall project accomplishments. Included as attachments are all published papers from year three. Note that the budget for this project was discontinued after year two, but that a residual budget from year two allowed minimal continuance into year three. Accomplishments include initial investigations into log-file based reverse engineering, service-based software reuse, and a source to XML generator.

Variable Cycle Intake for Reverse Core Engine

NASA Technical Reports Server (NTRS)

Chandler, Jesse M (Inventor); Staubach, Joseph B (Inventor); Suciu, Gabriel L (Inventor)

2016-01-01

A gas generator for a reverse core engine propulsion system has a variable cycle intake for the gas generator, which variable cycle intake includes a duct system. The duct system is configured for being selectively disposed in a first position and a second position, wherein free stream air is fed to the gas generator when in the first position, and fan stream air is fed to the gas generator when in the second position.

A new digitized reverse correction method for hypoid gears based on a one-dimensional probe

NASA Astrophysics Data System (ADS)

Li, Tianxing; Li, Jubo; Deng, Xiaozhong; Yang, Jianjun; Li, Genggeng; Ma, Wensuo

2017-12-01

In order to improve the tooth surface geometric accuracy and transmission quality of hypoid gears, a new digitized reverse correction method is proposed based on the measurement data from a one-dimensional probe. The minimization of tooth surface geometrical deviations is realized from the perspective of mathematical analysis and reverse engineering. Combining the analysis of complex tooth surface generation principles and the measurement mechanism of one-dimensional probes, the mathematical relationship between the theoretical designed tooth surface, the actual machined tooth surface and the deviation tooth surface is established, the mapping relation between machine-tool settings and tooth surface deviations is derived, and the essential connection between the accurate calculation of tooth surface deviations and the reverse correction method of machine-tool settings is revealed. Furthermore, a reverse correction model of machine-tool settings is built, a reverse correction strategy is planned, and the minimization of tooth surface deviations is achieved by means of the method of numerical iterative reverse solution. On this basis, a digitized reverse correction system for hypoid gears is developed by the organic combination of numerical control generation, accurate measurement, computer numerical processing, and digitized correction. Finally, the correctness and practicability of the digitized reverse correction method are proved through a reverse correction experiment. The experimental results show that the tooth surface geometric deviations meet the engineering requirements after two trial cuts and one correction.

Genetics Home Reference: nephronophthisis

MedlinePlus

... which can include liver fibrosis, heart abnormalities, or mirror image reversal of the position of one or ... Information from MedlinePlus (5 links) Diagnostic Tests Drug Therapy Genetic Counseling Palliative Care Surgery and Rehabilitation Related ...

Current achievements and future directions in genetic engineering of European plum (Prunus domestica L.).

PubMed

Petri, Cesar; Alburquerque, Nuria; Faize, Mohamed; Scorza, Ralph; Dardick, Chris

2018-06-01

In most woody fruit species, transformation and regeneration are difficult. However, European plum (Prunus domestica) has been shown to be amenable to genetic improvement technologies from classical hybridization, to genetic engineering, to rapid cycle crop breeding ('FasTrack' breeding). Since the first report on European plum transformation with marker genes in the early 90 s, numerous manuscripts have been published reporting the generation of new clones with agronomically interesting traits, such as pests, diseases and/or abiotic stress resistance, shorter juvenile period, dwarfing, continuous flowering, etc. This review focuses on the main advances in genetic transformation of European plum achieved to date, and the lines of work that are converting genetic engineering into a contemporary breeding tool for this species.

GMOs in Russia: Research, Society and Legislation.

PubMed

Korobko, I V; Georgiev, P G; Skryabin, K G; Kirpichnikov, M P

2016-01-01

Russian legislation lags behind the rapid developments witnessed in genetic engineering. Only a scientifically based and well-substantiated policy on the place of organisms that are created with the use of genetic engineering technologies and an assessment of the risks associated with them could guarantee that the breakthroughs achieved in modern genetic engineering technologies are effectively put to use in the real economy. A lack of demand for such breakthroughs in the practical field will lead to stagnation in scientific research and to a loss of expertise.

Tissue engineering of bladder using vascular endothelial growth factor gene-modified endothelial progenitor cells.

PubMed

Chen, Bai-Song; Xie, Hua; Zhang, Sheng-Li; Geng, Hong-Quan; Zhou, Jun-Mei; Pan, Jun; Chen, Fang

2011-12-01

This study assessed the use of vascular endothelial growth factor (VEGF) gene-modified endothelial progenitor cells (EPCs) seeded onto bladder acellular matrix grafts (BAMGs), to enhance the blood supply in tissue-engineered bladders in a porcine model. Autologous porcine peripheral EPCs were isolated, cultured, expanded, characterized, and modified with the VEGF gene using an adenovirus vector. The expression of VEGF was examined using reverse transcriptase polymerase chain reaction (RT-PCR) and an enzyme-linked immunosorbent assay (ELISA). VEGF gene modified EPCs were seeded onto BAMG and cultured for 3 days before implantation into pigs for bladder tissue engineering. A partial bladder cystectomy was performed in 12 pigs. The experimental group (6 pigs) received VEGF gene-modified EPC-seeded BAMG. The control group (6 pigs) received BAMG without seeded EPCs. The resulting tissue-engineered bladders were subject to a general and histological analysis. Microvessel density (MVD) was assessed using immunohistochemistry. The ex vivo transfection efficiency of EPCs was greater than 60%-70% when concentrated adenovirus was used. The genetically modified cells expressed both VEGF and green fluorescent protein (GFP). Scanning electron microscopy (SEM) and Masson's trichrome staining of cross sections of the cultured cells seeded to BAMG showed cell attachment and proliferation on the surface of the BAMG. Histological examination revealed bladder regeneration in a time-dependent fashion. Significant increases in MVD were observed in the experimental group, in comparison with the control group. VEGF-modified EPCs significantly enhanced neovascularization, compared with BAMG alone. These results indicate that EPCs, combined with VEGF gene therapy, may be a suitable approach for increasing blood supply in the tissue engineering of bladders. Thus, a useful strategy to achieve a tissue-engineered bladder is indicated.

Speciation reversal and biodiversity dynamics with hybridization in changing environments.

PubMed

Seehausen, Ole; Takimoto, Gaku; Roy, Denis; Jokela, Jukka

2008-01-01

A considerable fraction of the world's biodiversity is of recent evolutionary origin and has evolved as a by-product of, and is maintained by, divergent adaptation in heterogeneous environments. Conservationists have paid attention to genetic homogenization caused by human-induced translocations (e.g. biological invasions and stocking), and to the importance of environmental heterogeneity for the ecological coexistence of species. However, far less attention has been paid to the consequences of loss of environmental heterogeneity to the genetic coexistence of sympatric species. Our review of empirical observations and our theoretical considerations on the causes and consequences of interspecific hybridization suggest that a loss of environmental heterogeneity causes a loss of biodiversity through increased genetic admixture, effectively reversing speciation. Loss of heterogeneity relaxes divergent selection and removes ecological barriers to gene flow between divergently adapted species, promoting interspecific introgressive hybridization. Since heterogeneity of natural environments is rapidly deteriorating in most biomes, the evolutionary ecology of speciation reversal ought to be fully integrated into conservation biology.

Effect of synthetic auxin herbicides on seed development and viability in genetically-engineered glyphosate-resistant alfalfa

USDA-ARS?s Scientific Manuscript database

Feral populations of cultivated crops have the potential to function as bridges and reservoirs that contribute to the unwanted movement of novel genetically engineered (GE) traits. Recognizing that feral alfalfa has the potential to lower genetic purity in alfalfa seed production fields when it is g...

FLP-18 Functions through the G-Protein-Coupled Receptors NPR-1 and NPR-4 to Modulate Reversal Length in Caenorhabditis elegans.

PubMed

Bhardwaj, Ashwani; Thapliyal, Saurabh; Dahiya, Yogesh; Babu, Kavita

2018-05-16

Animal behavior is critically dependent on the activity of neuropeptides. Reversals, one of the most conspicuous behaviors in Caenorhabditis elegans , plays an important role in determining the navigation strategy of the animal. Our experiments on hermaphrodite C. elegans show the involvement of a neuropeptide FLP-18 in modulating reversal length in these hermaphrodites. We show that FLP-18 controls the reversal length by regulating the activity of AVA interneurons through the G-protein-coupled neuropeptide receptors, NPR-4 and NPR-1. We go on to show that the site of action of these receptors is the AVA interneuron for NPR-4 and the ASE sensory neurons for NPR-1. We further show that mutants in the neuropeptide, flp-18 , and its receptors show increased reversal lengths. Consistent with the behavioral data, calcium levels in the AVA neuron of freely reversing C. elegans were significantly higher and persisted for longer durations in flp-18 , npr-1 , npr-4 , and npr-1 npr-4 genetic backgrounds compared with wild-type control animals. Finally, we show that increasing FLP-18 levels through genetic and physiological manipulations causes shorter reversal lengths. Together, our analysis suggests that the FLP-18/NPR-1/NPR-4 signaling is a pivotal point in the regulation of reversal length under varied genetic and environmental conditions. SIGNIFICANCE STATEMENT In this study, we elucidate the circuit and molecular machinery required for normal reversal behavior in hermaphrodite Caenorhabditis elegans We delineate the circuit and the neuropeptide receptors required for maintaining reversal length in C. elegans Our work sheds light on the importance of a single neuropeptide, FLP-18, and how change in levels in this one peptide could allow the animal to change the length of its reversal, thereby modulating how the C. elegans explores its environment. We also go on to show that FLP-18 functions to maintain reversal length through the neuropeptide receptors NPR-4 and NPR-1. Our study will allow for a better understanding of the complete repertoire of behaviors shown by freely moving animals as they explore their environment. Copyright © 2018 the authors 0270-6474/18/384641-14$15.00/0.

Hybrid algorithms for fuzzy reverse supply chain network design.

PubMed

Che, Z H; Chiang, Tzu-An; Kuo, Y C; Cui, Zhihua

2014-01-01

In consideration of capacity constraints, fuzzy defect ratio, and fuzzy transport loss ratio, this paper attempted to establish an optimized decision model for production planning and distribution of a multiphase, multiproduct reverse supply chain, which addresses defects returned to original manufacturers, and in addition, develops hybrid algorithms such as Particle Swarm Optimization-Genetic Algorithm (PSO-GA), Genetic Algorithm-Simulated Annealing (GA-SA), and Particle Swarm Optimization-Simulated Annealing (PSO-SA) for solving the optimized model. During a case study of a multi-phase, multi-product reverse supply chain network, this paper explained the suitability of the optimized decision model and the applicability of the algorithms. Finally, the hybrid algorithms showed excellent solving capability when compared with original GA and PSO methods.

Hybrid Algorithms for Fuzzy Reverse Supply Chain Network Design

PubMed Central

Che, Z. H.; Chiang, Tzu-An; Kuo, Y. C.

2014-01-01

In consideration of capacity constraints, fuzzy defect ratio, and fuzzy transport loss ratio, this paper attempted to establish an optimized decision model for production planning and distribution of a multiphase, multiproduct reverse supply chain, which addresses defects returned to original manufacturers, and in addition, develops hybrid algorithms such as Particle Swarm Optimization-Genetic Algorithm (PSO-GA), Genetic Algorithm-Simulated Annealing (GA-SA), and Particle Swarm Optimization-Simulated Annealing (PSO-SA) for solving the optimized model. During a case study of a multi-phase, multi-product reverse supply chain network, this paper explained the suitability of the optimized decision model and the applicability of the algorithms. Finally, the hybrid algorithms showed excellent solving capability when compared with original GA and PSO methods. PMID:24892057

The strains recommended for use in the bacterial reverse mutation test (OECD guideline 471) can be certified as non-genetically modified organisms.

PubMed

Sugiyama, Kei-Ichi; Yamada, Masami; Awogi, Takumi; Hakura, Atsushi

2016-01-01

The bacterial reverse mutation test, commonly called Ames test, is used worldwide. In Japan, the genetically modified organisms (GMOs) are regulated under the Cartagena Domestic Law, and organisms obtained by self-cloning and/or natural occurrence would be exempted from the law case by case. The strains of Salmonella typhimurium and Escherichia coli recommended for use in the bacterial reverse mutation test (OECD guideline 471), have been considered as non-GMOs because they can be constructed by self-cloning or naturally occurring bacterial strains, or do not disturb the biological diversity. The present article explains the reasons why these tester strains should be classified as non-GMOs.

Techniques utilized in the simulated altitude testing of a 2D-CD vectoring and reversing nozzle

NASA Technical Reports Server (NTRS)

Block, H. Bruce; Bryant, Lively; Dicus, John H.; Moore, Allan S.; Burns, Maureen E.; Solomon, Robert F.; Sheer, Irving

1988-01-01

Simulated altitude testing of a two-dimensional, convergent-divergent, thrust vectoring and reversing exhaust nozzle was accomplished. An important objective of this test was to develop test hardware and techniques to properly operate a vectoring and reversing nozzle within the confines of an altitude test facility. This report presents detailed information on the major test support systems utilized, the operational performance of the systems and the problems encountered, and test equipment improvements recommended for future tests. The most challenging support systems included the multi-axis thrust measurement system, vectored and reverse exhaust gas collection systems, and infrared temperature measurement systems used to evaluate and monitor the nozzle. The feasibility of testing a vectoring and reversing nozzle of this type in an altitude chamber was successfully demonstrated. Supporting systems performed as required. During reverser operation, engine exhaust gases were successfully captured and turned downstream. However, a small amount of exhaust gas spilled out the collector ducts' inlet openings when the reverser was opened more than 60 percent. The spillage did not affect engine or nozzle performance. The three infrared systems which viewed the nozzle through the exhaust collection system worked remarkably well considering the harsh environment.

Generating Alternative Engineering Designs by Integrating Desktop VR with Genetic Algorithms

ERIC Educational Resources Information Center

Chandramouli, Magesh; Bertoline, Gary; Connolly, Patrick

2009-01-01

This study proposes an innovative solution to the problem of multiobjective engineering design optimization by integrating desktop VR with genetic computing. Although, this study considers the case of construction design as an example to illustrate the framework, this method can very much be extended to other engineering design problems as well.…

78 FR 13305 - Syngenta Seeds, Inc., and Bayer CropScience AG; Availability of Petition for Determination of...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-02-27

... Status of Soybean Genetically Engineered for Herbicide Tolerance AGENCY: Animal and Plant Health... SYHTOH2, which has been genetically engineered for tolerance to the herbicides glufosinate and mesotrione... engineered to tolerate exposure to the herbicides glufosinate and mesotrione. Glufosinate tolerance is not a...

75 FR 2845 - ArborGen, LLC; Availability of an Environmental Assessment for Controlled Release of a...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-01-19

... Engineered Eucalyptus Hybrid AGENCY: Animal and Plant Health Inspection Service, USDA. ACTION: Notice... for a proposed controlled field release of a genetically engineered clone of a Eucalyptus hybrid. This... proposed controlled field release of a genetically engineered clone of a Eucalyptus hybrid. \\1\\ To view the...

77 FR 40026 - 36(b)(1) Arms Sales Notification

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-06

... and contractor logistics, Quality Assurance Team support services, engineering and technical support..., engineering and technical support, and other related elements of program support. The estimated cost is $49..., maintenance, or training is Confidential. Reverse engineering could reveal Confidential information...

"Genetic Engineering" Gains Momentum (Science/Society Case Study).

ERIC Educational Resources Information Center

Moore, John W.; Moore, Elizabeth A., Eds.

1980-01-01

Reviews the benefits and hazards of genetic engineering, or "recombinant-DNA" research. Recent federal safety rules issued by NIH which ease the strict prohibitions on recombinant-DNA research are explained. (CS)

CD40 Agonists Alter Tumor Stroma and Show Efficacy Against Pancreatic Carcinoma in Mice and Humans

PubMed Central

Beatty, Gregory L.; Chiorean, Elena G.; Fishman, Matthew P.; Saboury, Babak; Teitelbaum, Ursina R.; Sun, Weijing; Huhn, Richard D.; Song, Wenru; Li, Dongguang; Sharp, Leslie L.; Torigian, Drew A.; O’Dwyer, Peter J.; Vonderheide, Robert H.

2012-01-01

Immunosuppressive tumor microenvironments can restrain antitumor immunity, particularly in pancreatic ductal adenocarcinoma (PDA). Because CD40 activation can reverse immune suppression and drive antitumor T cell responses, we tested the combination of an agonist CD40 antibody with gemcitabine chemotherapy in a small cohort of patients with surgically incurable PDA and observed tumor regressions in some patients. We reproduced this treatment effect in a genetically engineered mouse model of PDA and found unexpectedly that tumor regression required macrophages but not T cells or gemcitabine. CD40-activated macrophages rapidly infiltrated tumors, became tumoricidal, and facilitated the depletion of tumor stroma. Thus, cancer immune surveillance does not necessarily depend on therapy-induced T cells; rather, our findings demonstrate a CD40-dependent mechanism for targeting tumor stroma in the treatment of cancer. PMID:21436454

Computer-aided design for metabolic engineering.

PubMed

Fernández-Castané, Alfred; Fehér, Tamás; Carbonell, Pablo; Pauthenier, Cyrille; Faulon, Jean-Loup

2014-12-20

The development and application of biotechnology-based strategies has had a great socio-economical impact and is likely to play a crucial role in the foundation of more sustainable and efficient industrial processes. Within biotechnology, metabolic engineering aims at the directed improvement of cellular properties, often with the goal of synthesizing a target chemical compound. The use of computer-aided design (CAD) tools, along with the continuously emerging advanced genetic engineering techniques have allowed metabolic engineering to broaden and streamline the process of heterologous compound-production. In this work, we review the CAD tools available for metabolic engineering with an emphasis, on retrosynthesis methodologies. Recent advances in genetic engineering strategies for pathway implementation and optimization are also reviewed as well as a range of bionalytical tools to validate in silico predictions. A case study applying retrosynthesis is presented as an experimental verification of the output from Retropath, the first complete automated computational pipeline applicable to metabolic engineering. Applying this CAD pipeline, together with genetic reassembly and optimization of culture conditions led to improved production of the plant flavonoid pinocembrin. Coupling CAD tools with advanced genetic engineering strategies and bioprocess optimization is crucial for enhanced product yields and will be of great value for the development of non-natural products through sustainable biotechnological processes. Copyright © 2014 Elsevier B.V. All rights reserved.

Genetically Engineered Mouse Models for Studying Inflammatory Bowel Disease

PubMed Central

Mizoguchi, Atsushi; Takeuchi, Takahito; Himuro, Hidetomo; Okada, Toshiyuki; Mizoguchi, Emiko

2015-01-01

Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory condition that is mediated by very complex mechanisms controlled by genetic, immune, and environmental factors. More than 74 kinds of genetically engineered mouse strains have been established since 1993 for studying IBD. Although mouse models cannot fully reflect human IBD, they have provided significant contributions for not only understanding the mechanism, but also developing new therapeutic means for IBD. Indeed, 20 kinds of genetically engineered mouse models carry the susceptibility genes identified in human IBD, and the functions of some other IBD susceptibility genes have also been dissected out using mouse models. Cutting-edge technologies such as cell-specific and inducible knockout systems, which were recently employed to mouse IBD models, have further enhanced the ability of investigators to provide important and unexpected rationales for developing new therapeutic strategies for IBD. In this review article, we briefly introduce 74 kinds of genetically engineered mouse models that spontaneously develop intestinal inflammation. PMID:26387641

Perspectives for genetic engineering for the phytoremediation of arsenic-contaminated environments: from imagination to reality?

PubMed Central

Zhu, Yong-Guan; Rosen, Barry P

2015-01-01

Phytoremediation to clean up arsenic-contaminated environments has been widely hailed as environmentally friendly and cost effective, and genetic engineering is believed to improve the efficiency and versatility of phytoremediation. Successful genetic engineering requires the thorough understanding of the mechanisms involved in arsenic tolerance and accumulation by natural plant species. Key mechanisms include arsenate reduction, arsenic sequestration in vacuoles of root or shoot, arsenic loading to the xylem, and volatilization through the leaves. Key advances include the identification of arsenic (As) translocation from root to shoot in the As hyperaccumulator, Pteris vittata, and the characterization of related key genes from hyperaccumulator and nonaccumulators. In this paper we have proposed three pathways for genetic engineering: arsenic sequestration in the root, hyperaccumulation of arsenic in aboveground tissues, and phytovolatilization. PMID:19303764

Emergency deployment of genetically engineered veterinary vaccines in Europe.

PubMed

Ramezanpour, Bahar; de Foucauld, Jean; Kortekaas, Jeroen

2016-06-24

On the 9th of November 2015, preceding the World Veterinary Vaccine Congress, a workshop was held to discuss how veterinary vaccines can be deployed more rapidly to appropriately respond to future epizootics in Europe. Considering their potential and unprecedented suitability for surge production, the workshop focussed on vaccines based on genetically engineered viruses and replicon particles. The workshop was attended by academics and representatives from leading pharmaceutical companies, regulatory experts, the European Medicines Agency and the European Commission. We here outline the present regulatory pathways for genetically engineered vaccines in Europe and describe the incentive for the organization of the pre-congress workshop. The participants agreed that existing European regulations on the deliberate release of genetically engineered vaccines into the environment should be updated to facilitate quick deployment of these vaccines in emergency situations. Copyright © 2016.

CONDENSED MATTER: ELECTRONIC STRUCTURE, ELECTRICAL, MAGNETIC, AND OPTICAL PROPERTIES: Research on reverse recovery characteristics of SiGeC p-i-n diodes

NASA Astrophysics Data System (ADS)

Gao, Yong; Liu, Jing; Yang, Yuan

2008-12-01

This paper analyses the reverse recovery characteristics and mechanism of SiGeC p-i-n diodes. Based on the integrated systems engineering (ISE) data, the critical physical models of SiGeC diodes are proposed. Based on hetero-junction band gap engineering, the softness factor increases over six times, reverse recovery time is over 30% short and there is a 20% decrease in peak reverse recovery current for SiGeC diodes with 20% of germanium and 0.5% of carbon, compared to Si diodes. Those advantages of SiGeC p-i-n diodes are more obvious at high temperature. Compared to lifetime control, SiGeC technique is more suitable for improving diode properties and the tradeoff between reverse recovery time and forward voltage drop can be easily achieved in SiGeC diodes. Furthermore, the high thermal-stability of SiGeC diodes reduces the costs of further process steps and offers more freedoms to device design.

A novel technique for presurgical nasoalveolar molding using computer-aided reverse engineering and rapid prototyping.

PubMed

Yu, Quan; Gong, Xin; Wang, Guo-Min; Yu, Zhe-Yuan; Qian, Yu-Fen; Shen, Gang

2011-01-01

To establish a new method of presurgical nasoalveolar molding (NAM) using computer-aided reverse engineering and rapid prototyping technique in infants with unilateral cleft lip and palate (UCLP). Five infants (2 males and 3 females with mean age of 1.2 w) with complete UCLP were recruited. All patients were subjected to NAM before the cleft lip repair. The upper denture casts were recorded using a three-dimensional laser scanner within 2 weeks after birth in UCLP infants. A digital model was constructed and analyzed to simulate the NAM procedure with reverse engineering software. The digital geometrical data were exported to print the solid model with rapid prototyping system. The whole set of appliances was fabricated based on these solid models. Laser scanning and digital model construction simplified the NAM procedure and estimated the treatment objective. The appliances were fabricated based on the rapid prototyping technique, and for each patient, the complete set of appliances could be obtained at one time. By the end of presurgical NAM treatment, the cleft was narrowed, and the malformation of nasoalveolar segments was aligned normally. We have developed a novel technique of presurgical NAM based on a computer-aided design. The accurate digital denture model of UCLP infants could be obtained with laser scanning. The treatment design and appliance fabrication could be simplified with a computer-aided reverse engineering and rapid prototyping technique.

A biotin-triggered genetic switch in mammalian cells and mice.

PubMed

Weber, Wilfried; Lienhart, Cédric; Baba, Marie Daoud-El; Fussenegger, Martin

2009-03-01

Adjustable and reversible transgene expression systems enabling precise control of metabolic pathways and tunable production of specific target proteins have been essential for conditional reprogramming of mammalian cells to achieve progress in basic and applied bioengineering disciplines. Most of the currently available transgene control modalities have been designed to be responsive to clinically licensed pharmacologically active drugs which were expected to prevail in future clinical trials yet raised concerns about side effects when administered long term at subclinical doses. We have chosen vitamin H, also known as biotin, to control target gene transcription in mammalian cells in a potentially side effect-free manner. BirA, the Escherichia coli repressor of the biotin biosynthesis operon, was fused to the Herpes simplex transactivation domain to generate a biotin-dependent transactivator(BIT), which, in the presence of biotin, binds and activates chimeric target promoters (P(BIT)) harboring BirA-specific operator sites 5' of a minimal promoter. Biotin-inducible transgene expression was functional in a variety of rodent, monkey and human cell lines, showed excellent adjustability and reversibility in transgenic Chinese hamster ovary cell lines, provided precise product gene control in standard bioreactor cultures and enabled dose-dependent vitamin H control of a human glycoprotein in mice. The combination of a side effect-free inducer, precise and reversible transcription tunability and broad functionality in different cell types as well as in entire animals represents a unique asset for the use of biotin-inducible transgene control in future gene therapy, tissue engineering and biopharmaceutical manufacturing scenarios.

Reversing the Trend of Engineering Enrollment Declines with Innovative Outreach, Recruiting, and Retention Programs

ERIC Educational Resources Information Center

Davis, C. E.; Yeary, M. B.; Sluss, J. J., Jr.

2012-01-01

This paper discusses an all-encompassing approach to increase the number of students in engineering through innovative outreach, recruiting, and retention programs. Prior to adopting these programs, the School of Electrical and Computer Engineering (ECE) at the University of Oklahoma (OU), Norman, experienced a reduction in engineering enrollment…

Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies.

PubMed

Rickert, Keith W; Grinberg, Luba; Woods, Robert M; Wilson, Susan; Bowen, Michael A; Baca, Manuel

2016-01-01

The enormous diversity created by gene recombination and somatic hypermutation makes de novo protein sequencing of monoclonal antibodies a uniquely challenging problem. Modern mass spectrometry-based sequencing will rarely, if ever, provide a single unambiguous sequence for the variable domains. A more likely outcome is computation of an ensemble of highly similar sequences that can satisfy the experimental data. This outcome can result in the need for empirical testing of many candidate sequences, sometimes iteratively, to identity one which can replicate the activity of the parental antibody. Here we describe an improved approach to antibody protein sequencing by using phage display technology to generate a combinatorial library of sequences that satisfy the mass spectrometry data, and selecting for functional candidates that bind antigen. This approach was used to reverse engineer 2 commercially-obtained monoclonal antibodies against murine CD137. Proteomic data enabled us to assign the majority of the variable domain sequences, with the exception of 3-5% of the sequence located within or adjacent to complementarity-determining regions. To efficiently resolve the sequence in these regions, small phage-displayed libraries were generated and subjected to antigen binding selection. Following enrichment of antigen-binding clones, 2 clones were selected for each antibody and recombinantly expressed as antigen-binding fragments (Fabs). In both cases, the reverse-engineered Fabs exhibited identical antigen binding affinity, within error, as Fabs produced from the commercial IgGs. This combination of proteomic and protein engineering techniques provides a useful approach to simplifying the technically challenging process of reverse engineering monoclonal antibodies from protein material.

Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies

PubMed Central

Rickert, Keith W.; Grinberg, Luba; Woods, Robert M.; Wilson, Susan; Bowen, Michael A.; Baca, Manuel

2016-01-01

ABSTRACT The enormous diversity created by gene recombination and somatic hypermutation makes de novo protein sequencing of monoclonal antibodies a uniquely challenging problem. Modern mass spectrometry-based sequencing will rarely, if ever, provide a single unambiguous sequence for the variable domains. A more likely outcome is computation of an ensemble of highly similar sequences that can satisfy the experimental data. This outcome can result in the need for empirical testing of many candidate sequences, sometimes iteratively, to identity one which can replicate the activity of the parental antibody. Here we describe an improved approach to antibody protein sequencing by using phage display technology to generate a combinatorial library of sequences that satisfy the mass spectrometry data, and selecting for functional candidates that bind antigen. This approach was used to reverse engineer 2 commercially-obtained monoclonal antibodies against murine CD137. Proteomic data enabled us to assign the majority of the variable domain sequences, with the exception of 3–5% of the sequence located within or adjacent to complementarity-determining regions. To efficiently resolve the sequence in these regions, small phage-displayed libraries were generated and subjected to antigen binding selection. Following enrichment of antigen-binding clones, 2 clones were selected for each antibody and recombinantly expressed as antigen-binding fragments (Fabs). In both cases, the reverse-engineered Fabs exhibited identical antigen binding affinity, within error, as Fabs produced from the commercial IgGs. This combination of proteomic and protein engineering techniques provides a useful approach to simplifying the technically challenging process of reverse engineering monoclonal antibodies from protein material. PMID:26852694

Pluripotent stem cells and livestock genetic engineering

PubMed Central

Soto, Delia A.

2016-01-01

The unlimited proliferative ability and capacity to contribute to germline chimeras make pluripotent embryonic stem cells (ESCs) perfect candidates for complex genetic engineering. The utility of ESCs is best exemplified by the numerous genetic models that have been developed in mice, for which such cells are readily available. However, the traditional systems for mouse genetic engineering may not be practical for livestock species, as it requires several generations of mating and selection in order to establish homozygous founders. Nevertheless, the self-renewal and pluripotent characteristics of ESCs could provide advantages for livestock genetic engineering such as ease of genetic manipulation and improved efficiency of cloning by nuclear transplantation. These advantages have resulted in many attempts to isolate livestock ESCs, yet it has been generally concluded that the culture conditions tested so far are not supportive of livestock ESCs self-renewal and proliferation. In contrast, there are numerous reports of derivation of livestock induced pluripotent stem cells (iPSCs), with demonstrated capacity for long term proliferation and in vivo pluripotency, as indicated by teratoma formation assay. However, to what extent these iPSCs represent fully reprogrammed PSCs remains controversial, as most livestock iPSCs depend on continuous expression of reprogramming factors. Moreover, germline chimerism has not been robustly demonstrated, with only one successful report with very low efficiency. Therefore, even 34 years after derivation of mouse ESCs and their extensive use in the generation of genetic models, the livestock genetic engineering field can stand to gain enormously from continued investigations into the derivation and application of ESCs and iPSCs. PMID:26894405

Pluripotent stem cells and livestock genetic engineering.

PubMed

Soto, Delia A; Ross, Pablo J

2016-06-01

The unlimited proliferative ability and capacity to contribute to germline chimeras make pluripotent embryonic stem cells (ESCs) perfect candidates for complex genetic engineering. The utility of ESCs is best exemplified by the numerous genetic models that have been developed in mice, for which such cells are readily available. However, the traditional systems for mouse genetic engineering may not be practical for livestock species, as it requires several generations of mating and selection in order to establish homozygous founders. Nevertheless, the self-renewal and pluripotent characteristics of ESCs could provide advantages for livestock genetic engineering such as ease of genetic manipulation and improved efficiency of cloning by nuclear transplantation. These advantages have resulted in many attempts to isolate livestock ESCs, yet it has been generally concluded that the culture conditions tested so far are not supportive of livestock ESCs self-renewal and proliferation. In contrast, there are numerous reports of derivation of livestock induced pluripotent stem cells (iPSCs), with demonstrated capacity for long term proliferation and in vivo pluripotency, as indicated by teratoma formation assay. However, to what extent these iPSCs represent fully reprogrammed PSCs remains controversial, as most livestock iPSCs depend on continuous expression of reprogramming factors. Moreover, germline chimerism has not been robustly demonstrated, with only one successful report with very low efficiency. Therefore, even 34 years after derivation of mouse ESCs and their extensive use in the generation of genetic models, the livestock genetic engineering field can stand to gain enormously from continued investigations into the derivation and application of ESCs and iPSCs.

Static internal performance of a single-engine onaxisymmetric-nozzle vaned-thrust-reverser design with thrust modulation capabilities

NASA Technical Reports Server (NTRS)

Leavitt, L. D.; Burley, J. R., II

1985-01-01

An investigation has been conducted at wind-off conditions in the stati-test facility of the Langley 16-Foot Transonic Tunnel. The tests were conducted on a single-engine reverser configuration with partial and full reverse-thrust modulation capabilities. The reverser design had four ports with equal areas. These ports were angled outboard 30 deg from the vertical impart of a splay angle to the reverse exhaust flow. This splaying of reverser flow was intended to prevent impingement of exhaust flow on empennage surfaces and to help avoid inlet reingestion of exhaust gas when the reverser is integrated into an actual airplane configuration. External vane boxes were located directly over each of the four ports to provide variation of reverser efflux angle from 140 deg to 26 deg (measured forward from the horizontal reference axis). The reverser model was tested with both a butterfly-type inner door and an internal slider door to provide area control for each individual port. In addition, main nozzle throat area and vector angle were varied to examine various methods of modulating thrust levels. Other model variables included vane box configuration (four or six vanes per box), orientation of external vane boxes with respect to internal port walls (splay angle shims), and vane box sideplates. Nozzle pressure ratio was varied from 2.0 approximately 7.0.

The Significance of Content Knowledge for Informal Reasoning regarding Socioscientific Issues: Applying Genetics Knowledge to Genetic Engineering Issues

ERIC Educational Resources Information Center

Sadler, Troy D.; Zeidler, Dana L.

2005-01-01

This study focused on informal reasoning regarding socioscientific issues. It sought to explore how content knowledge influenced the negotiation and resolution of contentious and complex scenarios based on genetic engineering. Two hundred and sixty-nine students drawn from undergraduate natural science and nonnatural science courses completed a…

Teaching Applied Genetics and Molecular Biology to Agriculture Engineers. Application of the European Credit Transfer System

ERIC Educational Resources Information Center

Weiss, J.; Egea-Cortines, M.

2008-01-01

We have been teaching applied molecular genetics to engineers and adapted the teaching methodology to the European Credit Transfer System. We teach core principles of genetics that are universal and form the conceptual basis of most molecular technologies. The course then teaches widely used techniques and finally shows how different techniques…

Protection of genetic heritage in the era of cloning

PubMed Central

de Oliveira Júnior, Eudes Quintino; de Oliveira, Pedro Bellentani Quintino

2012-01-01

Research on human beings has expanded greatly due to progress and the evolution of society as well as customs. Not only the unceasing development of research on human beings, but also interference in the beginning and end of life with homologous and heterogonous human reproduction, surrogate motherhood, cloning, gene therapies, eugenics, euthanasia, dysthanasia, orthothanasia, assisted suicide, genetic engineering, reassignment surgery in cases of transsexuality, the use of recombinant DNA technology and embryonic stem cells, transplantation of human organs and tissues, biotechnology and many other scientific advances. Scientific progress goes faster than the real needs of human beings, who are the final recipient of the entire evolutionary progress. Hence, there is the need to scrutinize whether new technologies are necessary, suitable and timely so that humanity can achieve its postulate of bene vivere. Human cloning, as an abrupt scientific fact, has presented itself to the world community as a procedure that can be performed with relative success and with little difficulty, since it achieved its objectives with the cloning of Dolly the sheep. This issue became the topic of discussion not only in the scientific community but in the lay population, and it received from both, global disapproval. The conclusion is that the human being is unique, with a life cycle defined by the rules of nature. Reversal will cause a violation of the genetic heritage and, above all, will confront the constitutional principle of human dignity. PMID:23323071

Mediator Complex Subunits MED2, MED5, MED16, and MED23 Genetically Interact in the Regulation of Phenylpropanoid Biosynthesis.

PubMed

Dolan, Whitney L; Dilkes, Brian P; Stout, Jake M; Bonawitz, Nicholas D; Chapple, Clint

2017-12-01

The phenylpropanoid pathway is a major global carbon sink and is important for plant fitness and the engineering of bioenergy feedstocks. In Arabidopsis thaliana , disruption of two subunits of the transcriptional regulatory Mediator complex, MED5a and MED5b, results in an increase in phenylpropanoid accumulation. By contrast, the semidominant MED5b mutation reduced epidermal fluorescence4-3 ( ref4-3 ) results in dwarfism and constitutively repressed phenylpropanoid accumulation. Here, we report the results of a forward genetic screen for suppressors of ref4-3. We identified 13 independent lines that restore growth and/or phenylpropanoid accumulation in the ref4-3 background. Two of the suppressors restore growth without restoring soluble phenylpropanoid accumulation, indicating that the growth and metabolic phenotypes of the ref4-3 mutant can be genetically disentangled. Whole-genome sequencing revealed that all but one of the suppressors carry mutations in MED5b or other Mediator subunits. RNA-seq analysis showed that the ref4-3 mutation causes widespread changes in gene expression, including the upregulation of negative regulators of the phenylpropanoid pathway, and that the suppressors reverse many of these changes. Together, our data highlight the interdependence of individual Mediator subunits and provide greater insight into the transcriptional regulation of phenylpropanoid biosynthesis by the Mediator complex. © 2017 American Society of Plant Biologists. All rights reserved.

Protection of genetic heritage in the era of cloning.

PubMed

de Oliveira Júnior, Eudes Quintino; de Oliveira, Pedro Bellentani Quintino

2012-01-01

Research on human beings has expanded greatly due to progress and the evolution of society as well as customs. Not only the unceasing development of research on human beings, but also interference in the beginning and end of life with homologous and heterogonous human reproduction, surrogate motherhood, cloning, gene therapies, eugenics, euthanasia, dysthanasia, orthothanasia, assisted suicide, genetic engineering, reassignment surgery in cases of transsexuality, the use of recombinant DNA technology and embryonic stem cells, transplantation of human organs and tissues, biotechnology and many other scientific advances. Scientific progress goes faster than the real needs of human beings, who are the final recipient of the entire evolutionary progress. Hence, there is the need to scrutinize whether new technologies are necessary, suitable and timely so that humanity can achieve its postulate of bene vivere. Human cloning, as an abrupt scientific fact, has presented itself to the world community as a procedure that can be performed with relative success and with little difficulty, since it achieved its objectives with the cloning of Dolly the sheep.This issue became the topic of discussion not only in the scientific community but in the lay population, and it received from both, global disapproval. The conclusion is that the human being is unique, with a life cycle defined by the rules of nature. Reversal will cause a violation of the genetic heritage and, above all, will confront the constitutional principle of human dignity.

Registration of Dicamba for Use on Genetically Engineered Crops

EPA Pesticide Factsheets

EPA has registered a new dicamba formulation, Extendimax™ with VaporGrip™, specifically designed to have lower volatility, to control weeds in cotton and soybean plants that have been genetically engineered (GE) to resist dicamba.

Hybrid Neural-Network: Genetic Algorithm Technique for Aircraft Engine Performance Diagnostics Developed and Demonstrated

NASA Technical Reports Server (NTRS)

Kobayashi, Takahisa; Simon, Donald L.

2002-01-01

As part of the NASA Aviation Safety Program, a unique model-based diagnostics method that employs neural networks and genetic algorithms for aircraft engine performance diagnostics has been developed and demonstrated at the NASA Glenn Research Center against a nonlinear gas turbine engine model. Neural networks are applied to estimate the internal health condition of the engine, and genetic algorithms are used for sensor fault detection, isolation, and quantification. This hybrid architecture combines the excellent nonlinear estimation capabilities of neural networks with the capability to rank the likelihood of various faults given a specific sensor suite signature. The method requires a significantly smaller data training set than a neural network approach alone does, and it performs the combined engine health monitoring objectives of performance diagnostics and sensor fault detection and isolation in the presence of nominal and degraded engine health conditions.

Genetic correlations and the evolution of photoperiodic time measurement within a local population of the pitcher-plant mosquito, Wyeomyia smithii

PubMed Central

Bradshaw, W E; Emerson, K J; Holzapfel, C M

2012-01-01

The genetic relationship between the daily circadian clock and the seasonal photoperiodic timer remains a subject of intense controversy. In Wyeomyia smithii, the critical photoperiod (an overt expression of the photoperiodic timer) evolves independently of the rhythmic response to the Nanda–Hamner protocol (an overt expression of the daily circadian clock) over a wide geographical range in North America. Herein, we focus on these two processes within a single local population in which there is a negative genetic correlation between them. We show that antagonistic selection against this genetic correlation rapidly breaks it down and, in fact, reverses its sign, showing that the genetic correlation is due primarily to linkage and not to pleiotropy. This rapid reversal of the genetic correlation within a small, single population means that it is difficult to argue that circadian rhythmicity forms the necessary, causal basis for the adaptive divergence of photoperiodic time measurement within populations or for the evolution of photoperiodic time measurement among populations over a broad geographical gradient of seasonal selection. PMID:22072069

Efficient Reverse-Engineering of a Developmental Gene Regulatory Network

PubMed Central

Cicin-Sain, Damjan; Ashyraliyev, Maksat; Jaeger, Johannes

2012-01-01

Understanding the complex regulatory networks underlying development and evolution of multi-cellular organisms is a major problem in biology. Computational models can be used as tools to extract the regulatory structure and dynamics of such networks from gene expression data. This approach is called reverse engineering. It has been successfully applied to many gene networks in various biological systems. However, to reconstitute the structure and non-linear dynamics of a developmental gene network in its spatial context remains a considerable challenge. Here, we address this challenge using a case study: the gap gene network involved in segment determination during early development of Drosophila melanogaster. A major problem for reverse-engineering pattern-forming networks is the significant amount of time and effort required to acquire and quantify spatial gene expression data. We have developed a simplified data processing pipeline that considerably increases the throughput of the method, but results in data of reduced accuracy compared to those previously used for gap gene network inference. We demonstrate that we can infer the correct network structure using our reduced data set, and investigate minimal data requirements for successful reverse engineering. Our results show that timing and position of expression domain boundaries are the crucial features for determining regulatory network structure from data, while it is less important to precisely measure expression levels. Based on this, we define minimal data requirements for gap gene network inference. Our results demonstrate the feasibility of reverse-engineering with much reduced experimental effort. This enables more widespread use of the method in different developmental contexts and organisms. Such systematic application of data-driven models to real-world networks has enormous potential. Only the quantitative investigation of a large number of developmental gene regulatory networks will allow us to discover whether there are rules or regularities governing development and evolution of complex multi-cellular organisms. PMID:22807664

DEFINING THE PLAYERS IN HIGHER-ORDER NETWORKS: PREDICTIVE MODELING FOR REVERSE ENGINEERING FUNCTIONAL INFLUENCE NETWORKS

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

McDermott, Jason E.; Costa, Michelle N.; Stevens, S.L.

A difficult problem that is currently growing rapidly due to the sharp increase in the amount of high-throughput data available for many systems is that of determining useful and informative causative influence networks. These networks can be used to predict behavior given observation of a small number of components, predict behavior at a future time point, or identify components that are critical to the functioning of the system under particular conditions. In these endeavors incorporating observations of systems from a wide variety of viewpoints can be particularly beneficial, but has often been undertaken with the objective of inferring networks thatmore » are generally applicable. The focus of the current work is to integrate both general observations and measurements taken for a particular pathology, that of ischemic stroke, to provide improved ability to produce useful predictions of systems behavior. A number of hybrid approaches have recently been proposed for network generation in which the Gene Ontology is used to filter or enrich network links inferred from gene expression data through reverse engineering methods. These approaches have been shown to improve the biological plausibility of the inferred relationships determined, but still treat knowledge-based and machine-learning inferences as incommensurable inputs. In this paper, we explore how further improvements may be achieved through a full integration of network inference insights achieved through application of the Gene Ontology and reverse engineering methods with specific reference to the construction of dynamic models of transcriptional regulatory networks. We show that integrating two approaches to network construction, one based on reverse-engineering from conditional transcriptional data, one based on reverse-engineering from in situ hybridization data, and another based on functional associations derived from Gene Ontology, using probabilities can improve results of clustering as evaluated by a predictive model of transcriptional expression levels.« less

GMOs in Russia: Research, Society and Legislation

PubMed Central

Korobko, I. V.; Georgiev, P. G.; Skryabin, K. G.; Kirpichnikov, M. P.

2016-01-01

Russian legislation lags behind the rapid developments witnessed in genetic engineering. Only a scientifically based and well-substantiated policy on the place of organisms that are created with the use of genetic engineering technologies and an assessment of the risks associated with them could guarantee that the breakthroughs achieved in modern genetic engineering technologies are effectively put to use in the real economy. A lack of demand for such breakthroughs in the practical field will lead to stagnation in scientific research and to a loss of expertise. PMID:28050262

Engineering bacteria to solve the Burnt Pancake Problem

PubMed Central

Haynes, Karmella A; Broderick, Marian L; Brown, Adam D; Butner, Trevor L; Dickson, James O; Harden, W Lance; Heard, Lane H; Jessen, Eric L; Malloy, Kelly J; Ogden, Brad J; Rosemond, Sabriya; Simpson, Samantha; Zwack, Erin; Campbell, A Malcolm; Eckdahl, Todd T; Heyer, Laurie J; Poet, Jeffrey L

2008-01-01

Background We investigated the possibility of executing DNA-based computation in living cells by engineering Escherichia coli to address a classic mathematical puzzle called the Burnt Pancake Problem (BPP). The BPP is solved by sorting a stack of distinct objects (pancakes) into proper order and orientation using the minimum number of manipulations. Each manipulation reverses the order and orientation of one or more adjacent objects in the stack. We have designed a system that uses site-specific DNA recombination to mediate inversions of genetic elements that represent pancakes within plasmid DNA. Results Inversions (or "flips") of the DNA fragment pancakes are driven by the Salmonella typhimurium Hin/hix DNA recombinase system that we reconstituted as a collection of modular genetic elements for use in E. coli. Our system sorts DNA segments by inversions to produce different permutations of a promoter and a tetracycline resistance coding region; E. coli cells become antibiotic resistant when the segments are properly sorted. Hin recombinase can mediate all possible inversion operations on adjacent flippable DNA fragments. Mathematical modeling predicts that the system reaches equilibrium after very few flips, where equal numbers of permutations are randomly sorted and unsorted. Semiquantitative PCR analysis of in vivo flipping suggests that inversion products accumulate on a time scale of hours or days rather than minutes. Conclusion The Hin/hix system is a proof-of-concept demonstration of in vivo computation with the potential to be scaled up to accommodate larger and more challenging problems. Hin/hix may provide a flexible new tool for manipulating transgenic DNA in vivo. PMID:18492232

[Research progress of genetic engineering on medicinal plants].

PubMed

Teng, Zhong-qiu; Shen, Ye

2015-02-01

The application of genetic engineering technology in modern agriculture shows its outstanding role in dealing with food shortage. Traditional medicinal plant cultivation and collection have also faced with challenges, such as lack of resources, deterioration of environment, germplasm of recession and a series of problems. Genetic engineering can be used to improve the disease resistance, insect resistance, herbicides resistant ability of medicinal plant, also can improve the medicinal plant yield and increase the content of active substances in medicinal plants. Thus, the potent biotechnology can play an important role in protection and large area planting of medicinal plants. In the development of medicinal plant genetic engineering, the safety of transgenic medicinal plants should also be paid attention to. A set of scientific safety evaluation and judgment standard which is suitable for transgenic medicinal plants should be established based on the recognition of the particularity of medicinal plants.

Construction of Gene Regulatory Networks Using Recurrent Neural Networks and Swarm Intelligence.

PubMed

Khan, Abhinandan; Mandal, Sudip; Pal, Rajat Kumar; Saha, Goutam

2016-01-01

We have proposed a methodology for the reverse engineering of biologically plausible gene regulatory networks from temporal genetic expression data. We have used established information and the fundamental mathematical theory for this purpose. We have employed the Recurrent Neural Network formalism to extract the underlying dynamics present in the time series expression data accurately. We have introduced a new hybrid swarm intelligence framework for the accurate training of the model parameters. The proposed methodology has been first applied to a small artificial network, and the results obtained suggest that it can produce the best results available in the contemporary literature, to the best of our knowledge. Subsequently, we have implemented our proposed framework on experimental (in vivo) datasets. Finally, we have investigated two medium sized genetic networks (in silico) extracted from GeneNetWeaver, to understand how the proposed algorithm scales up with network size. Additionally, we have implemented our proposed algorithm with half the number of time points. The results indicate that a reduction of 50% in the number of time points does not have an effect on the accuracy of the proposed methodology significantly, with a maximum of just over 15% deterioration in the worst case.

A synthetic biology approach to engineer a functional reversal of the β-oxidation cycle.

PubMed

Clomburg, James M; Vick, Jacob E; Blankschien, Matthew D; Rodríguez-Moyá, María; Gonzalez, Ramon

2012-11-16

While we have recently constructed a functional reversal of the β-oxidation cycle as a platform for the production of fuels and chemicals by engineering global regulators and eliminating native fermentative pathways, the system-level approach used makes it difficult to determine which of the many deregulated enzymes are responsible for product synthesis. This, in turn, limits efforts to fine-tune the synthesis of specific products and prevents the transfer of the engineered pathway to other organisms. In the work reported here, we overcome the aforementioned limitations by using a synthetic biology approach to construct and functionally characterize a reversal of the β-oxidation cycle. This was achieved through the in vitro kinetic characterization of each functional unit of the core and termination pathways, followed by their in vivo assembly and functional characterization. With this approach, the four functional units of the core pathway, thiolase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydratase, and acyl-CoA dehydrogenase/trans-enoyl-CoA reductase, were purified and kinetically characterized in vitro. When these four functional units were assembled in vivo in combination with thioesterases as the termination pathway, the synthesis of a variety of 4-C carboxylic acids from a one-turn functional reversal of the β-oxidation cycle was realized. The individual expression and modular construction of these well-defined core components exerted the majority of control over product formation, with only highly selective termination pathways resulting in shifts in product formation. Further control over product synthesis was demonstrated by overexpressing a long-chain thiolase that enables the operation of multiple turns of the reversal of the β-oxidation cycle and hence the synthesis of longer-chain carboxylic acids. The well-defined and self-contained nature of each functional unit makes the engineered reversal of the β-oxidation cycle "chassis neutral" and hence transferrable to the host of choice for efficient fuel or chemical production.

Harnessing biodiesel-producing microbes: from genetic engineering of lipase to metabolic engineering of fatty acid biosynthetic pathway.

PubMed

Yan, Jinyong; Yan, Yunjun; Madzak, Catherine; Han, Bingnan

2017-02-01

Microbial production routes, notably whole-cell lipase-mediated biotransformation and fatty-acids-derived biosynthesis, offer new opportunities for synthesizing biodiesel. They compare favorably to immobilized lipase and chemically catalyzed processes. Genetically modified whole-cell lipase-mediated in vitro route, together with in vivo and ex vivo microbial biosynthesis routes, constitutes emerging and rapidly developing research areas for effective production of biodiesel. This review presents recent advances in customizing microorganisms for producing biodiesel, via genetic engineering of lipases and metabolic engineering (including system regulation) of fatty-acids-derived pathways. Microbial hosts used include Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris and Aspergillus oryzae. These microbial cells can be genetically modified to produce lipases under different forms: intracellularly expressed, secreted or surface-displayed. They can be metabolically redesigned and systematically regulated to obtain balanced biodiesel-producing cells, as highlighted in this study. Such genetically or metabolically modified microbial cells can support not only in vitro biotransformation of various common oil feedstocks to biodiesel, but also de novo biosynthesis of biodiesel from glucose, glycerol or even cellulosic biomass. We believe that the genetically tractable oleaginous yeast Yarrowia lipolytica could be developed to an effective biodiesel-producing microbial cell factory. For this purpose, we propose several engineered pathways, based on lipase and wax ester synthase, in this promising oleaginous host.

Intermediate-type vancomycin resistance (VISA) in genetically-distinct Staphylococcus aureus isolates is linked to specific, reversible metabolic alterations.

PubMed

Alexander, Elizabeth L; Gardete, Susana; Bar, Haim Y; Wells, Martin T; Tomasz, Alexander; Rhee, Kyu Y

2014-01-01

Intermediate (VISA-type) vancomycin resistance in Staphylococcus aureus has been associated with a range of physiologic and genetic alterations. Previous work described the emergence of VISA-type resistance in two clonally-distinct series of isolates. In both series (the first belonging to MRSA clone ST8-USA300, and the second to ST5-USA100), resistance was conferred by a single mutation in yvqF (a negative regulator of the vraSR two-component system associated with vancomycin resistance). In the USA300 series, resistance was reversed by a secondary mutation in vraSR. In this study, we combined systems-level metabolomic profiling with statistical modeling techniques to discover specific, reversible metabolic alterations associated with the VISA phenotype.

Engineering Strategies to Decode and Enhance the Genomes of Coral Symbionts.

PubMed

Levin, Rachel A; Voolstra, Christian R; Agrawal, Shobhit; Steinberg, Peter D; Suggett, David J; van Oppen, Madeleine J H

2017-01-01

Elevated sea surface temperatures from a severe and prolonged El Niño event (2014-2016) fueled by climate change have resulted in mass coral bleaching (loss of dinoflagellate photosymbionts, Symbiodinium spp., from coral tissues) and subsequent coral mortality, devastating reefs worldwide. Genetic variation within and between Symbiodinium species strongly influences the bleaching tolerance of corals, thus recent papers have called for genetic engineering of Symbiodinium to elucidate the genetic basis of bleaching-relevant Symbiodinium traits. However, while Symbiodinium has been intensively studied for over 50 years, genetic transformation of Symbiodinium has seen little success likely due to the large evolutionary divergence between Symbiodinium and other model eukaryotes rendering standard transformation systems incompatible. Here, we integrate the growing wealth of Symbiodinium next-generation sequencing data to design tailored genetic engineering strategies. Specifically, we develop a testable expression construct model that incorporates endogenous Symbiodinium promoters, terminators, and genes of interest, as well as an internal ribosomal entry site from a Symbiodinium virus. Furthermore, we assess the potential for CRISPR/Cas9 genome editing through new analyses of the three currently available Symbiodinium genomes. Finally, we discuss how genetic engineering could be applied to enhance the stress tolerance of Symbiodinium , and in turn, coral reefs.

Quiet Clean Short-Haul Experimental Engine (QCSEE) Over-The-Wing (OTW) propulsion system test report. Volume 2: Aerodynamics and performance. [engine performance tests to define propulsion system performance on turbofan engines

NASA Technical Reports Server (NTRS)

1978-01-01

The design and testing of the over the wing engine, a high bypass, geared turbofan engine, are discussed. The propulsion system performance is examined for uninstalled performance and installed performance. The fan aerodynamic performance and the D nozzle and reverser thrust performance are evaluated.

The Science of Solubility: Using Reverse Engineering to Brew a Perfect Cup of Coffee

ERIC Educational Resources Information Center

West, Andrew B.; Sickel, Aaron J.; Cribbs, Jennifer D.

2015-01-01

The Next Generation Science Standards call for the integration of science and engineering. Often, the introduction of engineering activities occurs after instruction in the science content. That is, engineering is used as a way for students to elaborate on science ideas that have already been explored. However, using only this sequence of…

Non-Genetic Engineering Approaches for Isolating and Generating Novel Yeasts for Industrial Applications

NASA Astrophysics Data System (ADS)

Chambers, P. J.; Bellon, J. R.; Schmidt, S. A.; Varela, C.; Pretorius, I. S.

Generating novel yeast strains for industrial applications should be quite straightforward; after all, research into the genetics, biochemistry and physiology of Baker's Yeast, Saccharomyces cerevisiae, has paved the way for many advances in the modern biological sciences. We probably know more about this humble eukaryote than any other, and it is the most tractable of organisms for manipulation using modern genetic engineering approaches. In many countries, however, there are restrictions on the use of genetically-modified organisms (GMOs), particularly in foods and beverages, and the level of consumer acceptance of GMOs is, at best, variable. Thus, many researchers working with industrial yeasts use genetic engineering techniques primarily as research tools, and strain development continues to rely on non-GM technologies. This chapter explores the non-GM tools and strategies available to such researchers.

Perception of risks and benefits of in vitro fertilization, genetic engineering and biotechnology.

PubMed

Macer, D R

1994-01-01

The use of new biotechnology in medicine has become an everyday experience, but many people still express concern about biotechnology. Concerns are evoked particularly by the phrases genetic engineering and in vitro fertilization (IVF), and these concerns persist despite more than a decade of their use in medicine. Mailed nationwide opinion surveys on attitudes to biotechnology were conducted in Japan, among samples of the public (N = 551), high school biology teachers (N = 228), scientists (N = 555) and nurses (N = 301). People do see more benefits coming from science than harm when balanced against the risks. There were especially mixed perceptions of benefit and risk about IVF and genetic engineering, and a relatively high degree of worry compared to other developments of science and technology. A discussion of assisted reproductive technologies and surrogacy in Japan is also made. The opinions of people in Japan were compared to the results of previous surveys conducted in Japan, and international surveys conducted in Australia, China, Europe, New Zealand, U.K. and U.S.A. Japanese have a very high awareness of biotechnology, 97% saying that they had heard of the word. They also have a high level of awareness of IVF and genetic engineering. Genetic engineering was said to be a worthwhile research area for Japan by 76%, while 58% perceived research on IVF as being worthwhile, however 61% were worried about research on IVF or genetic engineering. Japanese expressed more concern about IVF and genetic engineering than New Zealanders. The major reason cited for rejection of genetic manipulation research in Japan and New Zealand was that it was seen as interfering with nature, playing God or as unethical. The emotions concerning these technologies are complex, and we should avoid using simplistic public opinion data as measures of public perceptions. The level of concern expressed by scientists and teachers in Japan suggest that public education "technology promotion campaigns" will not reduce concern about science and technology. Such concern should be valued as discretion that is basic to increasing the bioethical maturity of a society, rather than being feared.

Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast.

PubMed

Huang, Mingtao; Bai, Yunpeng; Sjostrom, Staffan L; Hallström, Björn M; Liu, Zihe; Petranovic, Dina; Uhlén, Mathias; Joensson, Haakan N; Andersson-Svahn, Helene; Nielsen, Jens

2015-08-25

There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant α-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.

78 FR 25941 - Stine Seed Farm, Inc.; Extension of a Determination of Nonregulated Status of Corn Genetically...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-05-03

... Engineered for Herbicide Resistance AGENCY: Animal and Plant Health Inspection Service, USDA. ACTION: Notice... maize line HCEM485, which has been genetically engineered to be resistant to the herbicide glyphosate...

Notification: Evaluation of Office of Pesticide Programs’ Genetically Engineered Corn Insect Resistance Management

EPA Pesticide Factsheets

Project #OPE-FY15-0055, July 09, 2015. The EPA OIG plans to begin preliminary research on the EPA's ability to manage and prevent increased insect resistance to genetically engineered Bacillus thuringiensis (Bt) corn.

Germline Cas9 expression yields highly efficient genome engineering in a major worldwide disease vector, Aedes aegypti

PubMed Central

Li, Ming; Bui, Michelle; Yang, Ting; Bowman, Christian S.; White, Bradley J.; Akbari, Omar S.

2017-01-01

The development of CRISPR/Cas9 technologies has dramatically increased the accessibility and efficiency of genome editing in many organisms. In general, in vivo germline expression of Cas9 results in substantially higher activity than embryonic injection. However, no transgenic lines expressing Cas9 have been developed for the major mosquito disease vector Aedes aegypti. Here, we describe the generation of multiple stable, transgenic Ae. aegypti strains expressing Cas9 in the germline, resulting in dramatic improvements in both the consistency and efficiency of genome modifications using CRISPR. Using these strains, we disrupted numerous genes important for normal morphological development, and even generated triple mutants from a single injection. We have also managed to increase the rates of homology-directed repair by more than an order of magnitude. Given the exceptional mutagenic efficiency and specificity of the Cas9 strains we engineered, they can be used for high-throughput reverse genetic screens to help functionally annotate the Ae. aegypti genome. Additionally, these strains represent a step toward the development of novel population control technologies targeting Ae. aegypti that rely on Cas9-based gene drives. PMID:29138316

Germline Cas9 expression yields highly efficient genome engineering in a major worldwide disease vector, Aedes aegypti.

PubMed

Li, Ming; Bui, Michelle; Yang, Ting; Bowman, Christian S; White, Bradley J; Akbari, Omar S

2017-12-05

The development of CRISPR/Cas9 technologies has dramatically increased the accessibility and efficiency of genome editing in many organisms. In general, in vivo germline expression of Cas9 results in substantially higher activity than embryonic injection. However, no transgenic lines expressing Cas9 have been developed for the major mosquito disease vector Aedes aegypti Here, we describe the generation of multiple stable, transgenic Ae. aegypti strains expressing Cas9 in the germline, resulting in dramatic improvements in both the consistency and efficiency of genome modifications using CRISPR. Using these strains, we disrupted numerous genes important for normal morphological development, and even generated triple mutants from a single injection. We have also managed to increase the rates of homology-directed repair by more than an order of magnitude. Given the exceptional mutagenic efficiency and specificity of the Cas9 strains we engineered, they can be used for high-throughput reverse genetic screens to help functionally annotate the Ae. aegypti genome. Additionally, these strains represent a step toward the development of novel population control technologies targeting Ae. aegypti that rely on Cas9-based gene drives. Copyright © 2017 the Author(s). Published by PNAS.

Fruit metabolite networks in engineered and non-engineered tomato genotypes reveal fluidity in a hormone and agroecosystem specific manner.

PubMed

Fatima, Tahira; Sobolev, Anatoly P; Teasdale, John R; Kramer, Matthew; Bunce, Jim; Handa, Avtar K; Mattoo, Autar K

Metabolomics provides a view of endogenous metabolic patterns not only during plant growth, development and senescence but also in response to genetic events, environment and disease. The effects of the field environment on plant hormone-specific metabolite profiles are largely unknown. Few studies have analyzed useful phenotypes generated by introducing single or multiple gene events alongside the non-engineered wild type control at field scale to determine the robustness of the genetic trait and its modulation in the metabolome as a function of specific agroecosystem environments. We evaluated the influence of genetic background (high polyamine lines; low methyl jasmonate line; low ethylene line; and isogenic genotypes carrying double transgenic events) and environments (hairy vetch, rye, plastic black mulch and bare soil mulching systems) on the metabolomic profile of isogenic reverse genetic mutations and selected mulch based cropping systems in tomato fruit. Net photosynthesis and fruit yield were also determined. NMR spectroscopy was used for quantifying metabolites that are central to primary metabolism. We analyzed both the first moment (means) of metabolic response to genotypes and agroecosystems by traditional univariate/multivariate methods, and the second moment (covariances) of responses by creating networks that depicted changes in correlations of paired metabolites. This particular approach is novel and was necessary because our experimental material yielded highly variable metabolic responses that could not be easily understood using the traditional analytical approaches for first moment statistics. High endogenous spermidine and spermine content exhibited strong effects on amino acids, Krebs cycle intermediates and energy molecules (ADP + ATP) in ripening fruits of plants grown under different agroecosystem environments. The metabolic response to high polyamine genotypes was similar to the response to hairy vetch cover crop mulch; supported by the pattern of changes in correlation between metabolites. Changes in primary metabolites of genotypes mutated for the deficiency of ethylene or methyl jasmonate were unique under all growth conditions and opposite of high polyamine genotype results. The high polyamine trait was found to dominate the low ethylene and low jasmonate mutations under field conditions. For several metabolites low ethylene and low methyl jasmonate genotypes had an inverse relationship. Collectively, these results affirm that interactions between metabolite pathways and growth environments are affected by genotype, and influence the metabolite quality of a crop. This study portrays how metabolite relationships change, both in mean and in correlation, under different genotypic and environmental conditions. Although these networks are surprisingly dynamic, we also find examples of selectively conserved associations.

Mitigation of reversible self-association and viscosity in a human IgG1 monoclonal antibody by rational, structure-guided Fv engineering

PubMed Central

Geoghegan, James C.; Fleming, Ryan; Damschroder, Melissa; Bishop, Steven M.; Sathish, Hasige A.; Esfandiary, Reza

2016-01-01

ABSTRACT Undesired solution behaviors such as reversible self-association (RSA), high viscosity, and liquid-liquid phase separation can introduce substantial challenges during development of monoclonal antibody formulations. Although a global mechanistic understanding of RSA (i.e., native and reversible protein-protein interactions) is sufficient to develop robust formulation controls, its mitigation via protein engineering requires knowledge of the sites of protein-protein interactions. In the study reported here, we coupled our previous hydrogen-deuterium exchange mass spectrometry findings with structural modeling and in vitro screening to identify the residues responsible for RSA of a model IgG1 monoclonal antibody (mAb-C), and rationally engineered variants with improved solution properties (i.e., reduced RSA and viscosity). Our data show that mutation of either solvent-exposed aromatic residues within the heavy and light chain variable regions or buried residues within the heavy chain/light chain interface can significantly mitigate RSA and viscosity by reducing the IgG's surface hydrophobicity. The engineering strategy described here highlights the utility of integrating complementary experimental and in silico methods to identify mutations that can improve developability, in particular, high concentration solution properties, of candidate therapeutic antibodies. PMID:27050875

From Precaution to Peril: Public Relations Across Forty Years of Genetic Engineering.

PubMed

Hogan, Andrew J

2016-12-01

The Asilomar conference on genetic engineering in 1975 has long been pointed to by scientists as a model for internal regulation and public engagement. In 2015, the organizers of the International Summit on Human Gene Editing in Washington, DC looked to Asilomar as they sought to address the implications of the new CRISPR gene editing technique. Like at Asilomar, the conveners chose to limit the discussion to a narrow set of potential CRISPR applications, involving inheritable human genome editing. The adoption by scientists in 2015 of an Asilomar-like script for discussing genetic engineering offers historians the opportunity to analyze the adjustments that have been made since 1975, and to identify the blind spots that remain in public engagement. Scientists did take important lessons from the fallout of their limited engagement with public concerns at Asilomar. Nonetheless, the scientific community has continued to overlook some of the longstanding public concerns about genetic engineering, in particular the broad and often covert genetic modification of food products. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genetically engineered mouse models for studying inflammatory bowel disease.

PubMed

Mizoguchi, Atsushi; Takeuchi, Takahito; Himuro, Hidetomo; Okada, Toshiyuki; Mizoguchi, Emiko

2016-01-01

Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory condition that is mediated by very complex mechanisms controlled by genetic, immune, and environmental factors. More than 74 kinds of genetically engineered mouse strains have been established since 1993 for studying IBD. Although mouse models cannot fully reflect human IBD, they have provided significant contributions for not only understanding the mechanism, but also developing new therapeutic means for IBD. Indeed, 20 kinds of genetically engineered mouse models carry the susceptibility genes identified in human IBD, and the functions of some other IBD susceptibility genes have also been dissected out using mouse models. Cutting-edge technologies such as cell-specific and inducible knockout systems, which were recently employed to mouse IBD models, have further enhanced the ability of investigators to provide important and unexpected rationales for developing new therapeutic strategies for IBD. In this review article, we briefly introduce 74 kinds of genetically engineered mouse models that spontaneously develop intestinal inflammation. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Development of a reverse genetics system to generate a recombinant Ebola virus Makona expressing a green fluorescent protein

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

Albariño, César G., E-mail: calbarino@cdc.gov; Wiggleton Guerrero, Lisa; Lo, Michael K.

Previous studies have demonstrated the potential application of reverse genetics technology in studying a broad range of aspects of viral biology, including gene regulation, protein function, cell entry, and pathogenesis. Here, we describe a highly efficient reverse genetics system used to generate recombinant Ebola virus (EBOV) based on a recent isolate from a human patient infected during the 2014–2015 outbreak in Western Africa. We also rescued a recombinant EBOV expressing a fluorescent reporter protein from a cleaved VP40 protein fusion. Using this virus and an inexpensive method to quantitate the expression of the foreign gene, we demonstrate its potential usefulnessmore » as a tool for screening antiviral compounds and measuring neutralizing antibodies. - Highlights: • Recombinant Ebola virus (EBOV) derived from Makona variant was rescued. • New protocol for viral rescue allows 100% efficiency. • Modified EBOV expresses a green fluorescent protein from a VP40-fused protein. • Modified EBOV was tested as tool to screen antiviral compounds and measure neutralizing antibodies.« less

Science, law, and politics in the Food and Drug Administration's genetically engineered foods policy: FDA's 1992 policy statement.

PubMed

Pelletier, David L

2005-05-01

The US Food and Drug Administration's (FDA's) 1992 policy statement was developed in the context of critical gaps in scientific knowledge concerning the compositional effects of genetic transformation and severe limitations in methods for safety testing. FDA acknowledged that pleiotropy and insertional mutagenesis may cause unintended changes, but it was unknown whether this happens to a greater extent in genetic engineering compared with traditional breeding. Moreover, the agency was not able to identify methods by which producers could screen for unintended allergens and toxicants. Despite these uncertainties, FDA granted genetically engineered foods the presumption of GRAS (Generally Recognized As Safe) and recommended that producers use voluntary consultations before marketing them.

Notification: Evaluation of EPA's Management of Resistance Issues Related to Herbicide Tolerant Genetically Engineered Crops

EPA Pesticide Factsheets

Project #OPE-FY16-0023, March 25, 2016. The EPA OIG plans to begin preliminary research to assess the EPA's management and oversight of resistance issues related to herbicide tolerant genetically engineered crops.

The establishment of genetically engineered canola populations in the U.S.

EPA Science Inventory

Concerns regarding the commercial release of genetically engineered (GE) crops include naturalization, introgression to sexually compatible relatives and the transfer of beneficial traits to native and weedy species through hybridization. To date there have been few documented re...

Expanding and reprogramming the genetic code.

PubMed

Chin, Jason W

2017-10-04

Nature uses a limited, conservative set of amino acids to synthesize proteins. The ability to genetically encode an expanded set of building blocks with new chemical and physical properties is transforming the study, manipulation and evolution of proteins, and is enabling diverse applications, including approaches to probe, image and control protein function, and to precisely engineer therapeutics. Underpinning this transformation are strategies to engineer and rewire translation. Emerging strategies aim to reprogram the genetic code so that noncanonical biopolymers can be synthesized and evolved, and to test the limits of our ability to engineer the translational machinery and systematically recode genomes.

Recent advances in genetic modification systems for Actinobacteria.

PubMed

Deng, Yu; Zhang, Xi; Zhang, Xiaojuan

2017-03-01

Actinobacteria are extremely important to human health, agriculture, and forests. Because of the vast differences of the characteristics of Actinobacteria, a lot of genetic tools have been developed for efficiently manipulating the genetics. Although there are a lot of successful examples of engineering Actinobacteria, they are still more difficult to be genetically manipulated than other model microorganisms such as Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis etc. due to the diverse genomics and biochemical machinery. Here, we review the methods to introduce heterologous DNA into Actinobacteria and the available genetic modification tools. The trends and problems existing in engineering Actinobacteria are also covered.

Project-Based Teaching-Learning Computer-Aided Engineering Tools

ERIC Educational Resources Information Center

Simoes, J. A.; Relvas, C.; Moreira, R.

2004-01-01

Computer-aided design, computer-aided manufacturing, computer-aided analysis, reverse engineering and rapid prototyping are tools that play an important key role within product design. These are areas of technical knowledge that must be part of engineering and industrial design courses' curricula. This paper describes our teaching experience of…

Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis.

PubMed

Enyeart, Peter J; Mohr, Georg; Ellington, Andrew D; Lambowitz, Alan M

2014-01-13

Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into 'targetrons.' Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and 'cut-and-pastes' (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA-seq). The high processivity and fidelity of group II intron reverse transcriptases along with their novel template-switching activity, which can directly link RNA-seq adaptor sequences to cDNAs during reverse transcription, open new approaches for RNA-seq and the identification and profiling of non-coding RNAs, with potentially wide applications in research and biotechnology.

More than Just Hot Air: How Hairdryers and Role Models Inspire Girls in Engineering

ERIC Educational Resources Information Center

Kekelis, Linda; Larkin, Molly; Gomes, Lyn

2014-01-01

This article describes a reverse-engineering project where female students take a part a hair dryer--giving them an opportunity to see the many different kinds of engineering disciplines involved in making a hairdryer and that they work together. Mechanical Engineer, Lyn Gome, describes her experience leading a group of middle school girls through…

Profile of an Effective Engineering Manager at the Naval Avionics Center

DTIC Science & Technology

1991-06-01

GROUP Leadership ; Engineering Management Effectiveness; Engineers; Engineering Managers ; Naval Avionics Center 19 ABSTR. T (Continue on reverse if...Personnel. The purpose of the Institute is to support the implementation of the NAC Leadership / Management Principles throughout NAC. The Leadership ... Management Principles are as follows: - Develc 2 and Maintain a Corporate Outlook. - Communicate the Organizational Vision through Positive Leadership

Phenotypes on demand via switchable target protein degradation in multicellular organisms

PubMed Central

Faden, Frederik; Ramezani, Thomas; Mielke, Stefan; Almudi, Isabel; Nairz, Knud; Froehlich, Marceli S.; Höckendorff, Jörg; Brandt, Wolfgang; Hoehenwarter, Wolfgang; Dohmen, R. Jürgen; Schnittger, Arp; Dissmeyer, Nico

2016-01-01

Phenotypes on-demand generated by controlling activation and accumulation of proteins of interest are invaluable tools to analyse and engineer biological processes. While temperature-sensitive alleles are frequently used as conditional mutants in microorganisms, they are usually difficult to identify in multicellular species. Here we present a versatile and transferable, genetically stable system based on a low-temperature-controlled N-terminal degradation signal (lt-degron) that allows reversible and switch-like tuning of protein levels under physiological conditions in vivo. Thereby, developmental effects can be triggered and phenotypes on demand generated. The lt-degron was established to produce conditional and cell-type-specific phenotypes and is generally applicable in a wide range of organisms, from eukaryotic microorganisms to plants and poikilothermic animals. We have successfully applied this system to control the abundance and function of transcription factors and different enzymes by tunable protein accumulation. PMID:27447739

Elastin-like polypeptides: Therapeutic applications for an emerging class of nanomedicines.

PubMed

Despanie, Jordan; Dhandhukia, Jugal P; Hamm-Alvarez, Sarah F; MacKay, J Andrew

2016-10-28

Elastin-like polypeptides (ELPs) constitute a genetically engineered class of 'protein polymers' derived from human tropoelastin. They exhibit a reversible phase separation whereby samples remain soluble below a transition temperature (T t ) but form amorphous coacervates above T t . Their phase behavior has many possible applications in purification, sensing, activation, and nanoassembly. As humanized polypeptides, they are non-immunogenic, substrates for proteolytic biodegradation, and can be decorated with pharmacologically active peptides, proteins, and small molecules. Recombinant synthesis additionally allows precise control over ELP architecture and molecular weight, resulting in protein polymers with uniform physicochemical properties suited to the design of multifunctional biologics. As such, ELPs have been employed for various uses including as anti-cancer agents, ocular drug delivery vehicles, and protein trafficking modulators. This review aims to offer the reader a catalogue of ELPs, their various applications, and potential for commercialization across a broad spectrum of fields. Copyright © 2015. Published by Elsevier B.V.

Repurposing endogenous type I CRISPR-Cas systems for programmable gene repression

PubMed Central

Luo, Michelle L.; Mullis, Adam S.; Leenay, Ryan T.; Beisel, Chase L.

2015-01-01

CRISPR-Cas systems have shown tremendous promise as heterologous tools for genome editing and transcriptional regulation. Because these RNA-directed immune systems are found in most prokaryotes, an opportunity exists to harness the endogenous systems as convenient tools in these organisms. Here, we report that the Type I-E CRISPR-Cas system in Escherichia coli can be co-opted for programmable transcriptional repression. We found that deletion of the signature cas3 gene converted this immune system into a programmable gene regulator capable of reversible gene silencing of heterologous and endogenous genes. Targeting promoter regions yielded the strongest repression, whereas targeting coding regions showed consistent strand bias. Furthermore, multi-targeting CRISPR arrays could generate complex phenotypes. This strategy offers a simple approach to convert many endogenous Type I systems into transcriptional regulators, thereby expanding the available toolkit for CRISPR-mediated genetic control while creating new opportunities for genome-wide screens and pathway engineering. PMID:25326321

Hybrid grammar-based approach to nonlinear dynamical system identification from biological time series

NASA Astrophysics Data System (ADS)

McKinney, B. A.; Crowe, J. E., Jr.; Voss, H. U.; Crooke, P. S.; Barney, N.; Moore, J. H.

2006-02-01

We introduce a grammar-based hybrid approach to reverse engineering nonlinear ordinary differential equation models from observed time series. This hybrid approach combines a genetic algorithm to search the space of model architectures with a Kalman filter to estimate the model parameters. Domain-specific knowledge is used in a context-free grammar to restrict the search space for the functional form of the target model. We find that the hybrid approach outperforms a pure evolutionary algorithm method, and we observe features in the evolution of the dynamical models that correspond with the emergence of favorable model components. We apply the hybrid method to both artificially generated time series and experimentally observed protein levels from subjects who received the smallpox vaccine. From the observed data, we infer a cytokine protein interaction network for an individual’s response to the smallpox vaccine.

The Promise of Chimeric Antigen Receptor Engineered T cells in the Treatment of Hematologic Malignancies

PubMed Central

Nagle, Sarah J.; Garfall, Alfred L.; Stadtmauer, Edward A.

2015-01-01

Relapsed and refractory hematologic malignancies have a very poor prognosis. Chimeric antigen receptor (CAR) T cells are emerging as a powerful therapy in this setting. Early clinical trials of genetically modified T cells for the treatment of non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL) have shown high complete response rates in patients with few therapeutic options. Exploration is ongoing for other hematologic malignancies including multiple myeloma (MM), acute myeloid leukemia (AML) and Hodgkin lymphoma (HL). At the same time, the design and production of CAR T cells is being advanced so that this therapy can be more widely utilized. Cytokine release syndrome (CRS) and neurotoxicity are common, but they are treatable and fully reversible. This review will review currently available data as well as future developments and challenges in the field. PMID:26841014

Genetically Engineered Pig Models for Human Diseases

PubMed Central

Prather, Randall S.; Lorson, Monique; Ross, Jason W.; Whyte, Jeffrey J.; Walters, Eric

2015-01-01

Although pigs are used widely as models of human disease, their utility as models has been enhanced by genetic engineering. Initially, transgenes were added randomly to the genome, but with the application of homologous recombination, zinc finger nucleases, and transcription activator-like effector nuclease (TALEN) technologies, now most any genetic change that can be envisioned can be completed. To date these genetic modifications have resulted in animals that have the potential to provide new insights into human diseases for which a good animal model did not exist previously. These new animal models should provide the preclinical data for treatments that are developed for diseases such as Alzheimer's disease, cystic fibrosis, retinitis pigmentosa, spinal muscular atrophy, diabetes, and organ failure. These new models will help to uncover aspects and treatments of these diseases that were otherwise unattainable. The focus of this review is to describe genetically engineered pigs that have resulted in models of human diseases. PMID:25387017

Analysis of the flow field generated near an aircraft engine operating in reverse thrust. M.S. Thesis

NASA Technical Reports Server (NTRS)

Ledwith, W. A., Jr.

1972-01-01

A computer solution is developed to the exhaust gas reingestion problem for aircraft operating in the reverse thrust mode on a crosswind-free runway. The computer program determines the location of the inlet flow pattern, whether the exhaust efflux lies within the inlet flow pattern or not, and if so, the approximate time before the reversed flow reaches the engine inlet. The program is written so that the user is free to select discrete runway speeds or to study the entire aircraft deceleration process for both the far field and cross-ingestion problems. While developed with STOL applications in mind, the solution is equally applicable to conventional designs. The inlet and reversed jet flow fields involved in the problem are assumed to be noninteracting. The nacelle model used in determining the inlet flow field is generated using an iterative solution to the Neuman problem from potential flow theory while the reversed jet flow field is adapted using an empirical correlation from the literature. Sample results obtained using the program are included.

Synthetic alienation of microbial organisms by using genetic code engineering: Why and how?

PubMed

Kubyshkin, Vladimir; Budisa, Nediljko

2017-08-01

The main goal of synthetic biology (SB) is the creation of biodiversity applicable for biotechnological needs, while xenobiology (XB) aims to expand the framework of natural chemistries with the non-natural building blocks in living cells to accomplish artificial biodiversity. Protein and proteome engineering, which overcome limitation of the canonical amino acid repertoire of 20 (+2) prescribed by the genetic code by using non-canonic amino acids (ncAAs), is one of the main focuses of XB research. Ideally, estranging the genetic code from its current form via systematic introduction of ncAAs should enable the development of bio-containment mechanisms in synthetic cells potentially endowing them with a "genetic firewall" i.e. orthogonality which prevents genetic information transfer to natural systems. Despite rapid progress over the past two decades, it is not yet possible to completely alienate an organism that would use and maintain different genetic code associations permanently. In order to engineer robust bio-contained life forms, the chemical logic behind the amino acid repertoire establishment should be considered. Starting from recent proposal of Hartman and Smith about the genetic code establishment in the RNA world, here the authors mapped possible biotechnological invasion points for engineering of bio-contained synthetic cells equipped with non-canonical functionalities. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Induction of atherosclerosis in mice and hamsters without germline genetic engineering.

PubMed

Bjørklund, Martin Maeng; Hollensen, Anne Kruse; Hagensen, Mette Kallestrup; Dagnaes-Hansen, Frederik; Christoffersen, Christina; Mikkelsen, Jacob Giehm; Bentzon, Jacob Fog

2014-05-23

Atherosclerosis can be achieved in animals by germline genetic engineering, leading to hypercholesterolemia, but such models are constrained to few species and strains, and they are difficult to combine with other powerful techniques involving genetic manipulation or variation. To develop a method for induction of atherosclerosis without germline genetic engineering. Recombinant adeno-associated viral vectors were engineered to encode gain-of-function proprotein convertase subtilisin/kexin type 9 mutants, and mice were given a single intravenous vector injection followed by high-fat diet feeding. Plasma proprotein convertase subtilisin/kexin type 9 and total cholesterol increased rapidly and were maintained at high levels, and after 12 weeks, mice had atherosclerotic lesions in the aorta. Histology of the aortic root showed progression of lesions to the fibroatheromatous stage. To demonstrate the applicability of this method for rapid analysis of the atherosclerosis susceptibility of a mouse strain and for providing temporal control over disease induction, we demonstrated the accelerated atherosclerosis of mature diabetic Akita mice. Furthermore, the versatility of this approach for creating atherosclerosis models also in nonmurine species was demonstrated by inducing hypercholesterolemia and early atherosclerosis in Golden Syrian hamsters. Single injections of proprotein convertase subtilisin/kexin type 9-encoding recombinant adeno-associated viral vectors are a rapid and versatile method to induce atherosclerosis in animals. This method should prove useful for experiments that are high-throughput or involve genetic techniques, strains, or species that do not combine well with current genetically engineered models. © 2014 American Heart Association, Inc.

Genetic engineering for skeletal regenerative medicine.

PubMed

Gersbach, Charles A; Phillips, Jennifer E; García, Andrés J

2007-01-01

The clinical challenges of skeletal regenerative medicine have motivated significant advances in cellular and tissue engineering in recent years. In particular, advances in molecular biology have provided the tools necessary for the design of gene-based strategies for skeletal tissue repair. Consequently, genetic engineering has emerged as a promising method to address the need for sustained and robust cellular differentiation and extracellular matrix production. As a result, gene therapy has been established as a conventional approach to enhance cellular activities for skeletal tissue repair. Recent literature clearly demonstrates that genetic engineering is a principal factor in constructing effective methods for tissue engineering approaches to bone, cartilage, and connective tissue regeneration. This review highlights this literature, including advances in the development of efficacious gene carriers, novel cell sources, successful delivery strategies, and optimal target genes. The current status of the field and the challenges impeding the clinical realization of these approaches are also discussed.

Building of Reusable Reverse Logistics Model and its Optimization Considering the Decision of Backorder or Next Arrival of Goods

NASA Astrophysics Data System (ADS)

Lee, Jeong-Eun; Gen, Mitsuo; Rhee, Kyong-Gu; Lee, Hee-Hyol

This paper deals with the building of the reusable reverse logistics model considering the decision of the backorder or the next arrival of goods. The optimization method to minimize the transportation cost and to minimize the volume of the backorder or the next arrival of goods occurred by the Just in Time delivery of the final delivery stage between the manufacturer and the processing center is proposed. Through the optimization algorithms using the priority-based genetic algorithm and the hybrid genetic algorithm, the sub-optimal delivery routes are determined. Based on the case study of a distilling and sale company in Busan in Korea, the new model of the reusable reverse logistics of empty bottles is built and the effectiveness of the proposed method is verified.

copia-like retrotransposons are ubiquitous among plants.

PubMed Central

Voytas, D F; Cummings, M P; Koniczny, A; Ausubel, F M; Rodermel, S R

1992-01-01

Transposable genetic elements are assumed to be a feature of all eukaryotic genomes. Their identification, however, has largely been haphazard, limited principally to organisms subjected to molecular or genetic scrutiny. We assessed the phylogenetic distribution of copia-like retrotransposons, a class of transposable element that proliferates by reverse transcription, using a polymerase chain reaction assay designed to detect copia-like element reverse transcriptase sequences. copia-like retrotransposons were identified in 64 plant species as well as the photosynthetic protist Volvox carteri. The plant species included representatives from 9 of 10 plant divisions, including bryophytes, lycopods, ferns, gymnosperms, and angiosperms. DNA sequence analysis of 29 cloned PCR products and of a maize retrotransposon cDNA confirmed the identity of these sequences as copia-like reverse transcriptase sequences, thereby demonstrating that this class of retrotransposons is a ubiquitous component of plant genomes. Images PMID:1379734

Genetically Engineering Entomopathogenic Fungi.

PubMed

Zhao, H; Lovett, B; Fang, W

2016-01-01

Entomopathogenic fungi have been developed as environmentally friendly alternatives to chemical insecticides in biocontrol programs for agricultural pests and vectors of disease. However, mycoinsecticides currently have a small market share due to low virulence and inconsistencies in their performance. Genetic engineering has made it possible to significantly improve the virulence of fungi and their tolerance to adverse conditions. Virulence enhancement has been achieved by engineering fungi to express insect proteins and insecticidal proteins/peptides from insect predators and other insect pathogens, or by overexpressing the pathogen's own genes. Importantly, protein engineering can be used to mix and match functional domains from diverse genes sourced from entomopathogenic fungi and other organisms, producing insecticidal proteins with novel characteristics. Fungal tolerance to abiotic stresses, especially UV radiation, has been greatly improved by introducing into entomopathogens a photoreactivation system from an archaean and pigment synthesis pathways from nonentomopathogenic fungi. Conversely, gene knockout strategies have produced strains with reduced ecological fitness as recipients for genetic engineering to improve virulence; the resulting strains are hypervirulent, but will not persist in the environment. Coupled with their natural insect specificity, safety concerns can also be mitigated by using safe effector proteins with selection marker genes removed after transformation. With the increasing public concern over the continued use of synthetic chemical insecticides and growing public acceptance of genetically modified organisms, new types of biological insecticides produced by genetic engineering offer a range of environmentally friendly options for cost-effective control of insect pests. Copyright © 2016 Elsevier Inc. All rights reserved.

Combustion Light Gas Gun Technology Demonstration

DTIC Science & Technology

2007-01-23

J. G. Handbook of Cryogenic Engineering. Philadelphia: Taylor and Francis, 1998. ISBN 1-56032-332-9 Myth #2 from “Twenty Hydrogen Myths” by...the second using Helium-refrigerated reverse Brayton cycle manufactured by Linde. Neither system was designed specifically for naval applications...8 Since floor space is of a premium, the helium refrigerated reverse Brayton cycle is the system of primary current interest. The reverse Brayton

Reverse Engineering of Genome-wide Gene Regulatory Networks from Gene Expression Data

PubMed Central

Liu, Zhi-Ping

2015-01-01

Transcriptional regulation plays vital roles in many fundamental biological processes. Reverse engineering of genome-wide regulatory networks from high-throughput transcriptomic data provides a promising way to characterize the global scenario of regulatory relationships between regulators and their targets. In this review, we summarize and categorize the main frameworks and methods currently available for inferring transcriptional regulatory networks from microarray gene expression profiling data. We overview each of strategies and introduce representative methods respectively. Their assumptions, advantages, shortcomings, and possible improvements and extensions are also clarified and commented. PMID:25937810

Variable neighborhood search for reverse engineering of gene regulatory networks.

PubMed

Nicholson, Charles; Goodwin, Leslie; Clark, Corey

2017-01-01

A new search heuristic, Divided Neighborhood Exploration Search, designed to be used with inference algorithms such as Bayesian networks to improve on the reverse engineering of gene regulatory networks is presented. The approach systematically moves through the search space to find topologies representative of gene regulatory networks that are more likely to explain microarray data. In empirical testing it is demonstrated that the novel method is superior to the widely employed greedy search techniques in both the quality of the inferred networks and computational time. Copyright © 2016 Elsevier Inc. All rights reserved.

GRASP/Ada: Graphical Representations of Algorithms, Structures, and Processes for Ada. The development of a program analysis environment for Ada: Reverse engineering tools for Ada, task 2, phase 3

NASA Technical Reports Server (NTRS)

Cross, James H., II

1991-01-01

The main objective is the investigation, formulation, and generation of graphical representations of algorithms, structures, and processes for Ada (GRASP/Ada). The presented task, in which various graphical representations that can be extracted or generated from source code are described and categorized, is focused on reverse engineering. The following subject areas are covered: the system model; control structure diagram generator; object oriented design diagram generator; user interface; and the GRASP library.

Effects of an in-flight thrust reverser on the stability and control characteristics of a single-engine fighter airplane model

NASA Technical Reports Server (NTRS)

Mercer, C. E.; Maiden, D. L.

1972-01-01

The changes in thrust minus drag performance as well as longitudinal and directional stability and control characteristics of a single-engine jet aircraft attributable to an in-flight thrust reverser of the blocker-deflector door type were investigated in a 16-foot transonic wind tunnel. The longitudinal and directional stability data are presented. Test conditions simulated landing approach conditions as well as high speed maneuvering such as may be required for combat or steep descent from high altitude.

Development of high temperature liquid lubricants for low-heat rejection: Heavy duty diesel engines

NASA Technical Reports Server (NTRS)

Wiczynski, P. D.; Marolewski, T. A.

1993-01-01

The objective of this DOE program was to develop a liquid lubricant that will allow advanced diesel engines to operate at top ring reversal temperatures approaching 500 C and sump temperatures approaching 250 C. The lubricants developed demonstrated at marginal increase in sump temperature capability, approximately 15 C, and an increase in top ring reversal temperature. A 15W-40 synthetic lubricant designated HTL-4 was the best lubricant developed in terms of stability, wear control, deposit control dispersancy, and particulate emissions.

Silk Materials Functionalized via Genetic Engineering for Biomedical Applications.

PubMed

Deptuch, Tomasz; Dams-Kozlowska, Hanna

2017-12-12

The great mechanical properties, biocompatibility and biodegradability of silk-based materials make them applicable to the biomedical field. Genetic engineering enables the construction of synthetic equivalents of natural silks. Knowledge about the relationship between the structure and function of silk proteins enables the design of bioengineered silks that can serve as the foundation of new biomaterials. Furthermore, in order to better address the needs of modern biomedicine, genetic engineering can be used to obtain silk-based materials with new functionalities. Sequences encoding new peptides or domains can be added to the sequences encoding the silk proteins. The expression of one cDNA fragment indicates that each silk molecule is related to a functional fragment. This review summarizes the proposed genetic functionalization of silk-based materials that can be potentially useful for biomedical applications.

[Development of computer aided forming techniques in manufacturing scaffolds for bone tissue engineering].

PubMed

Wei, Xuelei; Dong, Fuhui

2011-12-01

To review recent advance in the research and application of computer aided forming techniques for constructing bone tissue engineering scaffolds. The literature concerning computer aided forming techniques for constructing bone tissue engineering scaffolds in recent years was reviewed extensively and summarized. Several studies over last decade have focused on computer aided forming techniques for bone scaffold construction using various scaffold materials, which is based on computer aided design (CAD) and bone scaffold rapid prototyping (RP). CAD include medical CAD, STL, and reverse design. Reverse design can fully simulate normal bone tissue and could be very useful for the CAD. RP techniques include fused deposition modeling, three dimensional printing, selected laser sintering, three dimensional bioplotting, and low-temperature deposition manufacturing. These techniques provide a new way to construct bone tissue engineering scaffolds with complex internal structures. With rapid development of molding and forming techniques, computer aided forming techniques are expected to provide ideal bone tissue engineering scaffolds.

Genetic Engineering and Crop Production.

ERIC Educational Resources Information Center

Jones, Helen C.; Frost, S.

1991-01-01

With a spotlight upon current agricultural difficulties and environmental dilemmas, this paper considers both the extant and potential applications of genetic engineering with respect to crop production. The nonagricultural factors most likely to sway the impact of this emergent technology upon future crop production are illustrated. (JJK)

A CAL Program to Teach the Basic Principles of Genetic Engineering--A Change from the Traditional Approach.

ERIC Educational Resources Information Center

Dewhurst, D. G.; And Others

1989-01-01

An interactive computer-assisted learning program written for the BBC microcomputer to teach the basic principles of genetic engineering is described. Discussed are the hardware requirements software, use of the program, and assessment. (Author/CW)

IMPROVING PLANT GENETIC ENGINEERING BY MANIPULATING THE HOST. (R829479C001)

EPA Science Inventory

Agrobacterium-mediated transformation is a major technique for the genetic engineering of plants. However, there are many economically important crop and tree species that remain highly recalcitrant to Agrobacterium infection. Although attempts have been made to ...

A portable expression resource for engineering cross-species genetic circuits and pathways

PubMed Central

Kushwaha, Manish; Salis, Howard M.

2015-01-01

Genetic circuits and metabolic pathways can be reengineered to allow organisms to process signals and manufacture useful chemicals. However, their functions currently rely on organism-specific regulatory parts, fragmenting synthetic biology and metabolic engineering into host-specific domains. To unify efforts, here we have engineered a cross-species expression resource that enables circuits and pathways to reuse the same genetic parts, while functioning similarly across diverse organisms. Our engineered system combines mixed feedback control loops and cross-species translation signals to autonomously self-regulate expression of an orthogonal polymerase without host-specific promoters, achieving nontoxic and tuneable gene expression in diverse Gram-positive and Gram-negative bacteria. Combining 50 characterized system variants with mechanistic modelling, we show how the cross-species expression resource's dynamics, capacity and toxicity are controlled by the control loops' architecture and feedback strengths. We also demonstrate one application of the resource by reusing the same genetic parts to express a biosynthesis pathway in both model and non-model hosts. PMID:26184393

Genetically Engineering Bacillus subtilis with a Heat-Resistant Arsenite Methyltransferase for Bioremediation of Arsenic-Contaminated Organic Waste

PubMed Central

Huang, Ke; Chen, Chuan; Shen, Qirong; Rosen, Barry P.

2015-01-01

Organic manures may contain high levels of arsenic (As) due to the use of As-containing growth-promoting substances in animal feed. To develop a bioremediation strategy to remove As from organic waste, Bacillus subtilis 168, a bacterial strain which can grow at high temperature but is unable to methylate and volatilize As, was genetically engineered to express the arsenite S-adenosylmethionine methyltransferase gene (CmarsM) from the thermophilic alga Cyanidioschyzon merolae. The genetically engineered B. subtilis 168 converted most of the inorganic As in the medium into dimethylarsenate and trimethylarsine oxide within 48 h and volatized substantial amounts of dimethylarsine and trimethylarsine. The rate of As methylation and volatilization increased with temperature from 37 to 50°C. When inoculated into an As-contaminated organic manure composted at 50°C, the modified strain significantly enhanced As volatilization. This study provides a proof of concept of using genetically engineered microorganisms for bioremediation of As-contaminated organic waste during composting. PMID:26187966

Effects of a fruit-vegetable dietary pattern on oxidative stress and genetic damage in coke oven workers: a cross-sectional study.

PubMed

Xie, Zheng; Lin, Haijiang; Fang, Renfei; Shen, Weiwei; Li, Shuguang; Chen, Bo

2015-05-06

Coke oven workers (COWs) are exposed to high level of genotoxic chemicals that induce oxidative stress and genetic damage. The dietary intake of certain types of foods may reverse these effects. We conducted a cross-sectional study with 51 topside COWs, 79 other COWs, and 67 controls, to assess the effects of dietary patterns on oxidative stress and genetic damage. Compared to the controls, both topside and other COWs had significantly higher urinary 1-hydroxypyrene levels, serum oxidant levels [malondialdehyde, (MDA)], and genetic damage [micronucleus (MN) frequency & 8-oxo-2'-deoxyguanosine (8-OH-dG)], but lower antioxidant levels [superoxide dismutase (SOD) and glutathione peroxidase, (GPx)]. The fruit-vegetable (FV) dietary pattern was positively correlated with serum SOD levels and negative correlated with serum MDA, MN frequency, and urinary 8-OH-dG. COWs with an FV patter in the highest quartile (Q4) had significantly increased antioxidant levels (SOD and GPx) and decreased oxidant levels (MDA) and genetic damage (MN frequency and 8-OH-dG) than those with an FV pattern in the lowest quartile (Q1). Compared to control subjects, COWs had increased oxidative stress and genetic damage. A FV dietary pattern may reverse oxidative stress and genetic damage in COWs.

Software engineering

NASA Technical Reports Server (NTRS)

Fridge, Ernest M., III; Hiott, Jim; Golej, Jim; Plumb, Allan

1993-01-01

Today's software systems generally use obsolete technology, are not integrated properly with other software systems, and are difficult and costly to maintain. The discipline of reverse engineering is becoming prominent as organizations try to move their systems up to more modern and maintainable technology in a cost effective manner. The Johnson Space Center (JSC) created a significant set of tools to develop and maintain FORTRAN and C code during development of the space shuttle. This tool set forms the basis for an integrated environment to reengineer existing code into modern software engineering structures which are then easier and less costly to maintain and which allow a fairly straightforward translation into other target languages. The environment will support these structures and practices even in areas where the language definition and compilers do not enforce good software engineering. The knowledge and data captured using the reverse engineering tools is passed to standard forward engineering tools to redesign or perform major upgrades to software systems in a much more cost effective manner than using older technologies. The latest release of the environment was in Feb. 1992.

Adaptable Hydrogel Networks with Reversible Linkages for Tissue Engineering

PubMed Central

Wang, Huiyuan

2015-01-01

Adaptable hydrogels have recently emerged as a promising platform for three-dimensional (3D) cell encapsulation and culture. In conventional, covalently crosslinked hydrogels, degradation is typically required to allow complex cellular functions to occur, leading to bulk material degradation. In contrast, adaptable hydrogels are formed by reversible crosslinks. Through breaking and re-forming of the reversible linkages, adaptable hydrogels can be locally modified to permit complex cellular functions while maintaining their long-term integrity. In addition, these adaptable materials can have biomimetic viscoelastic properties that make them well suited for several biotechnology and medical applications. In this review, adaptable hydrogel design considerations and linkage selections are overviewed, with a focus on various cell compatible crosslinking mechanisms that can be exploited to form adaptable hydrogels for tissue engineering. PMID:25989348

Pressure for a select committee on human embryo research and genetic engineering.

PubMed

McKie, David

1985-11-02

By a commanding majority of almost five million votes, this year's Labour Party conference agreed that Labour Members of Parliament should not be permitted to let their consciences decide their votes on "issues affecting the reproductive rights of women." The targets for this censure were the 44 Labour MPs who backed Enoch Powell's bill to outlaw experiments on embryos. Conservative supporters of the Powell bill are countering their defeat by advocating a Parliamentary select committee to examine "matters of human embryo research and human genetic engineering." McKie comments that they are thus shifting emphasis from "fertility," which has public support, to genetic engineering, which generates fear.

Physiological significance of ghrelin revealed by studies using genetically engineered mouse models with modifications in the ghrelin system.

PubMed

Ariyasu, Hiroyuki; Akamizu, Takashi

2015-01-01

Ghrelin, an endogenous ligand for the growth hormone (GH) secretagogue receptor (GHS-R or ghrelin receptor), is a 28-amino acid acylated peptide mainly produced in the stomach. The pharmacological administration of ghrelin is known to exert diverse effects, such as stimulating GH secretion, promoting food intake, and increasing adiposity. In recent years, genetically engineered mouse models have provided important insights into the physiology of various hormones. In this review, we discuss current knowledge regarding the physiological significance of ghrelin on the basis of studies using genetically engineered mouse models with modifications in the ghrelin system.

In-line stirling energy system

DOEpatents

Backhaus, Scott N [Espanola, NM; Keolian, Robert [State College, PA

2011-03-22

A high efficiency generator is provided using a Stirling engine to amplify an acoustic wave by heating the gas in the engine in a forward mode. The engine is coupled to an alternator to convert heat input to the engine into electricity. A plurality of the engines and respective alternators can be coupled to operate in a timed sequence to produce multi-phase electricity without the need for conversion. The engine system may be operated in a reverse mode as a refrigerator/heat pump.

Crop Genetics: The Seeds of Revolution.

ERIC Educational Resources Information Center

DeYoung, H. Garrett

1983-01-01

Current research in plant genetics is described. Benefits of this research (which includes genetic engineering applications) will include reduction/elimination of crop diseases, assurance of genetic stability, and the creation of new crop varieties. (JN)

Synthetic Biology and the U.S. Biotechnology Regulatory System: Challenges and Options

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

Carter, Sarah R.; Rodemeyer, Michael; Garfinkel, Michele S.

Synthetic Biology and the U.S. Biotechnology Regulatory System: Challenges and Options Sarah R. Carter, Ph.D., J. Craig Venter Institute; Michael Rodemeyer, J.D., University of Virginia; Michele S. Garfinkel, Ph.D., EMBO; Robert M. Friedman, Ph.D., J. Craig Venter Institute In recent years, a range of genetic engineering techniques referred to as “synthetic biology” has significantly expanded the tool kit available to scientists and engineers, providing them with far greater capabilities to engineer organisms than previous techniques allowed. The field of synthetic biology includes the relatively new ability to synthesize long pieces of DNA from chemicals, as well as improved methods formore » genetic manipulation and design of genetic pathways to achieve more precise control of biological systems. These advances will help usher in a new generation of genetically engineered microbes, plants, and animals. The JCVI Policy Center team, along with researchers at the University of Virginia and EMBO, examined how well the current U.S. regulatory system for genetically engineered products will handle the near-term introduction of organisms engineered using synthetic biology. In particular, the focus was on those organisms intended to be used or grown directly in the environment, outside of a contained facility. The study concludes that the U.S. regulatory agencies have adequate legal authority to address most, but not all, potential environmental, health and safety concerns posed by these organisms. Such near-term products are likely to represent incremental changes rather than a marked departure from previous genetically engineered organisms. However, the study also identified two key challenges for the regulatory system, which are detailed in the report. First, USDA’s authority over genetically engineered plants depends on the use of an older engineering technique that is no longer necessary for many applications. The shift to synthetic biology and other newer genetic engineering techniques will leave many engineered plants without any pre-market regulatory review. Second, the number and diversity of engineered microbes for commercial use will increase in the near future, challenging EPA’s resources, expertise, and perhaps authority to regulate them. For each of these challenges, the report sets out a series of options, including an analysis of the advantages and disadvantages of each option from a variety of perspectives, for policy makers to consider. Policy responses will depend on the trade-offs chosen among competing considerations. This report, funded by the Department of Energy with additional funds from the Alfred P. Sloan Foundation, is the result of a two-year process that included interviews, commissioned background papers, discussions, and two workshops that sought input from a wide range of experts, including U.S. federal agency regulators, legal and science policy experts, representatives from the biotechnology indus¬try, and non-governmental organiza¬tions. This cross-section of views informed this report, but the conclusions are solely those of the authors. An Executive Summary, full Report, and background papers are available at: http://www.jcvi.org/cms/research/projects/synthetic-biology-and-the-us-biotechnology-regulatory-system/overview/« less

Virus fitness differences observed between two naturally occurring isolates of Ebola virus Makona variant using a reverse genetics approach.

PubMed

Albariño, César G; Guerrero, Lisa Wiggleton; Chakrabarti, Ayan K; Kainulainen, Markus H; Whitmer, Shannon L M; Welch, Stephen R; Nichol, Stuart T

2016-09-01

During the large outbreak of Ebola virus disease that occurred in Western Africa from late 2013 to early 2016, several hundred Ebola virus (EBOV) genomes have been sequenced and the virus genetic drift analyzed. In a previous report, we described an efficient reverse genetics system designed to generate recombinant EBOV based on a Makona variant isolate obtained in 2014. Using this system, we characterized the replication and fitness of 2 isolates of the Makona variant. These virus isolates are nearly identical at the genetic level, but have single amino acid differences in the VP30 and L proteins. The potential effects of these differences were tested using minigenomes and recombinant viruses. The results obtained with this approach are consistent with the role of VP30 and L as components of the EBOV RNA replication machinery. Moreover, the 2 isolates exhibited clear fitness differences in competitive growth assays. Published by Elsevier Inc.

Paper Genetic Engineering.

ERIC Educational Resources Information Center

MacClintic, Scott D.; Nelson, Genevieve M.

Bacterial transformation is a commonly used technique in genetic engineering that involves transferring a gene of interest into a bacterial host so that the bacteria can be used to produce large quantities of the gene product. Although several kits are available for performing bacterial transformation in the classroom, students do not always…

Signature pathway expression of xylose utilization in the genetically engineered industrial yeast Saccharomyces cerevisiae

USDA-ARS?s Scientific Manuscript database

Background: The limited xylose utilizing ability of native Saccharomyces cerevisiae has been a major obstacle for efficient cellulosic ethanol production from lignocellulosic materials. Haploid laboratory strains of S. cerevisiae are commonly used for genetic engineering to enable its xylose utiliza...

University Students' Knowledge and Attitude about Genetic Engineering

ERIC Educational Resources Information Center

Bal, Senol; Samanci, Nilay Keskin; Bozkurt, Orçun

2007-01-01

Genetic engineering and biotechnology made possible of gene transfer without discriminating microorganism, plant, animal or human. However, although these scientific techniques have benefits, they cause arguments because of their ethical and social impacts. The arguments about ethical ad social impacts of biotechnology made clear that not only…

Gene flow in genetically engineered perennial grasses: Lessons for modification of dedicated bioenergy crops

EPA Science Inventory

The potential ecological consequences of the commercialization of genetically engineered (GD) crops have been the subject of intense debate, particularly when the GE crops are perennial and capable of outcrossing to wild relatives. The essential ecological impact issues for engi...

78 FR 1522 - Semiannual Regulatory Agenda, Fall 2012

Federal Register 2010, 2011, 2012, 2013, 2014

2013-01-08

..., interstate movement, and environmental release of certain genetically engineered organisms and update the... of Organisms 0579-AC31 and Products Altered or Produced Through Genetic Engineering. 139 Importation..., Riverdale, MD 20737-1231, Phone: 301 851-2286. RIN: 0579-AC05 138. Introduction of Organisms and Products...

Genetically-engineered pig-to-baboon liver xenotransplantation: histopathology of xenografts and native organs.

PubMed

Ekser, Burcin; Klein, Edwin; He, Jing; Stolz, Donna B; Echeverri, Gabriel J; Long, Cassandra; Lin, Chih Che; Ezzelarab, Mohamed; Hara, Hidetaka; Veroux, Massimiliano; Ayares, David; Cooper, David K C; Gridelli, Bruno

2012-01-01

Orthotopic liver transplantation was carried out in baboons using wild-type (WT, n = 1) or genetically-engineered pigs (α1,3-galactosyltransferase gene-knockout, GTKO), n = 1; GTKO pigs transgenic for human CD46, n = 7) and a clinically-acceptable immunosuppressive regimen. Biopsies were obtained from the WT pig liver pre-Tx and at 30 min, 1, 2, 3, 4 and 5 h post-transplantation. Biopsies of genetically-engineered livers were obtained pre-Tx, 2 h after reperfusion and at necropsy (4-7 days after transplantation). Tissues were examined by light, confocal, and electron microscopy. All major native organs were also examined. The WT pig liver underwent hyperacute rejection. After genetically-engineered pig liver transplantation, hyperacute rejection did not occur. Survival was limited to 4-7 days due to repeated spontaneous bleeding in the liver and native organs (as a result of profound thrombocytopenia) which necessitated euthanasia. At 2 h, graft histology was largely normal. At necropsy, genetically-engineered pig livers showed hemorrhagic necrosis, platelet aggregation, platelet-fibrin thrombi, monocyte/macrophage margination mainly in liver sinusoids, and vascular endothelial cell hypertrophy, confirmed by confocal and electron microscopy. Immunohistochemistry showed minimal deposition of IgM, and almost absence of IgG, C3, C4d, C5b-9, and of a cellular infiltrate, suggesting that neither antibody- nor cell-mediated rejection played a major role.

History and future of genetically engineered food animal regulation: an open request.

PubMed

Wells, Kevin D

2016-06-01

Modern biotechnology resulted from of a series of incremental improvements in the understanding of DNA and the enzymes that nature evolved to manipulate it. As the potential impact of genetic engineering became apparent, scientists began the process of trying to identify the potential unintended consequences. Restrictions to recombinant DNA experimentation were at first self-imposed. Collaborative efforts between scientists and lawyers formalized an initial set of guidelines. These guidelines have been used to promulgate regulations around world. However, the initial guidelines were only intended as a starting point and were motivated by a specific set of concerns. As new data became available, the guidelines and regulations should have been adapted to the new knowledge. Instead, other social drivers drove the development of regulations. For most species and most applications, the framework that was established has slowly allowed some products to reach the market. However, genetically engineered livestock that are intended for food have been left in a regulatory state of limbo. To date, no genetically engineered food animal is available in the marketplace. A short history and a U.S.-based genetic engineer's perspective are presented. In addition, a request to regulatory agencies is presented for consideration as regulation continues to evolve. Regulators appear to have shown preference for the slow, random progression of evolution over the efficiency of intentional design.

European Science Notes, Volume 40, Number 5.

DTIC Science & Technology

1986-05-01

microbiology , genetic engineering, and genetic engineering of plants is reviewed. Environmental Sciences -: ") EUROMECH 201-Aplications of the Jchanics...Si"co lo with the research activities at the Lab- Soil Microbiology oratory of Genetics at the University of It is now well documented that a Ghent...spec- tesi , for succeeding in running dedica- tacular observation of SFPM up to four " ted research under incredibly hard Stokes orders in a 300-m

Software reengineering

NASA Technical Reports Server (NTRS)

Fridge, Ernest M., III

1991-01-01

Today's software systems generally use obsolete technology, are not integrated properly with other software systems, and are difficult and costly to maintain. The discipline of reverse engineering is becoming prominent as organizations try to move their systems up to more modern and maintainable technology in a cost effective manner. JSC created a significant set of tools to develop and maintain FORTRAN and C code during development of the Space Shuttle. This tool set forms the basis for an integrated environment to re-engineer existing code into modern software engineering structures which are then easier and less costly to maintain and which allow a fairly straightforward translation into other target languages. The environment will support these structures and practices even in areas where the language definition and compilers do not enforce good software engineering. The knowledge and data captured using the reverse engineering tools is passed to standard forward engineering tools to redesign or perform major upgrades to software systems in a much more cost effective manner than using older technologies. A beta vision of the environment was released in Mar. 1991. The commercial potential for such re-engineering tools is very great. CASE TRENDS magazine reported it to be the primary concern of over four hundred of the top MIS executives.

Survival differences among freeze-dried genetically engineered and wild-type bacteria.

PubMed Central

Israeli, E; Shaffer, B T; Hoyt, J A; Lighthart, B; Ganio, L M

1993-01-01

Because the death mechanisms of freeze-dried and air-dried bacteria are thought to be similar, freeze-drying was used to investigate the survival differences between potentially airborne genetically engineered microorganisms and their wild types. To this end, engineered strains of Escherichia coli and Pseudomonas syringae were freeze-dried and exposed to air, visible light, or both. The death rates of all engineered strains were significantly higher than those of their parental strains. Light and air exposure were found to increase the death rates of all strains. Application of death rate models to freeze-dried engineered bacteria to be released into the environment is discussed. PMID:8434925

Critical evaluation of reverse engineering tool Imagix 4D!

PubMed

Yadav, Rashmi; Patel, Ravindra; Kothari, Abhay

2016-01-01

The comprehension of legacy codes is difficult to understand. Various commercial reengineering tools are available that have unique working styles, and are equipped with their inherent capabilities and shortcomings. The focus of the available tools is in visualizing static behavior not the dynamic one. Therefore, it is difficult for people who work in software product maintenance, code understanding reengineering/reverse engineering. Consequently, the need for a comprehensive reengineering/reverse engineering tool arises. We found the usage of Imagix 4D to be good as it generates the maximum pictorial representations in the form of flow charts, flow graphs, class diagrams, metrics and, to a partial extent, dynamic visualizations. We evaluated Imagix 4D with the help of a case study involving a few samples of source code. The behavior of the tool was analyzed on multiple small codes and a large code gcc C parser. Large code evaluation was performed to uncover dead code, unstructured code, and the effect of not including required files at preprocessing level. The utility of Imagix 4D to prepare decision density and complexity metrics for a large code was found to be useful in getting to know how much reengineering is required. At the outset, Imagix 4D offered limitations in dynamic visualizations, flow chart separation (large code) and parsing loops. The outcome of evaluation will eventually help in upgrading Imagix 4D and posed a need of full featured tools in the area of software reengineering/reverse engineering. It will also help the research community, especially those who are interested in the realm of software reengineering tool building.

Efficient targeted mutagenesis in the monarch butterfly using zinc-finger nucleases

PubMed Central

Merlin, Christine; Beaver, Lauren E.; Taylor, Orley R.; Wolfe, Scot A.; Reppert, Steven M.

2013-01-01

The development of reverse-genetic tools in “nonmodel” insect species with distinct biology is critical to establish them as viable model systems. The eastern North American monarch butterfly (Danaus plexippus), whose genome is sequenced, has emerged as a model to study animal clocks, navigational mechanisms, and the genetic basis of long-distance migration. Here, we developed a highly efficient gene-targeting approach in the monarch using zinc-finger nucleases (ZFNs), engineered nucleases that generate mutations at targeted genomic sequences. We focused our ZFN approach on targeting the type 2 vertebrate-like cryptochrome gene of the monarch (designated cry2), which encodes a putative transcriptional repressor of the monarch circadian clockwork. Co-injections of mRNAs encoding ZFNs targeting the second exon of monarch cry2 into “one nucleus” stage embryos led to high-frequency nonhomologous end-joining-mediated, mutagenic lesions in the germline (up to 50%). Heritable ZFN-induced lesions in two independent lines produced truncated, nonfunctional CRY2 proteins, resulting in the in vivo disruption of circadian behavior and the molecular clock mechanism. Our work genetically defines CRY2 as an essential transcriptional repressor of the monarch circadian clock and provides a proof of concept for the use of ZFNs for manipulating genes in the monarch butterfly genome. Importantly, this approach could be used in other lepidopterans and “nonmodel” insects, thus opening new avenues to decipher the molecular underpinnings of a variety of biological processes. PMID:23009861

Genetically engineered peptides for inorganics: study of an unconstrained bacterial display technology and bulk aluminum alloy.

PubMed

Adams, Bryn L; Finch, Amethist S; Hurley, Margaret M; Sarkes, Deborah A; Stratis-Cullum, Dimitra N

2013-09-06

The first-ever peptide biomaterial discovery using an unconstrained engineered bacterial display technology is reported. Using this approach, we have developed genetically engineered peptide binders for a bulk aluminum alloy and use molecular dynamics simulation of peptide conformational fluctuations to demonstrate sequence-dependent, structure-function relationships for metal and metal oxide interactions. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genetic Engineering--A Lesson on Bioethics for the Classroom.

ERIC Educational Resources Information Center

Armstrong, Kerri; Weber, Kurt

1991-01-01

A unit designed to cover the topic of genetic engineering and its ethical considerations is presented. Students are expected to learn the material while using a debate format. A list of objectives for the unit, the debate format, and the results from an opinion questionnaire are described. (KR)

Development and deregulation of the plum pox virus resistant transgenic plum 'HoneySweet'

USDA-ARS?s Scientific Manuscript database

We have demonstrated that genetic engineering can be an important source of high level and durable resistance against Plum pox virus (PPV). We have shown, through a number of field studies, the environmental safety of this genetically engineered plum. Nevertheless, the utilization of this demonstr...

78 FR 51706 - Bayer CropScience LP; Determination of Nonregulated Status of Soybean Genetically Engineered for...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-08-21

... Soybean Genetically Engineered for Herbicide Resistance AGENCY: Animal and Plant Health Inspection Service... for resistance to the herbicides glyphosate and isoxaflutole, is no longer considered a regulated... for resistance to the herbicides glyphosate and isoxaflutole. The petition states that this soybean is...

77 FR 41358 - Bayer CropScience LP; Availability of Petition, Plant Pest Risk Assessment, and Environmental...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-13

... for Determination of Nonregulated Status of Soybean Genetically Engineered for Herbicide Tolerance... genetically engineered for resistance to the herbicides glyphosate and isoxaflutole. The petition has been... herbicides glyphosate and isoxaflutole. The petition states that this soybean is unlikely to pose a plant...

Can Man Control His Biological Evolution? A Symposium on Genetic Engineering. Artificial Synthesis of New Life Forms

ERIC Educational Resources Information Center

Danielli, James F.

1972-01-01

Research in manipulation of genetic inheritance opens new vistas. Biologically-styled industrial synthesis is better in many respects than chemical engineering practices now in use. An approach for improving hereditary characters in living organisms without considering social implications is unwise. (PS)

78 FR 13302 - Syngenta Biotechnology, Inc.; Determination of Nonregulated Status of Corn Genetically Engineered...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-02-27

...] Syngenta Biotechnology, Inc.; Determination of Nonregulated Status of Corn Genetically Engineered for... are advising the public of our determination that a corn line developed by the Syngenta Biotechnology... evaluation of data submitted by Syngenta Biotechnology, Inc., in its petition for a determination of...

Gonadal sex differentiation and effects of dietary methyltestosterone treatment in sablefish (Anoplopoma fimbria).

PubMed

Luckenbach, J Adam; Fairgrieve, William T

2016-02-01

Methods for sex control are needed to establish monosex aquaculture of sablefish (Anoplopoma fimbria). Here we conducted the first characterization of sex differentiation by histology and hormonal sex reversal experiment in sablefish. Ovarian differentiation was first discernible at ~80 mm fork length (FL) and characterized by development of lamellar structures and onset of meiosis. Testes exhibited a dual-lobe appearance over much of their length and remained non-meiotic until males were ≥520 mm FL (2 years post-fertilization). Juveniles with undifferentiated gonads were provided diets containing 0 (control), 5 or 50 mg 17α-methyltestosterone (MT)/kg for 2 months. Following treatment, controls possessed either ovaries or non-meiotic testes, whereas MT-treated fish exhibited meiotic testes (60% of the fish), intersex gonads (~30%), or gonads that appeared sterile (~10%). A genetic sex marker revealed that all intersex fish were genetic females, although other females appeared to be completely sex reversed (i.e., neomales). One year after treatment, MT-treated fish possessed non-meiotic testes similar to control males or intersex gonads with reduced ovarian features, presumably due to atresia following MT withdrawal. Milt collected from neomales and genetic males 3 years post-treatment permitted sperm motility analyses; however, neomale sperm were virtually immotile. These results demonstrated that sablefish are differentiated gonochorists and that MT treatment from 76 to 196 mm FL induced permanent masculinization of a portion of the genetic females, but acquisition of sperm motility was impaired. Earlier administration of MT may be necessary to sex reverse a higher proportion of genetic females and reduce negative effects on fertility.

A Multi-Stage Reverse Logistics Network Problem by Using Hybrid Priority-Based Genetic Algorithm

NASA Astrophysics Data System (ADS)

Lee, Jeong-Eun; Gen, Mitsuo; Rhee, Kyong-Gu

Today remanufacturing problem is one of the most important problems regarding to the environmental aspects of the recovery of used products and materials. Therefore, the reverse logistics is gaining become power and great potential for winning consumers in a more competitive context in the future. This paper considers the multi-stage reverse Logistics Network Problem (m-rLNP) while minimizing the total cost, which involves reverse logistics shipping cost and fixed cost of opening the disassembly centers and processing centers. In this study, we first formulate the m-rLNP model as a three-stage logistics network model. Following for solving this problem, we propose a Genetic Algorithm pri (GA) with priority-based encoding method consisting of two stages, and introduce a new crossover operator called Weight Mapping Crossover (WMX). Additionally also a heuristic approach is applied in the 3rd stage to ship of materials from processing center to manufacturer. Finally numerical experiments with various scales of the m-rLNP models demonstrate the effectiveness and efficiency of our approach by comparing with the recent researches.

A shape-based inter-layer contours correspondence method for ICT-based reverse engineering

PubMed Central

Duan, Liming; Yang, Shangpeng; Zhang, Gui; Feng, Fei; Gu, Minghui

2017-01-01

The correspondence of a stack of planar contours in ICT (industrial computed tomography)-based reverse engineering, a key step in surface reconstruction, is difficult when the contours or topology of the object are complex. Given the regularity of industrial parts and similarity of the inter-layer contours, a specialized shape-based inter-layer contours correspondence method for ICT-based reverse engineering was presented to solve the above problem based on the vectorized contours. In this paper, the vectorized contours extracted from the slices consist of three graphical primitives: circles, arcs and segments. First, the correspondence of the inter-layer primitives is conducted based on the characteristics of the primitives. Second, based on the corresponded primitives, the inter-layer contours correspond with each other using the proximity rules and exhaustive search. The proposed method can make full use of the shape information to handle industrial parts with complex structures. The feasibility and superiority of this method have been demonstrated via the related experiments. This method can play an instructive role in practice and provide a reference for the related research. PMID:28489867

A shape-based inter-layer contours correspondence method for ICT-based reverse engineering.

PubMed

Duan, Liming; Yang, Shangpeng; Zhang, Gui; Feng, Fei; Gu, Minghui

2017-01-01

The correspondence of a stack of planar contours in ICT (industrial computed tomography)-based reverse engineering, a key step in surface reconstruction, is difficult when the contours or topology of the object are complex. Given the regularity of industrial parts and similarity of the inter-layer contours, a specialized shape-based inter-layer contours correspondence method for ICT-based reverse engineering was presented to solve the above problem based on the vectorized contours. In this paper, the vectorized contours extracted from the slices consist of three graphical primitives: circles, arcs and segments. First, the correspondence of the inter-layer primitives is conducted based on the characteristics of the primitives. Second, based on the corresponded primitives, the inter-layer contours correspond with each other using the proximity rules and exhaustive search. The proposed method can make full use of the shape information to handle industrial parts with complex structures. The feasibility and superiority of this method have been demonstrated via the related experiments. This method can play an instructive role in practice and provide a reference for the related research.

MORE: mixed optimization for reverse engineering--an application to modeling biological networks response via sparse systems of nonlinear differential equations.

PubMed

Sambo, Francesco; de Oca, Marco A Montes; Di Camillo, Barbara; Toffolo, Gianna; Stützle, Thomas

2012-01-01

Reverse engineering is the problem of inferring the structure of a network of interactions between biological variables from a set of observations. In this paper, we propose an optimization algorithm, called MORE, for the reverse engineering of biological networks from time series data. The model inferred by MORE is a sparse system of nonlinear differential equations, complex enough to realistically describe the dynamics of a biological system. MORE tackles separately the discrete component of the problem, the determination of the biological network topology, and the continuous component of the problem, the strength of the interactions. This approach allows us both to enforce system sparsity, by globally constraining the number of edges, and to integrate a priori information about the structure of the underlying interaction network. Experimental results on simulated and real-world networks show that the mixed discrete/continuous optimization approach of MORE significantly outperforms standard continuous optimization and that MORE is competitive with the state of the art in terms of accuracy of the inferred networks.

Silk Materials Functionalized via Genetic Engineering for Biomedical Applications

PubMed Central

Deptuch, Tomasz

2017-01-01

The great mechanical properties, biocompatibility and biodegradability of silk-based materials make them applicable to the biomedical field. Genetic engineering enables the construction of synthetic equivalents of natural silks. Knowledge about the relationship between the structure and function of silk proteins enables the design of bioengineered silks that can serve as the foundation of new biomaterials. Furthermore, in order to better address the needs of modern biomedicine, genetic engineering can be used to obtain silk-based materials with new functionalities. Sequences encoding new peptides or domains can be added to the sequences encoding the silk proteins. The expression of one cDNA fragment indicates that each silk molecule is related to a functional fragment. This review summarizes the proposed genetic functionalization of silk-based materials that can be potentially useful for biomedical applications. PMID:29231863

Engineering species-like barriers to sexual reproduction.

PubMed

Maselko, Maciej; Heinsch, Stephen C; Chacón, Jeremy M; Harcombe, William R; Smanski, Michael J

2017-10-12

Controlling the exchange of genetic information between sexually reproducing populations has applications in agriculture, eradication of disease vectors, control of invasive species, and the safe study of emerging biotechnology applications. Here we introduce an approach to engineer a genetic barrier to sexual reproduction between otherwise compatible populations. Programmable transcription factors drive lethal gene expression in hybrid offspring following undesired mating events. As a proof of concept, we target the ACT1 promoter of the model organism Saccharomyces cerevisiae using a dCas9-based transcriptional activator. Lethal overexpression of actin results from mating this engineered strain with a strain containing the wild-type ACT1 promoter.Genetic isolation of a genetically modified organism represents a useful strategy for biocontainment. Here the authors use dCas9-VP64-driven gene expression to construct a 'species-like' barrier to reproduction between two otherwise compatible populations.

Novel norovirus in dogs with diarrhea.

PubMed

Mesquita, João Rodrigo; Barclay, Leslie; Nascimento, Maria São José; Vinjé, Jan

2010-06-01

To identify the prevalence and genetic variability of noroviruses in dogs, we tested fecal samples by using reverse transcription-PCR. We found canine norovirus in 40% and 9% of dogs with and without diarrhea, respectively. The virus was genetically unrelated to other noroviruses and constitutes a tentative new genogroup.

A synthetic biology-based device prevents liver injury in mice.

PubMed

Bai, Peng; Ye, Haifeng; Xie, Mingqi; Saxena, Pratik; Zulewski, Henryk; Charpin-El Hamri, Ghislaine; Djonov, Valentin; Fussenegger, Martin

2016-07-01

The liver performs a panoply of complex activities coordinating metabolic, immunologic and detoxification processes. Despite the liver's robustness and unique self-regeneration capacity, viral infection, autoimmune disorders, fatty liver disease, alcohol abuse and drug-induced hepatotoxicity contribute to the increasing prevalence of liver failure. Liver injuries impair the clearance of bile acids from the hepatic portal vein which leads to their spill over into the peripheral circulation where they activate the G-protein-coupled bile acid receptor TGR5 to initiate a variety of hepatoprotective processes. By functionally linking activation of ectopically expressed TGR5 to an artificial promoter controlling transcription of the hepatocyte growth factor (HGF), we created a closed-loop synthetic signalling network that coordinated liver injury-associated serum bile acid levels to expression of HGF in a self-sufficient, reversible and dose-dependent manner. After implantation of genetically engineered human cells inside auto-vascularizing, immunoprotective and clinically validated alginate-poly-(L-lysine)-alginate beads into mice, the liver-protection device detected pathologic serum bile acid levels and produced therapeutic HGF levels that protected the animals from acute drug-induced liver failure. Genetically engineered cells containing theranostic gene circuits that dynamically interface with host metabolism may provide novel opportunities for preventive, acute and chronic healthcare. Liver diseases leading to organ failure may go unnoticed as they do not trigger any symptoms or significant discomfort. We have designed a synthetic gene circuit that senses excessive bile acid levels associated with liver injuries and automatically produces a therapeutic protein in response. When integrated into mammalian cells and implanted into mice, the circuit detects the onset of liver injuries and coordinates the production of a protein pharmaceutical which prevents liver damage. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions

PubMed Central

Eaton, Heather E.; Kobayashi, Takeshi; Dermody, Terence S.; Johnston, Randal N.

2017-01-01

ABSTRACT Reoviruses, like many eukaryotic viruses, contain an inverted 7-methylguanosine (m7G) cap linked to the 5′ nucleotide of mRNA. The traditional functions of capping are to promote mRNA stability, protein translation, and concealment from cellular proteins that recognize foreign RNA. To address the role of mRNA capping during reovirus replication, we assessed the benefits of adding the African swine fever virus NP868R capping enzyme during reovirus rescue. C3P3, a fusion protein containing T7 RNA polymerase and NP868R, was found to increase protein expression 5- to 10-fold compared to T7 RNA polymerase alone while enhancing reovirus rescue from the current reverse genetics system by 100-fold. Surprisingly, RNA stability was not increased by C3P3, suggesting a direct effect on protein translation. A time course analysis revealed that C3P3 increased protein synthesis within the first 2 days of a reverse genetics transfection. This analysis also revealed that C3P3 enhanced processing of outer capsid μ1 protein to μ1C, a previously described hallmark of reovirus assembly. Finally, to determine the rate of infectious-RNA incorporation into new virions, we developed a new recombinant reovirus S1 gene that expressed the fluorescent protein UnaG. Following transfection of cells with UnaG and infection with wild-type virus, passage of UnaG through progeny was significantly enhanced by C3P3. These data suggest that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation. IMPORTANCE Our findings expand our understanding of how viruses utilize capping, suggesting that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation, in addition to enhancing protein translation. Beyond providing mechanistic insight into reovirus replication, our findings also show that reovirus reverse genetics rescue is enhanced 100-fold by the NP868R capping enzyme. Since reovirus shows promise as a cancer therapy, efficient reovirus reverse genetics rescue will accelerate production of recombinant reoviruses as candidates to enhance therapeutic potency. NP868R-assisted reovirus rescue will also expedite production of recombinant reovirus for mechanistic insights into reovirus protein function and structure. PMID:28298603

Where Are All the Women Engineers? An Insider's View of Socialization and Power in Engineering Education

ERIC Educational Resources Information Center

Christman, Jeanne

2017-01-01

Despite more than thirty years of the underrepresentation of women in engineering being a persistent concern, research on the cause of the problem has not been successful in reversing the trend. A plethora of theories as to why females are not entering engineering exist, yet they only address issues on the surface and do not attend to a…

Natural tooth intrusion and reversal in implant-assisted prosthesis: evidence of and a hypothesis for the occurrence.

PubMed

Sheets, C G; Earthmann, J C

1993-12-01

Based on clinical observation, a hypothesis of the mechanism of intrusion of natural teeth in an implant-assisted prosthesis is suggested. Engineering principles are presented that establish an energy absorption model as it relates to the implant-assisted prosthesis. In addition, in the course of patient treatment it has been discovered that the intrusion of natural teeth can be reversed. Patient histories that demonstrate intrusion reversal are reviewed. The possible mechanisms for the intrusion/reversal phenomenon are presented and preventative recommendations are given.

A Tet-on and Cre-loxP Based Genetic Engineering System for Convenient Recycling of Selection Markers in Penicillium oxalicum

PubMed Central

Jiang, Baojie; Zhang, Ruiqin; Feng, Dan; Wang, Fangzhong; Liu, Kuimei; Jiang, Yi; Niu, Kangle; Yuan, Quanquan; Wang, Mingyu; Wang, Hailong; Zhang, Youming; Fang, Xu

2016-01-01

The lack of selective markers has been a key problem preventing multistep genetic engineering in filamentous fungi, particularly for industrial species such as the lignocellulose degrading Penicillium oxalicum JUA10-1(formerly named as Penicillium decumbens). To resolve this problem, we constructed a genetic manipulation system taking advantage of two established genetic systems: the Cre-loxP system and Tet-on system in P. oxalicum JUA10-1. This system is efficient and convenient. The expression of Cre recombinase was activated by doxycycline since it was controlled by Tet-on system. Using this system, two genes, ligD and bglI, were sequentially disrupted by loxP flanked ptrA. The successful application of this procedure will provide a useful tool for genetic engineering in filamentous fungi. This system will also play an important role in improving the productivity of interesting products and minimizing by-product when fermented by filamentous fungi. PMID:27148179

The role of genetically engineered pigs in xenotransplantation research.

PubMed

Cooper, David K C; Ekser, Burcin; Ramsoondar, Jagdeece; Phelps, Carol; Ayares, David

2016-01-01

There is a critical shortage in the number of deceased human organs that become available for the purposes of clinical transplantation. This problem might be resolved by the transplantation of organs from pigs genetically engineered to protect them from the human immune response. The pathobiological barriers to successful pig organ transplantation in primates include activation of the innate and adaptive immune systems, coagulation dysregulation and inflammation. Genetic engineering of the pig as an organ source has increased the survival of the transplanted pig heart, kidney, islet and corneal graft in non-human primates (NHPs) from minutes to months or occasionally years. Genetic engineering may also contribute to any physiological barriers that might be identified, as well as to reducing the risks of transfer of a potentially infectious micro-organism with the organ. There are now an estimated 40 or more genetic alterations that have been carried out in pigs, with some pigs expressing five or six manipulations. With the new technology now available, it will become increasingly common for a pig to express even more genetic manipulations, and these could be tested in the pig-to-NHP models to assess their efficacy and benefit. It is therefore likely that clinical trials of pig kidney, heart and islet transplantation will become feasible in the near future. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

New Algorithm and Software (BNOmics) for Inferring and Visualizing Bayesian Networks from Heterogeneous Big Biological and Genetic Data

PubMed Central

Gogoshin, Grigoriy; Boerwinkle, Eric

2017-01-01

Abstract Bayesian network (BN) reconstruction is a prototypical systems biology data analysis approach that has been successfully used to reverse engineer and model networks reflecting different layers of biological organization (ranging from genetic to epigenetic to cellular pathway to metabolomic). It is especially relevant in the context of modern (ongoing and prospective) studies that generate heterogeneous high-throughput omics datasets. However, there are both theoretical and practical obstacles to the seamless application of BN modeling to such big data, including computational inefficiency of optimal BN structure search algorithms, ambiguity in data discretization, mixing data types, imputation and validation, and, in general, limited scalability in both reconstruction and visualization of BNs. To overcome these and other obstacles, we present BNOmics, an improved algorithm and software toolkit for inferring and analyzing BNs from omics datasets. BNOmics aims at comprehensive systems biology—type data exploration, including both generating new biological hypothesis and testing and validating the existing ones. Novel aspects of the algorithm center around increasing scalability and applicability to varying data types (with different explicit and implicit distributional assumptions) within the same analysis framework. An output and visualization interface to widely available graph-rendering software is also included. Three diverse applications are detailed. BNOmics was originally developed in the context of genetic epidemiology data and is being continuously optimized to keep pace with the ever-increasing inflow of available large-scale omics datasets. As such, the software scalability and usability on the less than exotic computer hardware are a priority, as well as the applicability of the algorithm and software to the heterogeneous datasets containing many data types—single-nucleotide polymorphisms and other genetic/epigenetic/transcriptome variables, metabolite levels, epidemiological variables, endpoints, and phenotypes, etc. PMID:27681505

New Algorithm and Software (BNOmics) for Inferring and Visualizing Bayesian Networks from Heterogeneous Big Biological and Genetic Data.

PubMed

Gogoshin, Grigoriy; Boerwinkle, Eric; Rodin, Andrei S

2017-04-01

Bayesian network (BN) reconstruction is a prototypical systems biology data analysis approach that has been successfully used to reverse engineer and model networks reflecting different layers of biological organization (ranging from genetic to epigenetic to cellular pathway to metabolomic). It is especially relevant in the context of modern (ongoing and prospective) studies that generate heterogeneous high-throughput omics datasets. However, there are both theoretical and practical obstacles to the seamless application of BN modeling to such big data, including computational inefficiency of optimal BN structure search algorithms, ambiguity in data discretization, mixing data types, imputation and validation, and, in general, limited scalability in both reconstruction and visualization of BNs. To overcome these and other obstacles, we present BNOmics, an improved algorithm and software toolkit for inferring and analyzing BNs from omics datasets. BNOmics aims at comprehensive systems biology-type data exploration, including both generating new biological hypothesis and testing and validating the existing ones. Novel aspects of the algorithm center around increasing scalability and applicability to varying data types (with different explicit and implicit distributional assumptions) within the same analysis framework. An output and visualization interface to widely available graph-rendering software is also included. Three diverse applications are detailed. BNOmics was originally developed in the context of genetic epidemiology data and is being continuously optimized to keep pace with the ever-increasing inflow of available large-scale omics datasets. As such, the software scalability and usability on the less than exotic computer hardware are a priority, as well as the applicability of the algorithm and software to the heterogeneous datasets containing many data types-single-nucleotide polymorphisms and other genetic/epigenetic/transcriptome variables, metabolite levels, epidemiological variables, endpoints, and phenotypes, etc.

Genetic engineering of cyanobacteria as biodiesel feedstock.

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

Ruffing, Anne.; Trahan, Christine Alexandra; Jones, Howland D. T.

2013-01-01

Algal biofuels are a renewable energy source with the potential to replace conventional petroleum-based fuels, while simultaneously reducing greenhouse gas emissions. The economic feasibility of commercial algal fuel production, however, is limited by low productivity of the natural algal strains. The project described in this SAND report addresses this low algal productivity by genetically engineering cyanobacteria (i.e. blue-green algae) to produce free fatty acids as fuel precursors. The engineered strains were characterized using Sandias unique imaging capabilities along with cutting-edge RNA-seq technology. These tools are applied to identify additional genetic targets for improving fuel production in cyanobacteria. This proof-of-concept studymore » demonstrates successful fuel production from engineered cyanobacteria, identifies potential limitations, and investigates several strategies to overcome these limitations. This project was funded from FY10-FY13 through the President Harry S. Truman Fellowship in National Security Science and Engineering, a program sponsored by the LDRD office at Sandia National Laboratories.« less

Genome engineering and plant breeding: impact on trait discovery and development.

PubMed

Nogué, Fabien; Mara, Kostlend; Collonnier, Cécile; Casacuberta, Josep M

2016-07-01

New tools for the precise modification of crops genes are now available for the engineering of new ideotypes. A future challenge in this emerging field of genome engineering is to develop efficient methods for allele mining. Genome engineering tools are now available in plants, including major crops, to modify in a predictable manner a given gene. These new techniques have a tremendous potential for a spectacular acceleration of the plant breeding process. Here, we discuss how genetic diversity has always been the raw material for breeders and how they have always taken advantage of the best available science to use, and when possible, increase, this genetic diversity. We will present why the advent of these new techniques gives to the breeders extremely powerful tools for crop breeding, but also why this will require the breeders and researchers to characterize the genes underlying this genetic diversity more precisely. Tackling these challenges should permit the engineering of optimized alleles assortments in an unprecedented and controlled way.

Development of a Genetically Engineered Venezuelan Equine Encephalitis Virus Vaccine

DTIC Science & Technology

1988-12-20

immunization, the horses will be returned to the large animal biocontainment facility to be challenged with equine virulent VEE virus. The animals will be...AD £IT FiLE C p DEVELOPMENT OF A GENETICALLY ENGINEERED VENEZUELAN EQUINE ENCEPHALITIS VIRUS VACCINE ANNUAL REPORT to DENNIS W. TRENT 0DECEMBER 20...Engineered Venezuelan Equine Encephalitis Virus Vaccine 12. PERSONAL AUTHOR(S) Dennis W. Trent 13a. TYPE OF REPORT 13b. TIME COVERED 14. DATE OF REPORT

Sequestration of carbon dioxide with hydrogen to useful products

DOEpatents

Adams, Michael W. W.; Kelly, Robert M.; Hawkins, Aaron B.; Menon, Angeli Lal; Lipscomb, Gina Lynette Pries; Schut, Gerrit Jan

2017-03-07

Provided herein are genetically engineered microbes that include at least a portion of a carbon fixation pathway, and in one embodiment, use molecular hydrogen to drive carbon dioxide fixation. In one embodiment, the genetically engineered microbe is modified to convert acetyl CoA, molecular hydrogen, and carbon dioxide to 3-hydroxypropionate, 4-hydroxybutyrate, acetyl CoA, or the combination thereof at levels greater than a control microbe. Other products may also be produced. Also provided herein are cell free compositions that convert acetyl CoA, molecular hydrogen, and carbon dioxide to 3-hydroxypropionate, 4-hydroxybutyrate, acetyl CoA, or the combination thereof. Also provided herein are methods of using the genetically engineered microbes and the cell free compositions.

Thermoelectric energy converters under a trade-off figure of merit with broken time-reversal symmetry

NASA Astrophysics Data System (ADS)

Iyyappan, I.; Ponmurugan, M.

2017-09-01

We study the performance of a three-terminal thermoelectric device such as heat engine and refrigerator with broken time-reversal symmetry by applying the unified trade-off figure of merit (\\dotΩ criterion) which accounts for both useful energy and losses. For the heat engine, we find that a thermoelectric device working under the maximum \\dotΩ criterion gives a significantly better performance than a device working at maximum power output. Within the framework of linear irreversible thermodynamics such a direct comparison is not possible for refrigerators, however, our study indicates that, for refrigerator, the maximum cooling load gives a better performance than the maximum \\dotΩ criterion for a larger asymmetry. Our results can be useful to choose a suitable optimization criterion for operating a real thermoelectric device with broken time-reversal symmetry.

Sex Reversal in Reptiles: Reproductive Oddity or Powerful Driver of Evolutionary Change?

PubMed

Holleley, Clare E; Sarre, Stephen D; O'Meally, Denis; Georges, Arthur

2016-01-01

Is sex a product of genes, the environment, or both? In this review, we describe the diversity of sex-determining mechanisms in reptiles, with a focus on systems that display gene-environment interactions. We summarise the field and laboratory-based evidence for the occurrence of environmental sex reversal in reptiles and ask whether this is a widespread evolutionary mechanism affecting the evolution of sex chromosomes and speciation in vertebrates. Sex determination systems exist across a continuum of genetic and environmental influences, blurring the lines between what was once considered a strict dichotomy between genetic sex determination and temperature-dependent sex determination. Across this spectrum, we identify the potential for sex reversal in species with clearly differentiated heteromorphic sex chromosomes (Pogona vitticeps, Bassiana duperreyi, Eremias multiocellata, Gekko japonicus), weakly differentiated homomorphic sex chromosomes (Niveoscincus ocellatus), and species with only a weak heritable predisposition for sex (Emys orbicularis, Trachemys scripta). We argue that sex reversal is widespread in reptiles (Testudines, Lacertidae, Agamidae, Scincidae, Gekkonidae) and has the potential to have an impact on individual fitness, resulting in reproductively, morphologically, and behaviourally unique phenotypes. Sex reversal is likely to be a powerful evolutionary force responsible for generating and maintaining lability and diversity in reptile sex-determining modes. © 2016 S. Karger AG, Basel.

Thrust reverser design studies for an over-the-wing STOL transport

NASA Technical Reports Server (NTRS)

Ammer, R. C.; Sowers, H. D.

1977-01-01

Aerodynamic and acoustics analytical studies were conducted to evaluate three thrust reverser designs for potential use on commercial over-the-wing STOL transports. The concepts were: (1) integral D nozzle/target reverser, (2) integral D nozzle/top arc cascade reverser, and (3) post exit target reverser integral with wing. Aerodynamic flowpaths and kinematic arrangements for each concept were established to provide a 50% thrust reversal capability. Analytical aircraft stopping distance/noise trade studies conducted concurrently with flow path design showed that these high efficiency reverser concepts are employed at substantially reduced power settings to meet noise goals of 100 PNdB on a 152.4 m sideline and still meet 609.6 m landing runway length requirements. From an overall installation standpoint, only the integral D nozzle/target reverser concept was found to penalize nacelle cruise performance; for this concept a larger nacelle diameter was required to match engine cycle effective area demand in reverse thrust.

Climate-driven shifts in adult sex ratios via sex reversals: the type of sex determination matters.

PubMed

Bókony, Veronika; Kövér, Szilvia; Nemesházi, Edina; Liker, András; Székely, Tamás

2017-09-19

Sex reversals whereby individuals of one genetic sex develop the phenotype of the opposite sex occur in ectothermic vertebrates with genetic sex-determination systems that are sensitive to extreme temperatures during sexual differentiation. Recent rises in global temperatures have led researchers to predict that sex reversals will become more common, resulting in the distortion of many populations' sex ratios. However, it is unclear whether susceptibility to climate-driven sex-ratio shifts depends on the type of sex determination that varies across species. First, we show here using individual-based theoretical models that XX/XY (male-heterogametic) and ZZ/ZW (female-heterogametic) sex-determination systems can respond differentially to temperature-induced sex reversals. Interestingly, the impacts of climate warming on adult sex ratio (ASR) depend on the effects of both genotypic and phenotypic sex on survival and reproduction. Second, we analyse the temporal changes of ASR in natural amphibian populations using data from the literature, and find that ASR shifted towards males in ZZ/ZW species over the past 60 years, but did not change significantly in XX/XY species. Our results highlight the fact that we need a better understanding of the interactions between genetic and environmental sex-determining mechanisms to predict the responses of ectotherms to climate change and the associated extinction risks.This article is part of the themed issue 'Adult sex ratios and reproductive decisions: a critical re-examination of sex differences in human and animal societies'. © 2017 The Author(s).

Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein.

PubMed

Khamina, Kseniya; Lercher, Alexander; Caldera, Michael; Schliehe, Christopher; Vilagos, Bojan; Sahin, Mehmet; Kosack, Lindsay; Bhattacharya, Anannya; Májek, Peter; Stukalov, Alexey; Sacco, Roberto; James, Leo C; Pinschewer, Daniel D; Bennett, Keiryn L; Menche, Jörg; Bergthaler, Andreas

2017-12-01

RNA-dependent RNA polymerases (RdRps) play a key role in the life cycle of RNA viruses and impact their immunobiology. The arenavirus lymphocytic choriomeningitis virus (LCMV) strain Clone 13 provides a benchmark model for studying chronic infection. A major genetic determinant for its ability to persist maps to a single amino acid exchange in the viral L protein, which exhibits RdRp activity, yet its functional consequences remain elusive. To unravel the L protein interactions with the host proteome, we engineered infectious L protein-tagged LCMV virions by reverse genetics. A subsequent mass-spectrometric analysis of L protein pulldowns from infected human cells revealed a comprehensive network of interacting host proteins. The obtained LCMV L protein interactome was bioinformatically integrated with known host protein interactors of RdRps from other RNA viruses, emphasizing interconnected modules of human proteins. Functional characterization of selected interactors highlighted proviral (DDX3X) as well as antiviral (NKRF, TRIM21) host factors. To corroborate these findings, we infected Trim21-/- mice with LCMV and found impaired virus control in chronic infection. These results provide insights into the complex interactions of the arenavirus LCMV and other viral RdRps with the host proteome and contribute to a better molecular understanding of how chronic viruses interact with their host.

Autism-Associated Insertion Mutation (InsG) of Shank3 Exon 21 Causes Impaired Synaptic Transmission and Behavioral Deficits

PubMed Central

Speed, Haley E.; Kouser, Mehreen; Xuan, Zhong; Reimers, Jeremy M.; Ochoa, Christine F.; Gupta, Natasha; Liu, Shunan

2015-01-01

SHANK3 (also known as PROSAP2) is a postsynaptic scaffolding protein at excitatory synapses in which mutations and deletions have been implicated in patients with idiopathic autism, Phelan–McDermid (aka 22q13 microdeletion) syndrome, and other neuropsychiatric disorders. In this study, we have created a novel mouse model of human autism caused by the insertion of a single guanine nucleotide into exon 21 (Shank3G). The resulting frameshift causes a premature STOP codon and loss of major higher molecular weight Shank3 isoforms at the synapse. Shank3G/G mice exhibit deficits in hippocampus-dependent spatial learning, impaired motor coordination, altered response to novelty, and sensory processing deficits. At the cellular level, Shank3G/G mice also exhibit impaired hippocampal excitatory transmission and plasticity as well as changes in baseline NMDA receptor-mediated synaptic responses. This work identifies clear alterations in synaptic function and behavior in a novel, genetically accurate mouse model of autism mimicking an autism-associated insertion mutation. Furthermore, these findings lay the foundation for future studies aimed to validate and study region-selective and temporally selective genetic reversal studies in the Shank3G/G mouse that was engineered with such future experiments in mind. PMID:26134648

The Role of Nanobiotechnology in the Study of Dystrophin and B-Dystroglycan in Membrane Stability of Aging Skeletal Muscles

NASA Astrophysics Data System (ADS)

Vaseashta, Ashok

2005-03-01

Duchene muscular dystrophy (DMD) is one of nine types of muscular dystrophy, a group of genetic degenerative diseases, primarily affecting voluntary muscles, caused by absence of dystrophin. New experiments on mice with DMD has shown that gene therapy can reverse some symptoms of the disease. The ultimate goal of gene therapy for muscle diseases is improvement of strength and function, which will require treatment in multiple muscles simultaneously. A major limitation to gene therapy until now has been that no one had found a method by which a new gene could be delivered to all the muscles of an adult animal. Recent utilization of nanotechnology to life sciences has shown exciting promises in a wide range of disciplines, showing advances in the ability to manipulate, fabricate and alter tiny subjects at the nanometer scale. In the present investigation, we have employed such techniques to study single motors such as myosin and kinesin, as well elastic proteins viz. titin and nebulin, muscle filaments, cytoskeletal filaments, and receptors in cellular membranes and cellular organelles viz. myofibril, ribosome, and chromatin. Application of AFM to images and measures the elastic properties of single monomeric and oligomeric protein, genetically engineered titin, and nebulin molecules will be presented.

Quiet Clean Short-haul Experimental Engine (QCSEE) Under-The-Wing (UTW) engine composite nacelle test report. Volume 1: Summary, aerodynamic and mechanical performance

NASA Technical Reports Server (NTRS)

1979-01-01

The performance test results of the final under-the-wing engine configuration are presented. One hundred and six hours of engine operation were completed, including mechanical and performance checkout, baseline acoustic testing with a bellmouth inlet, reverse thrust testing, acoustic technology tests, and limited controls testing. The engine includes a variable pitch fan having advanced composite fan blades and using a ball-spline pitch actuation system.

PERSISTENCE OF A SURROGATE FOR A GENETICALLY ENGINEERED CELLULOLYTIC MICROORGANISM AND EFFECTS ON AQUATIC COMMUNITY AND ECOSYSTEM PROPERTIES: MICROCOSM AND STREAM COMPARISONS

EPA Science Inventory

Our research objectives were to: (1) determine the persistence of an introduced surrogate (Cellulomonas sp NRC 2406) for a genetically engineered microorganism (GEM) in three streamlined habitats; sediments, growths of Cladophora (Chlorophyta), and leaf packs, (2) test ommunity a...

77 FR 41363 - BASF Plant Science, LP; Availability of Petition for Determination of Nonregulated Status of...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-13

... Genetically Engineered for Herbicide Tolerance AGENCY: Animal and Plant Health Inspection Service, USDA... herbicides in the imidazolinone family. The petition has been submitted in accordance with our regulations... event BPS-CV127-9, which has been genetically engineered for tolerance to herbicides in the...

77 FR 41367 - Dow AgroSciences LLC; Availability of Petition, Plant Pest Risk Assessment, and Environmental...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-13

... Assessment for Determination of Nonregulated Status of Soybean Genetically Engineered for Herbicide Tolerance... been genetically engineered for tolerance to broadleaf herbicides in the phenoxy auxin group (such as the herbicide 2,4-D) and the herbicide glufosinate. The petition has been submitted in accordance with...

77 FR 41361 - Dow AgroSciences LLC; Availability of Petition for Determination of Nonregulated Status of...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-13

... Genetically Engineered for Herbicide Tolerance AGENCY: Animal and Plant Health Inspection Service, USDA... broadleaf herbicides in the phenoxy auxin group (such as the herbicide 2,4-D) and the herbicides glyphosate...-44406-6, which has been genetically engineered for tolerance to broadleaf herbicides in the phenoxy...

Genetic Engineering of Animals for Medical Research: Students' Views.

ERIC Educational Resources Information Center

Hill, Ruaraidh; Stanisstreet, Martin; O'Sullivan, Helen; Boyes, Edward

1999-01-01

Reports on the results of a survey meant to ascertain the views of 16- to 18-year-old students (n=778) on using animals in medical research. Suggests that students have no greater objection to the use of genetically engineered animals over naturally bred animals in medical research. Contains 16 references. (Author/WRM)

Projecting potential adoption of genetically engineered freeze-tolerant Eucalyptus in the United States

Treesearch

David N. Wear; Ernest Dixon IV; Robert C. Abt; Navinder Singh

2015-01-01

Development of commercial Eucalyptus plantations has been limited in the United States because of the species’ sensitivity to freezing temperatures. Recently developed genetically engineered clones of a Eucalyptus hybrid, which confer freeze tolerance, could expand the range of commercial plantations. This study explores how...

[The application of genetic engineering to the petroleum biodesulfurization].

PubMed

Tong, M Y; Fang, X C; Ma, T; Zhang, Q

2001-11-01

The developed course and reaction mechanisms of petroleum biodesulfurization were introduced. The recent development of genetic engineering technology, which used in desulfuration strain's construction, reconstruction and other fields, was summarized emphatically. Its current research situation internal and overseas and the developing prospect were simply analyzed, and our research designs were submitted.

'HoneySweet' plum - a valuable genetically engineered fruit-tree cultivar and germplasm resource

USDA-ARS?s Scientific Manuscript database

‘HoneySweet’ is a plum variety developed through genetic engineering to be highly resistant to plum pox potyvirus (PPV), the causal agent of sharka disease, that threatens stone-fruit industries world-wide and most specifically, in Europe. Field testing for over 15 years in Europe has demonstrated ...

Programmable cells: Interfacing natural and engineered gene networks

NASA Astrophysics Data System (ADS)

Kobayashi, Hideki; Kærn, Mads; Araki, Michihiro; Chung, Kristy; Gardner, Timothy S.; Cantor, Charles R.; Collins, James J.

2004-06-01

Novel cellular behaviors and characteristics can be obtained by coupling engineered gene networks to the cell's natural regulatory circuitry through appropriately designed input and output interfaces. Here, we demonstrate how an engineered genetic circuit can be used to construct cells that respond to biological signals in a predetermined and programmable fashion. We employ a modular design strategy to create Escherichia coli strains where a genetic toggle switch is interfaced with: (i) the SOS signaling pathway responding to DNA damage, and (ii) a transgenic quorum sensing signaling pathway from Vibrio fischeri. The genetic toggle switch endows these strains with binary response dynamics and an epigenetic inheritance that supports a persistent phenotypic alteration in response to transient signals. These features are exploited to engineer cells that form biofilms in response to DNA-damaging agents and cells that activate protein synthesis when the cell population reaches a critical density. Our work represents a step toward the development of "plug-and-play" genetic circuitry that can be used to create cells with programmable behaviors. heterologous gene expression | synthetic biology | Escherichia coli

Reverse engineering biological networks :applications in immune responses to bio-toxins.

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

Martino, Anthony A.; Sinclair, Michael B.; Davidson, George S.

Our aim is to determine the network of events, or the regulatory network, that defines an immune response to a bio-toxin. As a model system, we are studying T cell regulatory network triggered through tyrosine kinase receptor activation using a combination of pathway stimulation and time-series microarray experiments. Our approach is composed of five steps (1) microarray experiments and data error analysis, (2) data clustering, (3) data smoothing and discretization, (4) network reverse engineering, and (5) network dynamics analysis and fingerprint identification. The technological outcome of this study is a suite of experimental protocols and computational tools that reverse engineermore » regulatory networks provided gene expression data. The practical biological outcome of this work is an immune response fingerprint in terms of gene expression levels. Inferring regulatory networks from microarray data is a new field of investigation that is no more than five years old. To the best of our knowledge, this work is the first attempt that integrates experiments, error analyses, data clustering, inference, and network analysis to solve a practical problem. Our systematic approach of counting, enumeration, and sampling networks matching experimental data is new to the field of network reverse engineering. The resulting mathematical analyses and computational tools lead to new results on their own and should be useful to others who analyze and infer networks.« less

Genomic landscapes of endogenous retroviruses unveil intricate genetics of conventional and genetically-engineered laboratory mouse strains.

PubMed

Lee, Kang-Hoon; Lim, Debora; Chiu, Sophia; Greenhalgh, David; Cho, Kiho

2016-04-01

Laboratory strains of mice, both conventional and genetically engineered, have been introduced as critical components of a broad range of studies investigating normal and disease biology. Currently, the genetic identity of laboratory mice is primarily confirmed by surveying polymorphisms in selected sets of "conventional" genes and/or microsatellites in the absence of a single completely sequenced mouse genome. First, we examined variations in the genomic landscapes of transposable repetitive elements, named the TREome, in conventional and genetically engineered mouse strains using murine leukemia virus-type endogenous retroviruses (MLV-ERVs) as a probe. A survey of the genomes from 56 conventional strains revealed strain-specific TREome landscapes, and certain families (e.g., C57BL) of strains were discernible with defined patterns. Interestingly, the TREome landscapes of C3H/HeJ (toll-like receptor-4 [TLR4] mutant) inbred mice were different from its control C3H/HeOuJ (TLR4 wild-type) strain. In addition, a CD14 knock-out strain had a distinct TREome landscape compared to its control/backcross C57BL/6J strain. Second, an examination of superantigen (SAg, a "TREome gene") coding sequences of mouse mammary tumor virus-type ERVs in the genomes of the 46 conventional strains revealed a high diversity, suggesting a potential role of SAgs in strain-specific immune phenotypes. The findings from this study indicate that unexplored and intricate genomic variations exist in laboratory mouse strains, both conventional and genetically engineered. The TREome-based high-resolution genetics surveillance system for laboratory mice would contribute to efficient study design with quality control and accurate data interpretation. This genetics system can be easily adapted to other species ranging from plants to humans. Copyright © 2016 Elsevier Inc. All rights reserved.

Improving the selection efficiency of the counter-selection marker pheS* for the genetic engineering of Bacillus amyloliquefaciens.

PubMed

Kharchenko, Maria S; Teslya, Petr N; Babaeva, Maria N; Zakataeva, Natalia P

2018-05-01

Bacillus subtilis pheS was genetically modified to obtain a counter-selection marker with high selection efficiency in Bacillus amyloliquefaciens. The application of the new replication-thermosensitive integrative vector pNZTM1, containing this marker, pheS BsT255S/A309G , with a two-step replacement recombination procedure provides an effective tool for the genetic engineering of industrially important Bacillus species. Copyright © 2018. Published by Elsevier B.V.

Immersion of fry in 17-Alpha Methyltestosterone can be highly effective for sex reversal in rainbow trout

USDA-ARS?s Scientific Manuscript database

17-alpha methyltestosterone (MT) is currently used to sex reverse genetic female rainbow trout into phenotypic males, commonly referred to as neomales. Neomales are primarily generated to propagate all-female lines. The MT is most commonly administered orally, fed during the first 6-9 weeks after sw...

Novel Structure of Ty3 Reverse Transcriptase | Center for Cancer Research

Cancer.gov

Retrotransposons are mobile genetic elements that self amplify via a single-stranded RNA intermediate, which is converted to double-stranded DNA by an encoded reverse transcriptase (RT) with both DNA polymerase (pol) and ribonuclease H (RNase) activities. Categorized by whether they contain flanking long terminal repeat (LTR) sequences, retrotransposons play a critical role in

Current biotechnological developments in Belgium.

PubMed

Masschelein, C A; Callegari, J P; Laurent, M; Simon, J P; Taeymans, D

1989-01-01

In recent years, actions have been undertaken by the Belgian government to promote process innovation and technical diversification. Research programs are initiated and coordinated by the study committee for biotechnology setup within the Institute for Scientific Research in Industry and Agriculture (IRSIA). As a result of this action, the main areas where biotechnological processes are developed or commercially exploited include plant genetics, protein engineering, hybridoma technology, biopesticides, production by genetic engineering of vaccines and drugs, monoclonal detection of human and animal deseases, process reactors for aerobic and anaerobic wastewater treatment, and genetic modification of yeast and bacteria as a base for biomass and energy. Development research also includes new fermentation technologies principally based on immobilization of microorganisms, reactor design, and optimization of unit operations involved in downstream processing. Food, pharmaceutical, and chemical industries are involved in genetic engineering and biotechnology and each of these sectors is overviewed in this paper.

Genetic engineering of mesenchymal stem cells and its application in human disease therapy.

PubMed

Hodgkinson, Conrad P; Gomez, José A; Mirotsou, Maria; Dzau, Victor J

2010-11-01

The use of stem cells for tissue regeneration and repair is advancing both at the bench and bedside. Stem cells isolated from bone marrow are currently being tested for their therapeutic potential in a variety of clinical conditions including cardiovascular injury, kidney failure, cancer, and neurological and bone disorders. Despite the advantages, stem cell therapy is still limited by low survival, engraftment, and homing to damage area as well as inefficiencies in differentiating into fully functional tissues. Genetic engineering of mesenchymal stem cells is being explored as a means to circumvent some of these problems. This review presents the current understanding of the use of genetically engineered mesenchymal stem cells in human disease therapy with emphasis on genetic modifications aimed to improve survival, homing, angiogenesis, and heart function after myocardial infarction. Advancements in other disease areas are also discussed.

System Re-engineering Project Executive Summary

DTIC Science & Technology

1991-11-01

Management Information System (STAMIS) application. This project involved reverse engineering, evaluation of structured design and object-oriented design, and re- implementation of the system in Ada. This executive summary presents the approach to re-engineering the system, the lessons learned while going through the process, and issues to be considered in future tasks of this nature.... Computer-Aided Software Engineering (CASE), Distributed Software, Ada, COBOL, Systems Analysis, Systems Design, Life Cycle Development, Functional Decomposition, Object-Oriented

Production of human lactoferrin and lysozyme in the milk of transgenic dairy animals: past, present, and future.

PubMed

Cooper, Caitlin A; Maga, Elizabeth A; Murray, James D

2015-08-01

Genetic engineering, which was first developed in the 1980s, allows for specific additions to animals' genomes that are not possible through conventional breeding. Using genetic engineering to improve agricultural animals was first suggested when the technology was in the early stages of development by Palmiter et al. (Nature 300:611-615, 1982). One of the first agricultural applications identified was generating transgenic dairy animals that could produce altered or novel proteins in their milk. Human milk contains high levels of antimicrobial proteins that are found in low concentrations in the milk of ruminants, including the antimicrobial proteins lactoferrin and lysozyme. Lactoferrin and lysozyme are both part of the innate immune system and are secreted in tears, mucus, and throughout the gastrointestinal (GI) tract. Due to their antimicrobial properties and abundance in human milk, multiple lines of transgenic dairy animals that produce either human lactoferrin or human lysozyme have been developed. The focus of this review is to catalogue the different lines of genetically engineered dairy animals that produce either recombinant lactoferrin or lysozyme that have been generated over the years as well as compare the wealth of research that has been done on the in vitro and in vivo effects of the milk they produce. While recent advances including the development of CRISPRs and TALENs have removed many of the technical barriers to predictable and efficient genetic engineering in agricultural species, there are still many political and regulatory hurdles before genetic engineering can be used in agriculture. It is important to consider the substantial amount of work that has been done thus far on well established lines of genetically engineered animals evaluating both the animals themselves and the products they yield to identify the most effective path forward for future research and acceptance of this technology.

Sexual and somatic development of wood frog tadpoles along a thermal gradient.

PubMed

Lambert, Max R; Smylie, Meredith S; Roman, Amber J; Freidenburg, L Kealoha; Skelly, David K

2018-02-01

All amphibian species are known to have genetic sex determination. However, a variety of environmental conditions can moderate sexual differentiation, in some cases leading to sex reversal and skewed sex ratios. While there has been a recent focus on chemically-induced sex reversal in amphibians, temperature can also influence sexual differentiation. Building upon a classic 1929 study by Emil Witschi, we assessed temperature-mediated sex reversal. Witschi found that the wood frog sex ratio is 100% male at a high temperature (32°C) compared to a 50:50 sex ratio at 20°C. This pattern is consistent with multiple models of environmentally mediated sexual differentiation in vertebrates. To better understand thermally mediated sex reversal, we raised wood frogs at temperature increments of ∼1°C between 19 and 34°C. Mirroring earlier findings, wood frog metamorph sex ratios are indistinguishable from 50:50 at the lowest temperature and entirely male at the highest temperatures. In between, sex ratios become increasingly male-dominated as temperatures increase, implying a steadily increasing tendency toward female-to-male sex reversal in warmer environments. There was no evidence of a threshold temperature effect on reversal patterns. We also show that, compared to males, females metamorphose larger and later in cooler conditions but earlier and smaller under warmer conditions. While the ecological relevance in this species is unknown, these results conform to the Charnov-Bull model of sex determination (in which female-to-male sex reversal can increase fitness to genetic females at higher temperatures), suggesting the system would reward further study. © 2018 Wiley Periodicals, Inc.

Polygenic risk and the development and course of asthma: Evidence from a 4-decade longitudinal study

PubMed Central

Belsky, DW; Sears, MR; Hancox, RJ; Harrington, HL; Houts, R; Moffitt, TE; Sugden, K; Williams, B; Poulton, R; Caspi, A

2013-01-01

BACKGROUND Genome-wide association studies (GWAS) have discovered loci that predispose to asthma. To integrate these new discoveries with emerging models of asthma pathobiology, research is needed to test how genetic discoveries relate to developmental and biological characteristics of asthma. METHODS We derived a multi-locus profile of genetic risk from published GWAS of asthma case status. We then tested associations between this “genetic risk score” and developmental and biological characteristics of asthma in a population-based long-running birth cohort, the Dunedin Longitudinal Study (n=1,037). We evaluated asthma onset, persistence, atopy, airway hyperresponsiveness, incompletely reversible airflow obstruction, and asthma-related school and work absenteeism and hospitalization during 9 prospective assessments spanning ages 9–38 years, when 95% of surviving cohort members were seen. INTERPRETATION Cohort members at higher genetic risk experienced asthma onset earlier in life (HR=1.12 [1.01–1.26]). Childhood-onset asthma cases at higher genetic risk were more likely to become life-course-persistent asthma cases (RR=1.36 [1.14–1.63]). Asthma cases at higher genetic risk more often manifested atopy (RR=1.07 [1.01–1.14]), airway hyperresponsiveness (RR=1.16 [1.03–1.32]), and incompletely reversible airflow obstruction (RR=1.28 [1.04–1.57]). They were also more likely to miss school or work due to asthma (IRR=1.38 [1.02–1.86]) and to be hospitalized with breathing problems (HR=1.38 [1.07–1.79]). Genotypic information about asthma risk was independent of and additive to information derived from cohort members’ family histories of asthma. CONCLUSIONS Findings from this population study confirm that GWAS-discoveries for asthma associate with a childhood-onset phenotype and advance asthma genetics beyond the original GWAS-discoveries in three ways: (1) We show that genetic risks predict which childhood-onset asthma cases remit and which become life-course-persistent cases, although these predictions are not sufficiently sensitive or specific to support immediate clinical translation; (2) We elucidate a biological profile of the asthma that arises from these genetic risks: asthma characterized by atopy and airway hyperresponsiveness and leading to incompletely reversible airflow obstruction; and (3) We describe the real-life impact of GWAS-discoveries by quantifying genetic associations with missed school and work and hospitalization. PMID:24429243

Gender Differences in the Consistency of Middle School Students' Interest in Engineering and Science Careers

ERIC Educational Resources Information Center

Ing, Marsha; Aschbacher, Pamela R.; Tsai, Sherry M.

2014-01-01

This longitudinal study analyzes survey responses in seventh, eighth, and ninth grade from diverse public school students (n = 482) to explore gender differences in engineering and science career preferences. Females were far more likely to express interest in a science career (31%) than an engineering career (13%), while the reverse was true for…

Reverse Engineering and Software Products Reuse to Teach Collaborative Web Portals: A Case Study with Final-Year Computer Science Students

ERIC Educational Resources Information Center

Medina-Dominguez, Fuensanta; Sanchez-Segura, Maria-Isabel; Mora-Soto, Arturo; Amescua, Antonio

2010-01-01

The development of collaborative Web applications does not follow a software engineering methodology. This is because when university students study Web applications in general, and collaborative Web portals in particular, they are not being trained in the use of software engineering techniques to develop collaborative Web portals. This paper…

Foundations and Emerging Paradigms for Computing in Living Cells.

PubMed

Ma, Kevin C; Perli, Samuel D; Lu, Timothy K

2016-02-27

Genetic circuits, composed of complex networks of interacting molecular machines, enable living systems to sense their dynamic environments, perform computation on the inputs, and formulate appropriate outputs. By rewiring and expanding these circuits with novel parts and modules, synthetic biologists have adapted living systems into vibrant substrates for engineering. Diverse paradigms have emerged for designing, modeling, constructing, and characterizing such artificial genetic systems. In this paper, we first provide an overview of recent advances in the development of genetic parts and highlight key engineering approaches. We then review the assembly of these parts into synthetic circuits from the perspectives of digital and analog logic, systems biology, and metabolic engineering, three areas of particular theoretical and practical interest. Finally, we discuss notable challenges that the field of synthetic biology still faces in achieving reliable and predictable forward-engineering of artificial biological circuits. Copyright © 2016. Published by Elsevier Ltd.

File Compression and Expansion of the Genetic Code by the use of the Yin/Yang Directions to find its Sphered Cube

PubMed Central

Castro-Chavez, Fernando

2014-01-01

Objective The objective of this article is to demonstrate that the genetic code can be studied and represented in a 3-D Sphered Cube for bioinformatics and for education by using the graphical help of the ancient “Book of Changes” or I Ching for the comparison, pair by pair, of the three basic characteristics of nucleotides: H-bonds, molecular structure, and their tautomerism. Methods The source of natural biodiversity is the high plasticity of the genetic code, analyzable with a reverse engineering of its 2-D and 3-D representations (here illustrated), but also through the classical 64-hexagrams of the ancient I Ching, as if they were the 64-codons or words of the genetic code. Results In this article, the four elements of the Yin/Yang were found by correlating the 3×2=6 sets of Cartesian comparisons of the mentioned properties of nucleic acids, to the directionality of their resulting blocks of codons grouped according to their resulting amino acids and/or functions, integrating a 384-codon Sphered Cube whose function is illustrated by comparing six brain peptides and a promoter of osteoblasts from Humans versus Neanderthal, as well as to Negadi’s work on the importance of the number 384 within the genetic code. Conclusions Starting with the codon/anticodon correlation of Nirenberg, published in full here for the first time, and by studying the genetic code and its 3-D display, the buffers of reiteration within codons codifying for the same amino acid, displayed the two long (binary number one) and older Yin/Yang arrows that travel in opposite directions, mimicking the parental DNA strands, while annealing to the two younger and broken (binary number zero) Yin/Yang arrows, mimicking the new DNA strands; the graphic analysis of the of the genetic code and its plasticity was helpful to compare compatible sequences (human compatible to human versus neanderthal compatible to neanderthal), while further exploring the wondrous biodiversity of nature for educational purposes. PMID:25340175

Directed evolution and synthetic biology applications to microbial systems.

PubMed

Bassalo, Marcelo C; Liu, Rongming; Gill, Ryan T

2016-06-01

Biotechnology applications require engineering complex multi-genic traits. The lack of knowledge on the genetic basis of complex phenotypes restricts our ability to rationally engineer them. However, complex phenotypes can be engineered at the systems level, utilizing directed evolution strategies that drive whole biological systems toward desired phenotypes without requiring prior knowledge of the genetic basis of the targeted trait. Recent developments in the synthetic biology field accelerates the directed evolution cycle, facilitating engineering of increasingly complex traits in biological systems. In this review, we summarize some of the most recent advances in directed evolution and synthetic biology that allows engineering of complex traits in microbial systems. Then, we discuss applications that can be achieved through engineering at the systems level. Copyright © 2016 Elsevier Ltd. All rights reserved.

Engineering Delivery Vehicles for Genome Editing.

PubMed

Nelson, Christopher E; Gersbach, Charles A

2016-06-07

The field of genome engineering has created new possibilities for gene therapy, including improved animal models of disease, engineered cell therapies, and in vivo gene repair. The most significant challenge for the clinical translation of genome engineering is the development of safe and effective delivery vehicles. A large body of work has applied genome engineering to genetic modification in vitro, and clinical trials have begun using cells modified by genome editing. Now, promising preclinical work is beginning to apply these tools in vivo. This article summarizes the development of genome engineering platforms, including meganucleases, zinc finger nucleases, TALENs, and CRISPR/Cas9, and their flexibility for precise genetic modifications. The prospects for the development of safe and effective viral and nonviral delivery vehicles for genome editing are reviewed, and promising advances in particular therapeutic applications are discussed.

Biotechnological advancement in genetic improvement of broccoli (Brassica oleracea L. var. italica), an important vegetable crop.

PubMed

Kumar, Pankaj; Srivastava, Dinesh Kumar

2016-07-01

With the advent of molecular biotechnology, plant genetic engineering techniques have opened an avenue for the genetic improvement of important vegetable crops. Vegetable crop productivity and quality are seriously affected by various biotic and abiotic stresses which destabilize rural economies in many countries. Moreover, absence of proper post-harvest storage and processing facilities leads to qualitative and quantitative losses. In the past four decades, conventional breeding has significantly contributed to the improvement of vegetable yields, quality, post-harvest life, and resistance to biotic and abiotic stresses. However, there are many constraints in conventional breeding, which can only be overcome by advancements made in modern biology. Broccoli (Brassica oleracea L. var. italica) is an important vegetable crop, of the family Brassicaceae; however, various biotic and abiotic stresses cause enormous crop yield losses during the commercial cultivation of broccoli. Thus, genetic engineering can be used as a tool to add specific characteristics to existing cultivars. However, a pre-requisite for transferring genes into plants is the availability of efficient regeneration and transformation techniques. Recent advances in plant genetic engineering provide an opportunity to improve broccoli in many aspects. The goal of this review is to summarize genetic transformation studies on broccoli to draw the attention of researchers and scientists for its further genetic advancement.

Isolation of mtpim Proves Tnt1 a Useful Reverse Genetics Tool in Medicago truncatula and Uncovers New Aspects of AP1-Like Functions in Legumes1

PubMed Central

Benlloch, Reyes; d'Erfurth, Isabelle; Ferrandiz, Cristina; Cosson, Viviane; Beltrán, José Pío; Cañas, Luis Antonio; Kondorosi, Adam; Madueño, Francisco; Ratet, Pascal

2006-01-01

Comparative studies help shed light on how the huge diversity in plant forms found in nature has been produced. We use legume species to study developmental differences in inflorescence architecture and flower ontogeny with classical models such as Arabidopsis thaliana or Antirrhinum majus. Whereas genetic control of these processes has been analyzed mostly in pea (Pisum sativum), Medicago truncatula is emerging as a promising alternative system for these studies due to the availability of a range of genetic tools. To assess the use of the retrotransposon Tnt1 for reverse genetics in M. truncatula, we screened a small Tnt1-mutagenized population using degenerate primers for MADS-box genes, known controllers of plant development. We describe here the characterization of mtpim, a new mutant caused by the insertion of Tnt1 in a homolog to the PROLIFERATING INFLORESCENCE MERISTEM (PIM)/APETALA1 (AP1)/SQUAMOSA genes. mtpim shows flower-to-inflorescence conversion and altered flowers with sepals transformed into leaves, indicating that MtPIM controls floral meristem identity and flower development. Although more extreme, this phenotype resembles the pea pim mutants, supporting the idea that M. truncatula could be used to complement analysis of reproductive development already initiated in pea. In fact, our study reveals aspects not shown by analysis of pea mutants: that the mutation in the AP1 homolog interferes with the specification of floral organs from common primordia and causes conversion of sepals into leaves, in addition to true conversion of flowers into inflorescences. The isolation of mtpim represents a proof of concept demonstrating that Tnt1 populations can be efficiently used in reverse genetics screenings in M. truncatula. PMID:16963524

Genetic engineering of a mouse: Dr. Frank Ruddle and somatic cell genetics.

PubMed

Jones, Dennis

2011-06-01

Genetic engineering is the process of modifying an organism's genetic composition by adding foreign genes to produce desired traits or evaluate function. Dr. Jon W. Gordon and Sterling Professor Emeritus at Yale Dr. Frank H. Ruddle were pioneers in mammalian gene transfer research. Their research resulted in production of the first transgenic animals, which contained foreign DNA that was passed on to offspring. Transgenic mice have revolutionized biology, medicine, and biotechnology in the 21st century. In brief, this review revisits their creation of transgenic mice and discusses a few evolving applications of their transgenic technology used in biomedical research.

Dynamic role and importance of surrogate species for assessing potential adverse environmental impacts of genetically engineered insect-resistant plants on non-target organisms

USDA-ARS?s Scientific Manuscript database

Surrogate species have a long history of use in research and regulatory settings to understand the potentially harmful effects of toxic substances including pesticides. More recently, surrogate species have been used to evaluate the potential effects of proteins contained in genetically engineered ...

Can Man Control His Biological Evolution? A Symposium on Genetic Engineering. Probabilities and Practicalities

ERIC Educational Resources Information Center

Djerassi, Carl

1972-01-01

Manipulation of genes in human beings on a large scale is not possible under present conditions because it lacks economic potential and other attractions for industry. However, preventive'' genetic engineering may be a field for vast research in the future and will perhaps be approved by governments, parishes, people and industry. (PS)

Enhancing the Internationalisation of Distance Education in the Biological Sciences: The DUNE Project and Genetic Engineering.

ERIC Educational Resources Information Center

Leach, C. K.; And Others

1997-01-01

Describes the Distance Educational Network of Europe (DUNE) project that aims at enhancing the development of distance education in an international context. Highlights issues relating to the delivery of distance-learning courses in a transnational forum. Describes the genetic engineering course that aims at explaining the core techniques of…

77 FR 41357 - Monsanto Co.; Availability of Petition for Determination of Nonregulated Status of Canola...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-13

... Produced Through Genetic Engineering Which Are Plant Pests or Which There Is Reason to Believe Are Plant... into the environment) of organisms and products altered or produced through genetic engineering that... CP4 EPSPS expression in male reproductive tissues (i.e., pollen). Enhanced CP4 EPSPS expression in the...

Finding Patterns of Emergence in Science and Technology

DTIC Science & Technology

2012-09-24

formal evaluation scheduled – Case Studies, Eight Examples: Tissue Engineering, Cold Fusion, RF Metamaterials, DNA Microarrays, Genetic Algorithms, RNAi...emerging capabilities Case Studies, Eight Examples: • Tissue Engineering, Cold Fusion, RF Metamaterials, DNA Microarrays, Genetic Algorithms...Evidence Quality (i.e., the rubric ) and deliver comprehensible evidential support for nomination • Demonstrate proof-of-concept nomination for Chinese

77 FR 42693 - Monsanto Company and KWS SAAT AG; Determination of Nonregulated Status of Sugar Beet Genetically...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-07-20

... for Tolerance to the Herbicide Glyphosate AGENCY: Animal and Plant Health Inspection Service, USDA... engineered for tolerance to the herbicide glyphosate, designated as H7-1, is no longer considered a regulated... as event H7-1, which has been genetically engineered for tolerance to the herbicide glyphosate. The...