GAN: a platform of genomics and genetics analysis and application in Nicotiana

PubMed Central

Yang, Shuai; Zhang, Xingwei; Li, Huayang; Chen, Yudong

2018-01-01

Abstract Nicotiana is an important Solanaceae genus, and plays a significant role in modern biological research. Massive Nicotiana biological data have emerged from in-depth genomics and genetics studies. From big data to big discovery, large-scale analysis and application with new platforms is critical. Based on data accumulation, a comprehensive platform of Genomics and Genetics Analysis and Application in Nicotiana (GAN) has been developed, and is publicly available at http://biodb.sdau.edu.cn/gan/. GAN consists of four main sections: (i) Sources, a total of 5267 germplasm lines, along with detailed descriptions of associated characteristics, are all available on the Germplasm page, which can be queried using eight different inquiry modes. Seven fully sequenced species with accompanying sequences and detailed genomic annotation are available on the Genomics page. (ii) Genetics, detailed descriptions of 10 genetic linkage maps, constructed by different parents, 2239 KEGG metabolic pathway maps and 209 945 gene families across all catalogued genes, along with two co-linearity maps combining N. tabacum with available tomato and potato linkage maps are available here. Furthermore, 3 963 119 genome-SSRs, 10 621 016 SNPs, 12 388 PIPs and 102 895 reverse transcription-polymerase chain reaction primers, are all available to be used and searched on the Markers page. (iii) Tools, the genome browser JBrowse and five useful online bioinformatics softwares, Blast, Primer3, SSR-detect, Nucl-Protein and E-PCR, are provided on the JBrowse and Tools pages. (iv) Auxiliary, all the datasets are shown on a Statistics page, and are available for download on a Download page. In addition, the user’s manual is provided on a Manual page in English and Chinese languages. GAN provides a user-friendly Web interface for searching, browsing and downloading the genomics and genetics datasets in Nicotiana. As far as we can ascertain, GAN is the most comprehensive source of bio-data available, and the most applicable resource for breeding, gene mapping, gene cloning, the study of the origin and evolution of polyploidy, and related studies in Nicotiana. Database URL: http://biodb.sdau.edu.cn/gan/ PMID:29688356

First TILLING Platform in Cucurbita pepo: A New Mutant Resource for Gene Function and Crop Improvement

PubMed Central

Vicente-Dólera, Nelly; Troadec, Christelle; Moya, Manuel; del Río-Celestino, Mercedes; Pomares-Viciana, Teresa; Bendahmane, Abdelhafid; Picó, Belén; Román, Belén; Gómez, Pedro

2014-01-01

Although the availability of genetic and genomic resources for Cucurbita pepo has increased significantly, functional genomic resources are still limited for this crop. In this direction, we have developed a high throughput reverse genetic tool: the first TILLING (Targeting Induced Local Lesions IN Genomes) resource for this species. Additionally, we have used this resource to demonstrate that the previous EMS mutant population we developed has the highest mutation density compared with other cucurbits mutant populations. The overall mutation density in this first C. pepo TILLING platform was estimated to be 1/133 Kb by screening five additional genes. In total, 58 mutations confirmed by sequencing were identified in the five targeted genes, thirteen of which were predicted to have an impact on the function of the protein. The genotype/phenotype correlation was studied in a peroxidase gene, revealing that the phenotype of seedling homozygous for one of the isolated mutant alleles was albino. These results indicate that the TILLING approach in this species was successful at providing new mutations and can address the major challenge of linking sequence information to biological function and also the identification of novel variation for crop breeding. PMID:25386735

Single-Vector, Single-Injection Recombinant Vesicular Stomatitis Virus Vaccines Against High-Containment Viruses.

PubMed

Whitt, Michael A; Geisbert, Thomas W; Mire, Chad E

2016-01-01

There are many avenues for making an effective vaccine against viruses. Depending on the virus these can include one of the following: inactivation of whole virions; attenuation of viruses; recombinant viral proteins; non-replication-competent virus particles; or surrogate virus vector systems such as vesicular stomatitis virus (VSV). VSV is a prototypic enveloped animal virus that has been used for over four decades to study virus replication, entry, and assembly due to its ability to replicate to high titers in a wide variety of mammalian and insect cells. The use of reverse genetics to recover infectious and single-cycle replicating VSV from plasmid DNA transfected in cell culture began a revolution in the study of recombinant VSV (rVSV). This platform can be manipulated to study the viral genetic sequences and proteins important in the virus life cycle. Additionally, foreign genes can be inserted between naturally occurring or generated start/stop signals and polyadenylation sites within the VSV genome. VSV has a tolerance for foreign gene expression which has led to numerous rVSVs reported in the literature. Of particular interest are the very effective single-dose rVSV vaccine vectors against high-containment viruses such as filoviruses, henipaviruses, and arenaviruses. Herein we describe the methods for selecting foreign antigenic genes, selecting the location within the VSV genome for insertion, generation of rVSV using reverse genetics, and proper vaccine study designs.

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.

Novel throughput phenotyping platforms in plant genetic studies.

PubMed

Montes, Juan M; Melchinger, Albrecht E; Reif, Jochen C

2007-10-01

Unraveling the genetic basis of complex traits in plants is limited by the lack of appropriate phenotyping platforms that enable high-throughput screening of many genotypes in multilocation field trials. Near-infrared spectroscopy on agricultural harvesters and spectral reflectance of plant canopies have recently been reported as promising components of novel phenotyping platforms. Understanding the genetic basis of complex traits is now within reach with the use of these new techniques.

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.

Generation of recombinant rotaviruses expressing fluorescent proteins using an optimized reverse genetics system.

PubMed

Komoto, Satoshi; Fukuda, Saori; Ide, Tomihiko; Ito, Naoto; Sugiyama, Makoto; Yoshikawa, Tetsushi; Murata, Takayuki; Taniguchi, Koki

2018-04-18

An entirely plasmid-based reverse genetics system for rotaviruses was established very recently. We improved the reverse genetics system to generate recombinant rotavirus by transfecting only 11 cDNA plasmids for its 11 gene segments under the condition of increasing the ratio of the cDNA plasmids for NSP2 and NSP5 genes. Utilizing this highly efficient system, we then engineered infectious recombinant rotaviruses expressing bioluminescent (NanoLuc luciferase) and fluorescent (EGFP and mCherry) reporters. These recombinant rotaviruses expressing reporters remained genetically stable during serial passages. Our reverse genetics approach and recombinant rotaviruses carrying reporter genes will be great additions to the tool kit for studying the molecular virology of rotavirus, and for developing future next-generation vaccines and expression vectors. IMPORTANCE Rotavirus is one of the most important pathogens causing severe gastroenteritis in young children worldwide. In this paper, we describe a robust and simple reverse genetics system based on only rotavirus cDNAs, and its application for engineering infectious recombinant rotaviruses harboring bioluminescent (NanoLuc) and fluorescent (EGFP and mCherry) protein genes. This highly efficient reverse genetics system and recombinant RVAs expressing reporters could be powerful tools for the study of different aspects of rotavirus replication. Furthermore, they may be useful for next-generation vaccine production for this medically important virus. Copyright © 2018 American Society for Microbiology.

A universal TagModule collection for parallel genetic analysis of microorganisms

PubMed Central

Oh, Julia; Fung, Eula; Price, Morgan N.; Dehal, Paramvir S.; Davis, Ronald W.; Giaever, Guri; Nislow, Corey; Arkin, Adam P.; Deutschbauer, Adam

2010-01-01

Systems-level analyses of non-model microorganisms are limited by the existence of numerous uncharacterized genes and a corresponding over-reliance on automated computational annotations. One solution to this challenge is to disrupt gene function using DNA tag technology, which has been highly successful in parallelizing reverse genetics in Saccharomyces cerevisiae and has led to discoveries in gene function, genetic interactions and drug mechanism of action. To extend the yeast DNA tag methodology to a wide variety of microorganisms and applications, we have created a universal, sequence-verified TagModule collection. A hallmark of the 4280 TagModules is that they are cloned into a Gateway entry vector, thus facilitating rapid transfer to any compatible genetic system. Here, we describe the application of the TagModules to rapidly generate tagged mutants by transposon mutagenesis in the metal-reducing bacterium Shewanella oneidensis MR-1 and the pathogenic yeast Candida albicans. Our results demonstrate the optimal hybridization properties of the TagModule collection, the flexibility in applying the strategy to diverse microorganisms and the biological insights that can be gained from fitness profiling tagged mutant collections. The publicly available TagModule collection is a platform-independent resource for the functional genomics of a wide range of microbial systems in the post-genome era. PMID:20494978

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...

Resealable, optically accessible, PDMS-free fluidic platform for ex vivo interrogation of pancreatic islets.

PubMed

Lenguito, Giovanni; Chaimov, Deborah; Weitz, Jonathan R; Rodriguez-Diaz, Rayner; Rawal, Siddarth A K; Tamayo-Garcia, Alejandro; Caicedo, Alejandro; Stabler, Cherie L; Buchwald, Peter; Agarwal, Ashutosh

2017-02-28

We report the design and fabrication of a robust fluidic platform built out of inert plastic materials and micromachined features that promote optimized convective fluid transport. The platform is tested for perfusion interrogation of rodent and human pancreatic islets, dynamic secretion of hormones, concomitant live-cell imaging, and optogenetic stimulation of genetically engineered islets. A coupled quantitative fluid dynamics computational model of glucose stimulated insulin secretion and fluid dynamics was first utilized to design device geometries that are optimal for complete perfusion of three-dimensional islets, effective collection of secreted insulin, and minimization of system volumes and associated delays. Fluidic devices were then fabricated through rapid prototyping techniques, such as micromilling and laser engraving, as two interlocking parts from materials that are non-absorbent and inert. Finally, the assembly was tested for performance using both rodent and human islets with multiple assays conducted in parallel, such as dynamic perfusion, staining and optogenetics on standard microscopes, as well as for integration with commercial perfusion machines. The optimized design of convective fluid flows, use of bio-inert and non-absorbent materials, reversible assembly, manual access for loading and unloading of islets, and straightforward integration with commercial imaging and fluid handling systems proved to be critical for perfusion assay, and particularly suited for time-resolved optogenetics studies.

[Exploration and practice of genetics teaching assisted by network technology platform].

PubMed

Li, Ya-Xuan; Zhang, Fei-Xiong; Zhao, Xin; Cai, Min-Hua; Yan, Yue-Ming; Hu, Ying-Kao

2010-04-01

More teaching techniques have been brought out gradually along with the development of new technologies. On the basis of those traditional teaching methods, a new platform has been set up by the network technology for teaching process. In genetics teaching, it is possible to use the network platform to guide student studying, promote student's learning interest and study independently by themselves. It has been proved, after exploring and applying for many years, that network teaching is one of the most useful methods and has inimitable advantage comparing to the traditional ones in genetics teaching. The establishment of network teaching platform, the advantage and deficiency and relevant strategies were intro-duced in this paper.

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

Generation of EMS-Mutagenized Populations of Arabidopsis thaliana for Polyamine Genetics.

PubMed

Atanasov, Kostadin E; Liu, Changxin; Tiburcio, Antonio F; Alcázar, Rubén

2018-01-01

In the recent years, genetic engineering of polyamine biosynthetic genes has provided evidence for their involvement in plant stress responses and different aspects of plant development. Such approaches are being complemented with the use of reverse genetics, in which mutants affected on a particular trait, tightly associated with polyamines, are isolated and the causal genes mapped. Reverse genetics enables the identification of novel genes in the polyamine pathway, which may be involved in downstream signaling, transport, homeostasis, or perception. Here, we describe a basic protocol for the generation of ethyl methanesulfonate (EMS) mutagenized populations of Arabidopsis thaliana for its use in reverse genetics applied to polyamines.

Characterization of cis-Acting RNA Elements of Zika Virus by Using a Self-Splicing Ribozyme-Dependent Infectious Clone.

PubMed

Liu, Zhong-Yu; Yu, Jiu-Yang; Huang, Xing-Yao; Fan, Hang; Li, Xiao-Feng; Deng, Yong-Qiang; Ji, Xue; Cheng, Meng-Li; Ye, Qing; Zhao, Hui; Han, Jian-Feng; An, Xiao-Ping; Jiang, Tao; Zhang, Bo; Tong, Yi-Gang; Qin, Cheng-Feng

2017-11-01

Zika virus (ZIKV) has caused significant outbreaks and epidemics in the Americas recently, raising global concern due to its ability to cause microcephaly and other neurological complications. A stable and efficient infectious clone of ZIKV is urgently needed. However, the instability and toxicity of flavivirus cDNA clones in Escherichia coli hosts has hindered the development of ZIKV infectious clones. Here, using a novel self-splicing ribozyme-based strategy, we generated a stable infectious cDNA clone of a contemporary ZIKV strain imported from Venezuela to China in 2016. The constructed clone contained a modified version of the group II self-splicing intron P.li.LSUI2 near the junction between the E and NS1 genes, which were removed from the RNA transcripts by an easy-to-establish in vitro splicing reaction. Transfection of the spliced RNAs into BHK-21 cells led to the production of infectious progeny virus that resembled the parental virus. Finally, potential cis -acting RNA elements in ZIKV genomic RNA were identified based on this novel reverse genetics system, and the critical role of 5'-SLA promoter and 5'-3' cyclization sequences were characterized by a combination of different assays. Our results provide another stable and reliable reverse genetics system for ZIKV that will help study ZIKV infection and pathogenesis, and the novel self-splicing intron-based strategy could be further expanded for the construction of infectious clones from other emerging and reemerging flaviviruses. IMPORTANCE The ongoing Zika virus (ZIKV) outbreaks have drawn global concern due to the unexpected causal link to fetus microcephaly and other severe neurological complications. The infectious cDNA clones of ZIKV are critical for the research community to study the virus, understand the disease, and inform vaccine design and antiviral screening. A panel of existing technologies have been utilized to develop ZIKV infectious clones. Here, we successfully generated a stable infectious clone of a 2016 ZIKV strain using a novel self-splicing ribozyme-based technology that abolished the potential toxicity of ZIKV cDNA clones to the E. coli host. Moreover, two crucial cis -acting replication elements (5'-SLA and 5'-CS) of ZIKV were first identified using this novel reverse genetics system. This novel self-splicing ribozyme-based reverse genetics platform will be widely utilized in future ZIKV studies and provide insight for the development of infectious clones of other emerging viruses. Copyright © 2017 American Society for Microbiology.

Characterization of cis-Acting RNA Elements of Zika Virus by Using a Self-Splicing Ribozyme-Dependent Infectious Clone

PubMed Central

Liu, Zhong-Yu; Yu, Jiu-Yang; Huang, Xing-Yao; Fan, Hang; Li, Xiao-Feng; Deng, Yong-Qiang; Ji, Xue; Cheng, Meng-Li; Ye, Qing; Zhao, Hui; Han, Jian-Feng; An, Xiao-Ping; Jiang, Tao; Zhang, Bo; Tong, Yi-Gang

2017-01-01

ABSTRACT Zika virus (ZIKV) has caused significant outbreaks and epidemics in the Americas recently, raising global concern due to its ability to cause microcephaly and other neurological complications. A stable and efficient infectious clone of ZIKV is urgently needed. However, the instability and toxicity of flavivirus cDNA clones in Escherichia coli hosts has hindered the development of ZIKV infectious clones. Here, using a novel self-splicing ribozyme-based strategy, we generated a stable infectious cDNA clone of a contemporary ZIKV strain imported from Venezuela to China in 2016. The constructed clone contained a modified version of the group II self-splicing intron P.li.LSUI2 near the junction between the E and NS1 genes, which were removed from the RNA transcripts by an easy-to-establish in vitro splicing reaction. Transfection of the spliced RNAs into BHK-21 cells led to the production of infectious progeny virus that resembled the parental virus. Finally, potential cis-acting RNA elements in ZIKV genomic RNA were identified based on this novel reverse genetics system, and the critical role of 5′-SLA promoter and 5′-3′ cyclization sequences were characterized by a combination of different assays. Our results provide another stable and reliable reverse genetics system for ZIKV that will help study ZIKV infection and pathogenesis, and the novel self-splicing intron-based strategy could be further expanded for the construction of infectious clones from other emerging and reemerging flaviviruses. IMPORTANCE The ongoing Zika virus (ZIKV) outbreaks have drawn global concern due to the unexpected causal link to fetus microcephaly and other severe neurological complications. The infectious cDNA clones of ZIKV are critical for the research community to study the virus, understand the disease, and inform vaccine design and antiviral screening. A panel of existing technologies have been utilized to develop ZIKV infectious clones. Here, we successfully generated a stable infectious clone of a 2016 ZIKV strain using a novel self-splicing ribozyme-based technology that abolished the potential toxicity of ZIKV cDNA clones to the E. coli host. Moreover, two crucial cis-acting replication elements (5′-SLA and 5′-CS) of ZIKV were first identified using this novel reverse genetics system. This novel self-splicing ribozyme-based reverse genetics platform will be widely utilized in future ZIKV studies and provide insight for the development of infectious clones of other emerging viruses. PMID:28814522

pTcGW plasmid vectors 1.1 version: a versatile tool for Trypanosoma cruzi gene characterisation

PubMed Central

Kugeratski, Fernanda G; Batista, Michel; Inoue, Alexandre Haruo; Ramos, Bruno Dias; Krieger, Marco Aurelio; Marchini/, Fabricio K

2015-01-01

The functional characterisation of thousands of Trypanosoma cruzi genes remains a challenge. Reverse genetics approaches compatible with high-throughput cloning strategies can provide the tool needed to tackle this challenge. We previously published the pTcGW platform, composed by plasmid vectors carrying different options of N-terminal fusion tags based on Gateway® technology. Here, we present an improved 1.1 version of pTcGW vectors, which is characterised by a fully flexible structure allowing an easy customisation of each element of the vectors in a single cloning step. Additionally, both N and C-terminal fusions are available with new tag options for protein complexes purification. Three of the newly created vectors were successfully used to determine the cellular localisation of four T. cruzi proteins. The 1.1 version of pTcGW platform can be used in a variety of assays, such as protein overexpression, identification of protein-protein interaction and protein localisation. This powerful and versatile tool allows adding valuable functional information to T. cruzi genes and is freely available for scientific community. PMID:26200713

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.

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

Perumalla, Kalyan S.; Yoginath, Srikanth B.

Problems such as fault tolerance and scalable synchronization can be efficiently solved using reversibility of applications. Making applications reversible by relying on computation rather than on memory is ideal for large scale parallel computing, especially for the next generation of supercomputers in which memory is expensive in terms of latency, energy, and price. In this direction, a case study is presented here in reversing a computational core, namely, Basic Linear Algebra Subprograms, which is widely used in scientific applications. A new Reversible BLAS (RBLAS) library interface has been designed, and a prototype has been implemented with two modes: (1) amore » memory-mode in which reversibility is obtained by checkpointing to memory in forward and restoring from memory in reverse, and (2) a computational-mode in which nothing is saved in the forward, but restoration is done entirely via inverse computation in reverse. The article is focused on detailed performance benchmarking to evaluate the runtime dynamics and performance effects, comparing reversible computation with checkpointing on both traditional CPU platforms and recent GPU accelerator platforms. For BLAS Level-1 subprograms, data indicates over an order of magnitude better speed of reversible computation compared to checkpointing. For BLAS Level-2 and Level-3, a more complex tradeoff is observed between reversible computation and checkpointing, depending on computational and memory complexities of the subprograms.« less

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.

GeNets: a unified web platform for network-based genomic analyses.

PubMed

Li, Taibo; Kim, April; Rosenbluh, Joseph; Horn, Heiko; Greenfeld, Liraz; An, David; Zimmer, Andrew; Liberzon, Arthur; Bistline, Jon; Natoli, Ted; Li, Yang; Tsherniak, Aviad; Narayan, Rajiv; Subramanian, Aravind; Liefeld, Ted; Wong, Bang; Thompson, Dawn; Calvo, Sarah; Carr, Steve; Boehm, Jesse; Jaffe, Jake; Mesirov, Jill; Hacohen, Nir; Regev, Aviv; Lage, Kasper

2018-06-18

Functional genomics networks are widely used to identify unexpected pathway relationships in large genomic datasets. However, it is challenging to compare the signal-to-noise ratios of different networks and to identify the optimal network with which to interpret a particular genetic dataset. We present GeNets, a platform in which users can train a machine-learning model (Quack) to carry out these comparisons and execute, store, and share analyses of genetic and RNA-sequencing datasets.

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.

Towards reversible basic linear algebra subprograms: A performance study

DOE PAGES

Perumalla, Kalyan S.; Yoginath, Srikanth B.

2014-12-06

Problems such as fault tolerance and scalable synchronization can be efficiently solved using reversibility of applications. Making applications reversible by relying on computation rather than on memory is ideal for large scale parallel computing, especially for the next generation of supercomputers in which memory is expensive in terms of latency, energy, and price. In this direction, a case study is presented here in reversing a computational core, namely, Basic Linear Algebra Subprograms, which is widely used in scientific applications. A new Reversible BLAS (RBLAS) library interface has been designed, and a prototype has been implemented with two modes: (1) amore » memory-mode in which reversibility is obtained by checkpointing to memory in forward and restoring from memory in reverse, and (2) a computational-mode in which nothing is saved in the forward, but restoration is done entirely via inverse computation in reverse. The article is focused on detailed performance benchmarking to evaluate the runtime dynamics and performance effects, comparing reversible computation with checkpointing on both traditional CPU platforms and recent GPU accelerator platforms. For BLAS Level-1 subprograms, data indicates over an order of magnitude better speed of reversible computation compared to checkpointing. For BLAS Level-2 and Level-3, a more complex tradeoff is observed between reversible computation and checkpointing, depending on computational and memory complexities of the subprograms.« less

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.

Effects of the GluN2B-NMDA receptor antagonist Ro 25-6981 on two types of behavioral flexibility in rats.

PubMed

Clark, Emma; Antoniak, Kristen; Feniquito, Alyssandra; Dringenberg, Hans C

2017-02-15

Recent evidence has implicated N-methyl-d-aspartate receptors (NMDARs) in several aspects of learning and behavioral flexibility in rodents. Here, we examined the effects of treatment with Ro 25-6981, a selective antagonist of NMDARs containing GluN2B subunits, on two types of behavioral flexibility in rats, spatial reversal learning and set-shifting (spatial vs. motor strategy). To examine spatial reversal learning, rats were trained to swim to a hidden platform in a water maze over four days. On the following day, the platform was moved to a new location in the maze. Administration of Ro 25-6981 (10mg/kg) selectively impaired the early phase of reversal learning, but all rats learned to navigate to the new platform location over 12 trials. To examine set-shifting, independent groups of rats were trained to either swim to a fixed location (spatial strategy) or use a motor response (e.g., "turn left"; motor strategy) to find a hidden escape platform in a cross-shaped water maze apparatus; after task acquisition, rats were trained on the second, novel strategy (set-shift) following treatment with either Ro 25-6981 (10mg/kg) or saline. Administration of Ro 25-6981 had no effect on the ability of rats to perform the set-shift and use the new strategy to locate the escape platform. These results suggest that, in rats, spatial reversal learning, but not set-shifting, is sensitive to Ro-25-6981 treatment. Thus, NMDARs-GluN2B signaling may play a selective role in some forms of behavioral plasticity, particularly for situations involving the updating of information in the spatial domain. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

Age-dependent effects of neonatal methamphetamine exposure on spatial learning

PubMed Central

Vorhees, Charles V.; Skelton, Matthew R.; Williams, Michael T.

2009-01-01

Neonatal rats exposed to (+)-methamphetamine (MA) display spatial learning and reference memory deficits in the Morris water maze. In separate experiments the emergence and permanence of these effects were determined. Twenty litters were used in each experiment, and two male/female pairs/litter received saline or MA (5 mg/kg four times a day) on postnatal days (P) 11–20. In experiment 1, one MA and one saline pair from each litter began testing on either P30 or P40, whereas in experiment 2, testing began on P180 or P360. Animals received trials in a straight swimming channel and then in the Morris maze (acquisition, reversal, and reduced platform phases). In both experiments, MA-treated groups showed impaired learning in the platform trials and impaired reference memory in the probe trials, which were largely independent of age. The P30 and P40 MA impairments were seen on acquisition and reduced platform trials but not on reversal. In the probe trials, MA effects were seen during all phases. The P180 and P360 MA-induced deficits were seen in all phases of the platform trials. In probe trials, deficits were only seen during the reversal and reduced platform phases. The results demonstrate that neonatal MA treatment induces spatial learning and reference memory deficits that emerge early and persist until at least 1 year of age, suggesting permanence. PMID:17762523

An automated microfluidic DNA microarray platform for genetic variant detection in inherited arrhythmic diseases.

PubMed

Huang, Shu-Hong; Chang, Yu-Shin; Juang, Jyh-Ming Jimmy; Chang, Kai-Wei; Tsai, Mong-Hsun; Lu, Tzu-Pin; Lai, Liang-Chuan; Chuang, Eric Y; Huang, Nien-Tsu

2018-03-12

In this study, we developed an automated microfluidic DNA microarray (AMDM) platform for point mutation detection of genetic variants in inherited arrhythmic diseases. The platform allows for automated and programmable reagent sequencing under precise conditions of hybridization flow and temperature control. It is composed of a commercial microfluidic control system, a microfluidic microarray device, and a temperature control unit. The automated and rapid hybridization process can be performed in the AMDM platform using Cy3 labeled oligonucleotide exons of SCN5A genetic DNA, which produces proteins associated with sodium channels abundant in the heart (cardiac) muscle cells. We then introduce a graphene oxide (GO)-assisted DNA microarray hybridization protocol to enable point mutation detection. In this protocol, a GO solution is added after the staining step to quench dyes bound to single-stranded DNA or non-perfectly matched DNA, which can improve point mutation specificity. As proof-of-concept we extracted the wild-type and mutant of exon 12 and exon 17 of SCN5A genetic DNA from patients with long QT syndrome or Brugada syndrome by touchdown PCR and performed a successful point mutation discrimination in the AMDM platform. Overall, the AMDM platform can greatly reduce laborious and time-consuming hybridization steps and prevent potential contamination. Furthermore, by introducing the reciprocating flow into the microchannel during the hybridization process, the total assay time can be reduced to 3 hours, which is 6 times faster than the conventional DNA microarray. Given the automatic assay operation, shorter assay time, and high point mutation discrimination, we believe that the AMDM platform has potential for low-cost, rapid and sensitive genetic testing in a simple and user-friendly manner, which may benefit gene screening in medical practice.

Compact and lightweight support platform with electromagnetic disturbance elimination for interferometer on reversed field pinch Keda Torus eXperiment

NASA Astrophysics Data System (ADS)

Mao, Wenzhe; Yuan, Peng; Zheng, Jian; Ding, Weixing; Li, Hong; Lan, Tao; Liu, Adi; Liu, Wandong; Xie, Jinlin

2016-11-01

A compact and lightweight support platform has been used as a holder for the interferometer system on the Keda Torus eXperiment (KTX), which is a reversed field pinch device. The vibration caused by the interaction between the time-varying magnetic field and the induced current driven in the metal optical components has been measured and, following comparison with the mechanical vibration of the KTX device and the refraction effect of the ambient turbulent air flow, has been identified as the primary vibration source in this case. To eliminate this electromagnetic disturbance, nonmetallic epoxy resin has been selected as the material for the support platform and the commercially available metal optical mounts are replaced. Following these optimization steps and mechanical reinforcements, the stability of the interferometer platform has improved significantly. The phase shift caused by the vibration has been reduced to the level of background noise.

A CRISPR Cas9-based gene drive platform for genetic interaction analysis in Candida albicans

PubMed Central

Shapiro, Rebecca S.; Chavez, Alejandro; Porter, Caroline B. M.; Hamblin, Meagan; Kaas, Christian S.; DiCarlo, James E.; Zeng, Guisheng; Xu, Xiaoli; Revtovich, Alexey V.; Kirienko, Natalia V.; Wang, Yue; Church, George M.; Collins, James J.

2018-01-01

Candida albicans is the leading cause of fungal infections; yet, complex genetic interaction analysis remains cumbersome in this diploid pathogen. Here, we developed a CRISPR-Cas9-based ‘gene drive array’ (GDA) platform to facilitate efficient genetic analysis in C. albicans. In our system, a modified DNA donor molecule acts as a selfish genetic element, replaces the targeted site, and propagates to replace additional wild-type loci. Using mating-competent C. albicans haploids, each carrying a different gene drive disabling a gene of interest, we are able to create diploid strains that are homozygous double-deletion mutants. We generate double-gene deletion libraries to demonstrate this technology, targeting antifungal efflux and biofilm adhesion factors. We screen these libraries to identify virulence regulators and determine how genetic networks shift under diverse conditions. This platform transforms our ability to perform genetic interaction analysis in C. albicans and is readily extended to other fungal pathogens. PMID:29062088

Gene expression platform for synthetic biology in the human pathogen Streptococcus pneumoniae.

PubMed

Sorg, Robin A; Kuipers, Oscar P; Veening, Jan-Willem

2015-03-20

The human pathogen Streptococcus pneumoniae (pneumococcus) is a bacterium that owes its success to complex gene expression regulation patterns on both the cellular and the population level. Expression of virulence factors enables a mostly hazard-free presence of the commensal, in balance with the host and niche competitors. Under specific circumstances, changes in this expression can result in a more aggressive behavior and the reversion to the invasive form as pathogen. These triggering conditions are very difficult to study due to the fact that environmental cues are often unknown or barely possible to simulate outside the host (in vitro). An alternative way of investigating expression patterns is found in synthetic biology approaches of reconstructing regulatory networks that mimic an observed behavior with orthogonal components. Here, we created a genetic platform suitable for synthetic biology approaches in S. pneumoniae and characterized a set of standardized promoters and reporters. We show that our system allows for fast and easy cloning with the BglBrick system and that reliable and robust gene expression after integration into the S. pneumoniae genome is achieved. In addition, the cloning system was extended to allow for direct linker-based assembly of ribosome binding sites, peptide tags, and fusion proteins, and we called this new generally applicable standard "BglFusion". The gene expression platform and the methods described in this study pave the way for employing synthetic biology approaches in S. pneumoniae.

Complete Protection against Influenza Virus H1N1 Strain A/PR/8/34 Challenge in Mice Immunized with Non-Adjuvanted Novirhabdovirus Vaccines

PubMed Central

Rouxel, Ronan N.; Mérour, Emilie; Biacchesi, Stéphane; Brémont, Michel

2016-01-01

Novirhabdoviruses like Viral Hemorrhagic Septicemia Virus (VHSV) and Infectious Hematopoietic Necrosis Virus (IHNV) are fish-infecting Rhabdoviruses belonging to the Mononegavirales order. By reverse genetics, we previously showed that a recombinant VHSV expressing the West Nile Virus (WNV) E glycoprotein could serve as a vaccine platform against WNV. In the current study, we aimed to evaluate the potential of the Novirhabdovirus platform as a vaccine against influenza virus. Recombinant Novirhabdoviruses, rVHSV-HA and rIHNV-HA, expressing at the viral surface the hemagglutinin HA ectodomain were generated and used to immunized mice. We showed that mice immunized with either, rVHSV-HA or rIHNV-HA, elicited a strong neutralizing antibody response against influenza virus. A complete protection was conferred to the immunized mice when challenged with a lethal dose of influenza H1N1 A/PR/8/34 virus. Furthermore we showed that although acting as inert antigen in mice, since naturally inactivated over 20°C, mice immunized with rVHSV-HA or rIHNV-HA in the absence of adjuvant were also completely protected from a lethal challenge. Novirhabdoviruses platform are of particular interest as vaccines for mammals since they are cost effective to produce, relatively easy to generate and very effective to protect immunized animals. PMID:27711176

High-speed RNA microextraction technology using magnetic oligo-dT beads and lateral magnetophoresis.

PubMed

Lee, Hwanyong; Jung, Jinhee; Han, Song-I; Han, Ki-Ho

2010-10-21

This paper presents a high-speed RNA microextractor for the direct isolation of RNA from peripheral blood lysate using magnetic oligo-dT beads. The extraction is achieved through lateral magnetophoresis, generated by a ferromagnetic wire array inlaid on a glass substrate. This RNA microextractor separated more than 80% of magnetic beads with a flow rate up to 20 ml h(-1), and the overall extraction procedure was completed within 1 min. The absorbance ratio of RNA to protein (A(260)/A(280)) was >1.7, indicating that the extraction technology yielded nearly pure RNA. The feasibility of this technique was evaluated further for its applicability to reverse transcription polymerase chain reaction (RT-PCR) procedures by performing cDNA synthesis and PCR. The analysis verified that the RNA microextractor is a practical method for easy, rapid, and high-precision RT-PCR using minimal reagent volumes without requiring highly trained personnel. In addition, it can be readily incorporated into genetic analysis procedures for realizing automated on-chip genetic platforms in a micro format.

Stage-Specific Human Induced Pluripotent Stem Cells Map the Progression of Myeloid Transformation to Transplantable Leukemia.

PubMed

Kotini, Andriana G; Chang, Chan-Jung; Chow, Arthur; Yuan, Han; Ho, Tzu-Chieh; Wang, Tiansu; Vora, Shailee; Solovyov, Alexander; Husser, Chrystel; Olszewska, Malgorzata; Teruya-Feldstein, Julie; Perumal, Deepak; Klimek, Virginia M; Spyridonidis, Alexandros; Rampal, Raajit K; Silverman, Lewis; Reddy, E Premkumar; Papaemmanuil, Elli; Parekh, Samir; Greenbaum, Benjamin D; Leslie, Christina S; Kharas, Michael G; Papapetrou, Eirini P

2017-03-02

Myeloid malignancy is increasingly viewed as a disease spectrum, comprising hematopoietic disorders that extend across a phenotypic continuum ranging from clonal hematopoiesis to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In this study, we derived a collection of induced pluripotent stem cell (iPSC) lines capturing a range of disease stages encompassing preleukemia, low-risk MDS, high-risk MDS, and secondary AML. Upon their differentiation, we found hematopoietic phenotypes of graded severity and/or stage specificity that together delineate a phenotypic roadmap of disease progression culminating in serially transplantable leukemia. We also show that disease stage transitions, both reversal and progression, can be modeled in this system using genetic correction or introduction of mutations via CRISPR/Cas9 and that this iPSC-based approach can be used to uncover disease-stage-specific responses to drugs. Our study therefore provides insight into the cellular events demarcating the initiation and progression of myeloid transformation and a new platform for testing genetic and pharmacological interventions. Copyright © 2017 Elsevier Inc. All rights reserved.

Covering Chemical Diversity of Genetically-Modified Tomatoes Using Metabolomics for Objective Substantial Equivalence Assessment

PubMed Central

Hirai, Tadayoshi; Oikawa, Akira; Matsuda, Fumio; Fukushima, Atsushi; Arita, Masanori; Watanabe, Shin; Yano, Megumu; Hiwasa-Tanase, Kyoko; Ezura, Hiroshi; Saito, Kazuki

2011-01-01

As metabolomics can provide a biochemical snapshot of an organism's phenotype it is a promising approach for charting the unintended effects of genetic modification. A critical obstacle for this application is the inherently limited metabolomic coverage of any single analytical platform. We propose using multiple analytical platforms for the direct acquisition of an interpretable data set of estimable chemical diversity. As an example, we report an application of our multi-platform approach that assesses the substantial equivalence of tomatoes over-expressing the taste-modifying protein miraculin. In combination, the chosen platforms detected compounds that represent 86% of the estimated chemical diversity of the metabolites listed in the LycoCyc database. Following a proof-of-safety approach, we show that % had an acceptable range of variation while simultaneously indicating a reproducible transformation-related metabolic signature. We conclude that multi-platform metabolomics is an approach that is both sensitive and robust and that it constitutes a good starting point for characterizing genetically modified organisms. PMID:21359231

Genetic connectivity in scleractinian corals across the Northern Gulf of Mexico: oil/gas platforms, and relationship to the Flower Garden Banks.

PubMed

Sammarco, Paul W; Brazeau, Daniel A; Sinclair, James

2012-01-01

The 3,000 oil/gas structures currently deployed in the northern Gulf of Mexico (GOM) provide hard substratum for marine organisms in a region where such has been rare since the Holocene. The major exception to this are the Flower Garden Banks (FGB). Corals are known to have colonized oil/gas platforms around the FGB, facilitating biogeographic expansion. We ask the question, what are the patterns of genetic affinity in these coral populations. We sampled coral tissue from populations of two species occurring on oil and gas platforms: Madracis decactis (hermatype) and Tubastraea coccinea (invasive ahermatype). We sampled 28 platforms along four transects from 20 km offshore to the continental shelf edge off 1) Matagorda Island, TX; 2) Lake Sabine, TX; 3) Terrebonne Bay, LA; and 4) Mobile, AL. The entire population of M. decactis was sampled between depths of 5 m and 37 m. T. coccinea populations were sub-sampled. Genetic variation was assessed using the PCR-based Amplified Fragment Length Polymorphisms (AFLPs). Data were analyzed via AFLPOP and STRUCTURE. Genetic connectivity among M. decactis platform populations was highest near the FGB and decreased to the east. Connectivity increased again in the eastern sector, indicating isolation between the populations from different sides of the Mississippi River (Transects 3 and 4). A point-drop in genetic affinity (relatedness) at the shelf edge south of Terrebonne Bay, LA indicated a population differing from all others in the northern GOM. Genetic affinities among T. coccinea were highest in the west and decreased to the east. Very low genetic affinities off Mobile, AL indicated a dramatic difference between those populations and those west of the Mississippi River, apparently a formidable barrier to larval dispersal.

Genetic Connectivity in Scleractinian Corals across the Northern Gulf of Mexico: Oil/Gas Platforms, and Relationship to the Flower Garden Banks

PubMed Central

Sammarco, Paul W.; Brazeau, Daniel A.; Sinclair, James

2012-01-01

The 3,000 oil/gas structures currently deployed in the northern Gulf of Mexico (GOM) provide hard substratum for marine organisms in a region where such has been rare since the Holocene. The major exception to this are the Flower Garden Banks (FGB). Corals are known to have colonized oil/gas platforms around the FGB, facilitating biogeographic expansion. We ask the question, what are the patterns of genetic affinity in these coral populations. We sampled coral tissue from populations of two species occurring on oil and gas platforms: Madracis decactis (hermatype) and Tubastraea coccinea (invasive ahermatype). We sampled 28 platforms along four transects from 20 km offshore to the continental shelf edge off 1) Matagorda Island, TX; 2) Lake Sabine, TX; 3) Terrebonne Bay, LA; and 4) Mobile, AL. The entire population of M. decactis was sampled between depths of 5 m and 37 m. T. coccinea populations were sub-sampled. Genetic variation was assessed using the PCR-based Amplified Fragment Length Polymorphisms (AFLPs). Data were analyzed via AFLPOP and STRUCTURE. Genetic connectivity among M. decactis platform populations was highest near the FGB and decreased to the east. Connectivity increased again in the eastern sector, indicating isolation between the populations from different sides of the Mississippi River (Transects 3 and 4). A point-drop in genetic affinity (relatedness) at the shelf edge south of Terrebonne Bay, LA indicated a population differing from all others in the northern GOM. Genetic affinities among T. coccinea were highest in the west and decreased to the east. Very low genetic affinities off Mobile, AL indicated a dramatic difference between those populations and those west of the Mississippi River, apparently a formidable barrier to larval dispersal. PMID:22558066

A Distributed Parallel Genetic Algorithm of Placement Strategy for Virtual Machines Deployment on Cloud Platform

PubMed Central

Dong, Yu-Shuang; Xu, Gao-Chao; Fu, Xiao-Dong

2014-01-01

The cloud platform provides various services to users. More and more cloud centers provide infrastructure as the main way of operating. To improve the utilization rate of the cloud center and to decrease the operating cost, the cloud center provides services according to requirements of users by sharding the resources with virtualization. Considering both QoS for users and cost saving for cloud computing providers, we try to maximize performance and minimize energy cost as well. In this paper, we propose a distributed parallel genetic algorithm (DPGA) of placement strategy for virtual machines deployment on cloud platform. It executes the genetic algorithm parallelly and distributedly on several selected physical hosts in the first stage. Then it continues to execute the genetic algorithm of the second stage with solutions obtained from the first stage as the initial population. The solution calculated by the genetic algorithm of the second stage is the optimal one of the proposed approach. The experimental results show that the proposed placement strategy of VM deployment can ensure QoS for users and it is more effective and more energy efficient than other placement strategies on the cloud platform. PMID:25097872

A distributed parallel genetic algorithm of placement strategy for virtual machines deployment on cloud platform.

PubMed

Dong, Yu-Shuang; Xu, Gao-Chao; Fu, Xiao-Dong

2014-01-01

The cloud platform provides various services to users. More and more cloud centers provide infrastructure as the main way of operating. To improve the utilization rate of the cloud center and to decrease the operating cost, the cloud center provides services according to requirements of users by sharding the resources with virtualization. Considering both QoS for users and cost saving for cloud computing providers, we try to maximize performance and minimize energy cost as well. In this paper, we propose a distributed parallel genetic algorithm (DPGA) of placement strategy for virtual machines deployment on cloud platform. It executes the genetic algorithm parallelly and distributedly on several selected physical hosts in the first stage. Then it continues to execute the genetic algorithm of the second stage with solutions obtained from the first stage as the initial population. The solution calculated by the genetic algorithm of the second stage is the optimal one of the proposed approach. The experimental results show that the proposed placement strategy of VM deployment can ensure QoS for users and it is more effective and more energy efficient than other placement strategies on the cloud platform.

Genetic Platforms of blaCTX-M in Carbapenemase-Producing Strains of K. pneumoniae Isolated in Chile

PubMed Central

Carrasco-Anabalón, Sergio; Vera-Leiva, Alejandra; Quezada-Aguiluz, Mario; Morales-Rivera, María F.; Lima, Celia A.; Fernández, Jorge; Ulloa, Soledad; Domínguez, Mariana; González-Rocha, Gerardo; Bello-Toledo, Helia

2018-01-01

Objective: To elucidate whether the genetic platforms of blaCTX-M contribute to the phenotypes of multi-drug-resistance (MDR) in the first carbapenemase-producing K. pneumoniae strains isolated in Chile. Method: Twenty-two carbapenemase-producing K. pneumoniae strains isolated from different Chilean patients and hospitals were studied. Their genetic relatedness was assessed by PFGE and MLST. The levels of antibiotic resistance were evaluated by determining the minimum inhibitory concentration of various antimicrobials. In addition, several antibiotic resistance genes of clinical relevance in Chile were investigated. The prevalence, allelic variants, and genetic platforms of blaCTX-M were determined by PCR and sequencing. Results: Out of the 22 strains studied, 20 carry KPC, one carries NDM-1, and one carries OXA-370. The PFGE analysis showed three clades with a genetic relatedness >85%, two formed by four strains and one by eight strains. The other strains are not genetically related, and a total of 17 different pulse types were detected. Ten different STs were identified, the main ones being ST258 (five strains) and ST1161 (seven strains). The isolates presented different percentages of resistance, and 82% were resistant to all the β-lactams tested, 91% to ciprofloxacin, 73% to colistin, 59% to gentamicin, 50% to amikacin, and only 9% to tigecycline. All isolates carried blaTEM and blaSHV, whereas 71% carried aac(6′)Ib-cr, and 57% one qnr gene (A, B, C, D, or S). The blaCTX-M gene was found in 10 of the isolates (4 blaCTX-M−15 and 6 blaCTX-M−2). The characterization of the platform, in seven selected strains, revealed that the gene is associated with unusual class 1 integrons and insertion sequences such as ISCR1, ISECp1, and IS26. Conclusion: In the first carbapenemase-producing K. pneumoniae strains isolated in Chile the genetic platform of blaCTX-M−2 corresponds to an unusual class 1 integron that can be responsible for the MDR phenotype, whereas the genetic platforms of blaCTX-M−15 are associated with different IS and do not contribute to multi-drug resistance. PMID:29593660

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

Validation of Biomarker Proteins Using Reverse Capture Protein Microarrays.

PubMed

Jozwik, Catherine; Eidelman, Ofer; Starr, Joshua; Pollard, Harvey B; Srivastava, Meera

2017-01-01

Genomics has revolutionized large-scale and high-throughput sequencing and has led to the discovery of thousands of new proteins. Protein chip technology is emerging as a miniaturized and highly parallel platform that is suited to rapid, simultaneous screening of large numbers of proteins and the analysis of various protein-binding activities, enzyme substrate relationships, and posttranslational modifications. Specifically, reverse capture protein microarrays provide the most appropriate platform for identifying low-abundance, disease-specific biomarker proteins in a sea of high-abundance proteins from biological fluids such as blood, serum, plasma, saliva, urine, and cerebrospinal fluid as well as tissues and cells obtained by biopsy. Samples from hundreds of patients can be spotted in serial dilutions on many replicate glass slides. Each slide can then be probed with one specific antibody to the biomarker of interest. That antibody's titer can then be determined quantitatively for each patient, allowing for the statistical assessment and validation of the diagnostic or prognostic utility of that particular antigen. As the technology matures and the availability of validated, platform-compatible antibodies increases, the platform will move further into the desirable realm of discovery science for detecting and quantitating low-abundance signaling proteins. In this chapter, we describe methods for the successful application of the reverse capture protein microarray platform for which we have made substantial contributions to the development and application of this method, particularly in the use of body fluids other than serum/plasma.

Systematic Comparison of Reverse Phase and Hydrophilic Interaction Liquid Chromatography Platforms for the Analysis of N-linked Glycans

PubMed Central

Walker, S. Hunter; Carlisle, Brandon C.; Muddiman, David C.

2013-01-01

Due to the hydrophilic nature of glycans, reverse phase chromatography has not been widely used as a glycomic separation technique coupled to mass spectrometry. Other approaches such as hydrophilic interaction chromatography and porous graphitized carbon chromatography are often employed, though these strategies frequently suffer from decreased chromatographic resolution, long equilibration times, indefinite retention, and column bleed. Herein, it is shown that through an efficient hydrazone formation derivatization of N-linked glycans (∼4 hr of additional sample preparation time which is carried out in parallel), numerous experimental and practical advantages are gained when analyzing the glycans by online reverse phase chromatography. These benefits include an increased number of glycans detected, increased peak capacity of the separation, and the ability to analyze glycans on the identical liquid chromatography-mass spectrometry platform commonly used for proteomic analyses. The data presented show that separation of derivatized N-linked glycans by reverse phase chromatography significantly out-performs traditional separation of native or derivatized glycans by hydrophilic interaction chromatography. Furthermore, the movement to a more ubiquitous separation technique will afford numerous research groups the opportunity to analyze both proteomic and glycomic samples on the same platform with minimal time and physical change between experiments, increasing the efficiency of ‘multi-omic’ biological approaches. PMID:22954204

Traditional and New Influenza Vaccines

PubMed Central

Wong, Sook-San

2013-01-01

SUMMARY The challenges in successful vaccination against influenza using conventional approaches lie in their variable efficacy in different age populations, the antigenic variability of the circulating virus, and the production and manufacturing limitations to ensure safe, timely, and adequate supply of vaccine. The conventional influenza vaccine platform is based on stimulating immunity against the major neutralizing antibody target, hemagglutinin (HA), by virus attenuation or inactivation. Improvements to this conventional system have focused primarily on improving production and immunogenicity. Cell culture, reverse genetics, and baculovirus expression technology allow for safe and scalable production, while adjuvants, dose variation, and alternate routes of delivery aim to improve vaccine immunogenicity. Fundamentally different approaches that are currently under development hope to signal new generations of influenza vaccines. Such approaches target nonvariable regions of antigenic proteins, with the idea of stimulating cross-protective antibodies and thus creating a “universal” influenza vaccine. While such approaches have obvious benefits, there are many hurdles yet to clear. Here, we discuss the process and challenges of the current influenza vaccine platform as well as new approaches that are being investigated based on the same antigenic target and newer technologies based on different antigenic targets. PMID:23824369

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.

An Overview of Coral Community Development on Offshore Platforms in the Gulf of Mexico

NASA Astrophysics Data System (ADS)

Sammarco, P. W.

2016-02-01

Oil platforms in the N. Gulf of Mexico (GOM) number 1,750, providing hard substratum for settlement of marine fauna, including corals, where little existed in shallow water prior to the 1940s. The introduction of hard substratum has facilitated the biogeographic expansion of Caribbean reef corals. We surveyed scleractinian corals, hermatypic and ahermatypic, on 48 platforms around the Flower Garden Banks (FGB) and across the continental shelf, from Corpus Christi, Texas to Mobile, Alabama, USA. We also assessed coral populations on platforms and the FGB for genetic affinities using AFLPs. The western limit for hermatypes was the shelf edge off Corpus Christi. The highest hermatypic densities occurred at the shelf edge, 200 km from shore in the north-central GOM. Mobile was the eastern limit for some hermatypic corals. Ahermatypes (i.e., Tubastraea coccinea, Oculina diffusa, Phyllangia americana) were absent inshore and in the north-central GOM. Species richness of hermatypic corals peaked near the FGB. Genetic analyses revealed high self-recognition and site fidelity on the platforms and the FGB in Madracis decactis, particularly in the eastern GOM. Platform populations exhibited a strong genetic affinity to those on the FGB, indicating that the FGB are the likely larval source for many corals on the platforms. There was little or no genetic affinity of coral populations across the mouth of the Mississippi River, although in T. coccinea (invasive species), cross-recognition between populations was higher between platforms on a given side. The Mississippi is a strong geographic barrier to east-west dispersal. Brooders were found to be more effective at colonizing patchy habitats at this meso-scale than broadcasters (Diploria strigosa and Montastraea cavernosa). Broadcaster recruits were rarely found, indicating less effective dispersal capabilities. Oil/gas platforms have facilitated the development of coral communities across the northern GOM.

Comparative aerial- and ground-based high-throughput phenotyping for the genetic dissection of NDVI as a proxy for drought-adaptive traits in durum wheat

USDA-ARS?s Scientific Manuscript database

High-throughput phenotyping platforms (HTPPs) provide novel opportunities to more effectively dissect the genetic basis of drought-adaptive traits. This genome-wide association study (GWAS) compares the results obtained with two Unmanned Aerial Vehicles (UAVs) and a ground-based platform used to mea...

MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms.

PubMed

Kumar, Sudhir; Stecher, Glen; Li, Michael; Knyaz, Christina; Tamura, Koichiro

2018-06-01

The Molecular Evolutionary Genetics Analysis (Mega) software implements many analytical methods and tools for phylogenomics and phylomedicine. Here, we report a transformation of Mega to enable cross-platform use on Microsoft Windows and Linux operating systems. Mega X does not require virtualization or emulation software and provides a uniform user experience across platforms. Mega X has additionally been upgraded to use multiple computing cores for many molecular evolutionary analyses. Mega X is available in two interfaces (graphical and command line) and can be downloaded from www.megasoftware.net free of charge.

Genetic tests in major psychiatric disorders-integrating molecular medicine with clinical psychiatry-why is it so difficult?

PubMed

Demkow, U; Wolańczyk, T

2017-06-13

With the advent of post-genomic era, new technologies create extraordinary possibilities for diagnostics and personalized therapy, transforming todays' medicine. Rooted in both medical genetics and clinical psychiatry, the paper is designed as an integrated source of information of the current and potential future application of emerging genomic technologies as diagnostic tools in psychiatry, moving beyond the classical concept of patient approach. Selected approaches are presented, starting from currently used technologies (next-generation sequencing (NGS) and microarrays), followed by newer options (reverse phenotyping). Next, we describe an old concept in a new light (endophenotypes), subsequently coming up with a sophisticated and complex approach (gene networks) ending by a nascent field (computational psychiatry). The challenges and barriers that exist to translate genomic research to real-world patient assessment are further discussed. We emphasize the view that only a paradigm shift can bring a fundamental change in psychiatric practice, allowing to disentangle the intricacies of mental diseases. All the diagnostic methods, as described, are directed at uncovering the integrity of the system including many types of relations within a complex structure. The integrative system approach offers new opportunity to connect genetic background with specific diseases entities, or concurrently, with symptoms regardless of a diagnosis. To advance the field, we propose concerted cross-disciplinary effort to provide a diagnostic platform operating at the general level of genetic pathogenesis of complex-trait psychiatric disorders rather than at the individual level of a specific disease.

Genetic tests in major psychiatric disorders—integrating molecular medicine with clinical psychiatry—why is it so difficult?

PubMed Central

Demkow, U; Wolańczyk, T

2017-01-01

With the advent of post-genomic era, new technologies create extraordinary possibilities for diagnostics and personalized therapy, transforming todays’ medicine. Rooted in both medical genetics and clinical psychiatry, the paper is designed as an integrated source of information of the current and potential future application of emerging genomic technologies as diagnostic tools in psychiatry, moving beyond the classical concept of patient approach. Selected approaches are presented, starting from currently used technologies (next-generation sequencing (NGS) and microarrays), followed by newer options (reverse phenotyping). Next, we describe an old concept in a new light (endophenotypes), subsequently coming up with a sophisticated and complex approach (gene networks) ending by a nascent field (computational psychiatry). The challenges and barriers that exist to translate genomic research to real-world patient assessment are further discussed. We emphasize the view that only a paradigm shift can bring a fundamental change in psychiatric practice, allowing to disentangle the intricacies of mental diseases. All the diagnostic methods, as described, are directed at uncovering the integrity of the system including many types of relations within a complex structure. The integrative system approach offers new opportunity to connect genetic background with specific diseases entities, or concurrently, with symptoms regardless of a diagnosis. To advance the field, we propose concerted cross-disciplinary effort to provide a diagnostic platform operating at the general level of genetic pathogenesis of complex-trait psychiatric disorders rather than at the individual level of a specific disease. PMID:28608853

Combining field performance with controlled environment plant imaging to identify the genetic control of growth and transpiration underlying yield response to water-deficit stress in wheat

PubMed Central

Parent, Boris; Shahinnia, Fahimeh; Maphosa, Lance; Berger, Bettina; Rabie, Huwaida; Chalmers, Ken; Kovalchuk, Alex; Langridge, Peter; Fleury, Delphine

2015-01-01

Crop yield in low-rainfall environments is a complex trait under multigenic control that shows significant genotype×environment (G×E) interaction. One way to understand and track this trait is to link physiological studies to genetics by using imaging platforms to phenotype large segregating populations. A wheat population developed from parental lines contrasting in their mechanisms of yield maintenance under water deficit was studied in both an imaging platform and in the field. We combined phenotyping methods in a common analysis pipeline to estimate biomass and leaf area from images and then inferred growth and relative growth rate, transpiration, and water-use efficiency, and applied these to genetic analysis. From the 20 quantitative trait loci (QTLs) found for several traits in the platform, some showed strong effects, accounting for between 26 and 43% of the variation on chromosomes 1A and 1B, indicating that the G×E interaction could be reduced in a controlled environment and by using dynamic variables. Co-location of QTLs identified in the platform and in the field showed a possible common genetic basis at some loci. Co-located QTLs were found for average growth rate, leaf expansion rate, transpiration rate, and water-use efficiency from the platform with yield, spike number, grain weight, grain number, and harvest index in the field. These results demonstrated that imaging platforms are a suitable alternative to field-based screening and may be used to phenotype recombinant lines for positional cloning. PMID:26179580

Benchmarking of TALE- and CRISPR/dCas9-Based Transcriptional Regulators in Mammalian Cells for the Construction of Synthetic Genetic Circuits.

PubMed

Lebar, Tina; Jerala, Roman

2016-10-21

Transcriptional activator-like effector (TALE)- and CRISPR/Cas9-based designable recognition domains represent a technological breakthrough not only for genome editing but also for building designed genetic circuits. Both platforms are able to target rarely occurring DNA segments, even within complex genomes. TALE and dCas9 domains, genetically fused to transcriptional regulatory domains, can be used for the construction of engineered logic circuits. Here we benchmarked the performance of the two platforms, targeting the same DNA sequences, to compare their advantages for the construction of designed circuits in mammalian cells. Optimal targeting strands for repression and activation of dCas9-based designed transcription factors were identified; both platforms exhibited good orthogonality and were used to construct functionally complete NOR gates. Although the CRISPR/dCas9 system is clearly easier to construct, TALE-based activators were significantly stronger, and the TALE-based platform performed better, especially for the construction of layered circuits.

Inversion of left-right asymmetry alters performance of Xenopus tadpoles in nonlateralized cognitive tasks.

PubMed

Blackiston, Douglas J; Levin, Michael

2013-08-01

Left-right behavioural biases are well documented across the animal kingdom, and handedness has long been associated with cognitive performance. However, the relationship between body laterality and cognitive ability is poorly understood. The embryonic pathways dictating normal left-right patterning have been molecularly dissected in model vertebrates, and numerous genetic and pharmacological treatments now facilitate experimental randomization or reversal of the left-right axis in these animals. Several recent studies showed a link between brain asymmetry and strongly lateralized behaviours such as eye use preference. However, links between laterality of the body and performance on cognitive tasks utilizing nonlateralized cues remain unknown. Xenopus tadpoles are an established model for the study of early left-right patterning, and protocols were recently developed to quantitatively evaluate learning and memory in these animals. Using an automated testing and training platform, we tested wild-type, left-right-randomized and left-right-reversed tadpoles for their ability to learn colour cues in an automated assay. Our results indicate that animals with either randomization or reversal of somatic left-right patterning learned more slowly than wild-type siblings, although all groups were able to reach the same performance optimum given enough training sessions. These results are the first analysis of the link between body laterality and learning of nonlateralized cues, and they position the Xenopus tadpole as an attractive and tractable model for future studies of the links between asymmetry of the body, lateralization of the brain and behaviour.

Modeling Molecular and Cellular Aspects of Human Disease using the Nematode Caenorhabditis elegans

PubMed Central

Silverman, Gary A.; Luke, Cliff J.; Bhatia, Sangeeta R.; Long, Olivia S.; Vetica, Anne C.; Perlmutter, David H.; Pak, Stephen C.

2009-01-01

As an experimental system, Caenorhabditis elegans, offers a unique opportunity to interrogate in vivo the genetic and molecular functions of human disease-related genes. For example, C. elegans has provided crucial insights into fundamental biological processes such as cell death and cell fate determinations, as well as pathological processes such as neurodegeneration and microbial susceptibility. The C. elegans model has several distinct advantages including a completely sequenced genome that shares extensive homology with that of mammals, ease of cultivation and storage, a relatively short lifespan and techniques for generating null and transgenic animals. However, the ability to conduct unbiased forward and reverse genetic screens in C. elegans remains one of the most powerful experimental paradigms for discovering the biochemical pathways underlying human disease phenotypes. The identification of these pathways leads to a better understanding of the molecular interactions that perturb cellular physiology, and forms the foundation for designing mechanism-based therapies. To this end, the ability to process large numbers of isogenic animals through automated work stations suggests that C. elegans, manifesting different aspects of human disease phenotypes, will become the platform of choice for in vivo drug discovery and target validation using high-throughput/content screening technologies. PMID:18852689

Dynamic Gaming Platform (DGP)

DTIC Science & Technology

2009-04-01

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

Visuospatial asymmetries and interocular transfer in the split-brain rat.

PubMed

Adelstein, A; Crowne, D P

1991-06-01

Interocular transfer (IOT), hemispheric superiority, and cerebral dominance were examined in split-brain female albino rats. Callosum-sectioned and intact animals were monocularly trained in the Morris water maze and tested in IOT and reversal phases. In the IOT phase, split-brain rats entered more nontarget quadrants and headed less accurately toward the platform than did controls. For both split-brain animals and controls, right-eye training resulted in shorter latencies and fewer nontarget entries than did left-eye training. Analyses of cerebral dominance showed shorter latencies and smaller heading errors over all 3 phases in rats that were trained with the nondominant eye. Right-eye dominant controls were less affected by platform reversal. Split-brain rats were inferior to controls in latency to find the platform and in target quadrant entries. This finding establishes a spatial cognitive deficit from callosum section.

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

Reverse Genetics Approaches for the Development of Influenza Vaccines

PubMed Central

Nogales, Aitor; Martínez-Sobrido, Luis

2016-01-01

Influenza viruses cause annual seasonal epidemics and occasional pandemics of human respiratory disease. Influenza virus infections represent a serious public health and economic problem, which are most effectively prevented through vaccination. However, influenza viruses undergo continual antigenic variation, which requires either the annual reformulation of seasonal influenza vaccines or the rapid generation of vaccines against potential pandemic virus strains. The segmented nature of influenza virus allows for the reassortment between two or more viruses within a co-infected cell, and this characteristic has also been harnessed in the laboratory to generate reassortant viruses for their use as either inactivated or live-attenuated influenza vaccines. With the implementation of plasmid-based reverse genetics techniques, it is now possible to engineer recombinant influenza viruses entirely from full-length complementary DNA copies of the viral genome by transfection of susceptible cells. These reverse genetics systems have provided investigators with novel and powerful approaches to answer important questions about the biology of influenza viruses, including the function of viral proteins, their interaction with cellular host factors and the mechanisms of influenza virus transmission and pathogenesis. In addition, reverse genetics techniques have allowed the generation of recombinant influenza viruses, providing a powerful technology to develop both inactivated and live-attenuated influenza vaccines. In this review, we will summarize the current knowledge of state-of-the-art, plasmid-based, influenza reverse genetics approaches and their implementation to provide rapid, convenient, safe and more effective influenza inactivated or live-attenuated vaccines. PMID:28025504

Integrated electrochemical microsystems for genetic detection of pathogens at the point of care.

PubMed

Hsieh, Kuangwen; Ferguson, B Scott; Eisenstein, Michael; Plaxco, Kevin W; Soh, H Tom

2015-04-21

The capacity to achieve rapid, sensitive, specific, quantitative, and multiplexed genetic detection of pathogens via a robust, portable, point-of-care platform could transform many diagnostic applications. And while contemporary technologies have yet to effectively achieve this goal, the advent of microfluidics provides a potentially viable approach to this end by enabling the integration of sophisticated multistep biochemical assays (e.g., sample preparation, genetic amplification, and quantitative detection) in a monolithic, portable device from relatively small biological samples. Integrated electrochemical sensors offer a particularly promising solution to genetic detection because they do not require optical instrumentation and are readily compatible with both integrated circuit and microfluidic technologies. Nevertheless, the development of generalizable microfluidic electrochemical platforms that integrate sample preparation and amplification as well as quantitative and multiplexed detection remains a challenging and unsolved technical problem. Recognizing this unmet need, we have developed a series of microfluidic electrochemical DNA sensors that have progressively evolved to encompass each of these critical functionalities. For DNA detection, our platforms employ label-free, single-step, and sequence-specific electrochemical DNA (E-DNA) sensors, in which an electrode-bound, redox-reporter-modified DNA "probe" generates a current change after undergoing a hybridization-induced conformational change. After successfully integrating E-DNA sensors into a microfluidic chip format, we subsequently incorporated on-chip genetic amplification techniques including polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) to enable genetic detection at clinically relevant target concentrations. To maximize the potential point-of-care utility of our platforms, we have further integrated sample preparation via immunomagnetic separation, which allowed the detection of influenza virus directly from throat swabs and developed strategies for the multiplexed detection of related bacterial strains from the blood of septic mice. Finally, we developed an alternative electrochemical detection platform based on real-time LAMP, which not is only capable of detecting across a broad dynamic range of target concentrations, but also greatly simplifies quantitative measurement of nucleic acids. These efforts represent considerable progress toward the development of a true sample-in-answer-out platform for genetic detection of pathogens at the point of care. Given the many advantages of these systems, and the growing interest and innovative contributions from researchers in this field, we are optimistic that iterations of these systems will arrive in clinical settings in the foreseeable future.

Preimplantation genetic testing for aneuploidy: what technology should you use and what are the differences?

PubMed

Brezina, Paul R; Anchan, Raymond; Kearns, William G

2016-07-01

The purpose of the review was to define the various diagnostic platforms currently available to perform preimplantation genetic testing for aneuploidy and describe in a clear and balanced manner the various strengths and weaknesses of these technologies. A systematic literature review was conducted. We used the terms "preimplantation genetic testing," "preimplantation genetic diagnosis," "preimplantation genetic screening," "preimplantation genetic diagnosis for aneuploidy," "PGD," "PGS," and "PGD-A" to search through PubMed, ScienceDirect, and Google Scholar from the year 2000 to April 2016. Bibliographies of articles were also searched for relevant studies. When possible, larger randomized controlled trials were used. However, for some emerging data, only data from meeting abstracts were available. PGS is emerging as one of the most valuable tools to enhance pregnancy success with assisted reproductive technologies. While all of the current diagnostic platforms currently available have various advantages and disadvantages, some platforms, such as next-generation sequencing (NGS), are capable of evaluating far more data points than has been previously possible. The emerging complexity of different technologies, especially with the utilization of more sophisticated tools such as NGS, requires an understanding by clinicians in order to request the best test for their patients.. Ultimately, the choice of which diagnostic platform is utilized should be individualized to the needs of both the clinic and the patient. Such a decision must incorporate the risk tolerance of both the patient and provider, fiscal considerations, and other factors such as the ability to counsel patients on their testing results and how these may or may not impact clinical outcomes.

Genetic Constructor: An Online DNA Design Platform.

PubMed

Bates, Maxwell; Lachoff, Joe; Meech, Duncan; Zulkower, Valentin; Moisy, Anaïs; Luo, Yisha; Tekotte, Hille; Franziska Scheitz, Cornelia Johanna; Khilari, Rupal; Mazzoldi, Florencio; Chandran, Deepak; Groban, Eli

2017-12-15

Genetic Constructor is a cloud Computer Aided Design (CAD) application developed to support synthetic biologists from design intent through DNA fabrication and experiment iteration. The platform allows users to design, manage, and navigate complex DNA constructs and libraries, using a new visual language that focuses on functional parts abstracted from sequence. Features like combinatorial libraries and automated primer design allow the user to separate design from construction by focusing on functional intent, and design constraints aid iterative refinement of designs. A plugin architecture enables contributions from scientists and coders to leverage existing powerful software and connect to DNA foundries. The software is easily accessible and platform agnostic, free for academics, and available in an open-source community edition. Genetic Constructor seeks to democratize DNA design, manufacture, and access to tools and services from the synthetic biology community.

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.

Combining field performance with controlled environment plant imaging to identify the genetic control of growth and transpiration underlying yield response to water-deficit stress in wheat.

PubMed

Parent, Boris; Shahinnia, Fahimeh; Maphosa, Lance; Berger, Bettina; Rabie, Huwaida; Chalmers, Ken; Kovalchuk, Alex; Langridge, Peter; Fleury, Delphine

2015-09-01

Crop yield in low-rainfall environments is a complex trait under multigenic control that shows significant genotype×environment (G×E) interaction. One way to understand and track this trait is to link physiological studies to genetics by using imaging platforms to phenotype large segregating populations. A wheat population developed from parental lines contrasting in their mechanisms of yield maintenance under water deficit was studied in both an imaging platform and in the field. We combined phenotyping methods in a common analysis pipeline to estimate biomass and leaf area from images and then inferred growth and relative growth rate, transpiration, and water-use efficiency, and applied these to genetic analysis. From the 20 quantitative trait loci (QTLs) found for several traits in the platform, some showed strong effects, accounting for between 26 and 43% of the variation on chromosomes 1A and 1B, indicating that the G×E interaction could be reduced in a controlled environment and by using dynamic variables. Co-location of QTLs identified in the platform and in the field showed a possible common genetic basis at some loci. Co-located QTLs were found for average growth rate, leaf expansion rate, transpiration rate, and water-use efficiency from the platform with yield, spike number, grain weight, grain number, and harvest index in the field. These results demonstrated that imaging platforms are a suitable alternative to field-based screening and may be used to phenotype recombinant lines for positional cloning. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

eSensor: an electrochemical detection-based DNA microarray technology enabling sample-to-answer molecular diagnostics

NASA Astrophysics Data System (ADS)

Liu, Robin H.; Longiaru, Mathew

2009-05-01

DNA microarrays are becoming a widespread tool used in life science and drug screening due to its many benefits of miniaturization and integration. Microarrays permit a highly multiplexed DNA analysis. Recently, the development of new detection methods and simplified methodologies has rapidly expanded the use of microarray technologies from predominantly gene expression analysis into the arena of diagnostics. Osmetech's eSensor® is an electrochemical detection platform based on a low-to- medium density DNA hybridization array on a cost-effective printed circuit board substrate. eSensor® has been cleared by FDA for Warfarin sensitivity test and Cystic Fibrosis Carrier Detection. Other genetic-based diagnostic and infectious disease detection tests are under development. The eSensor® platform eliminates the need for an expensive laser-based optical system and fluorescent reagents. It allows one to perform hybridization and detection in a single and small instrument without any fluidic processing and handling. Furthermore, the eSensor® platform is readily adaptable to on-chip sample-to-answer genetic analyses using microfluidics technology. The eSensor® platform provides a cost-effective solution to direct sample-to-answer genetic analysis, and thus have a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.

Molecular Dissection of Xyloglucan Recognition in a Prominent Human Gut Symbiont

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

Tauzin, Alexandra S.; Kwiatkowski, Kurt J.; Orlovsky, Nicole I.

Polysaccharide utilization loci (PUL) within the genomes of resident human gutBacteroidetesare central to the metabolism of the otherwise indigestible complex carbohydrates known as “dietary fiber.” However, functional characterization of PUL lags significantly behind sequencing efforts, which limits physiological understanding of the human-bacterial symbiosis. In particular, the molecular basis of complex polysaccharide recognition, an essential prerequisite to hydrolysis by cell surface glycosidases and subsequent metabolism, is generally poorly understood. Here, we present the biochemical, structural, and reverse genetic characterization of two unique cell surface glycan-binding proteins (SGBPs) encoded by a xyloglucan utilization locus (XyGUL) fromBacteroides ovatus, which are integral to growthmore » on this key dietary vegetable polysaccharide. Biochemical analysis reveals that these outer membrane-anchored proteins are in fact exquisitely specific for the highly branched xyloglucan (XyG) polysaccharide. The crystal structure of SGBP-A, a SusD homolog, with a bound XyG tetradecasaccharide reveals an extended carbohydrate-binding platform that primarily relies on recognition of the β-glucan backbone. The unique, tetra-modular structure of SGBP-B is comprised of tandem Ig-like folds, with XyG binding mediated at the distal C-terminal domain. Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Finally, together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture byB. ovatusand elaborate a model of how vegetable xyloglucans are accessed by theBacteroidetes.« less

Molecular Dissection of Xyloglucan Recognition in a Prominent Human Gut Symbiont

DOE PAGES

Tauzin, Alexandra S.; Kwiatkowski, Kurt J.; Orlovsky, Nicole I.; ...

2016-04-26

Polysaccharide utilization loci (PUL) within the genomes of resident human gutBacteroidetesare central to the metabolism of the otherwise indigestible complex carbohydrates known as “dietary fiber.” However, functional characterization of PUL lags significantly behind sequencing efforts, which limits physiological understanding of the human-bacterial symbiosis. In particular, the molecular basis of complex polysaccharide recognition, an essential prerequisite to hydrolysis by cell surface glycosidases and subsequent metabolism, is generally poorly understood. Here, we present the biochemical, structural, and reverse genetic characterization of two unique cell surface glycan-binding proteins (SGBPs) encoded by a xyloglucan utilization locus (XyGUL) fromBacteroides ovatus, which are integral to growthmore » on this key dietary vegetable polysaccharide. Biochemical analysis reveals that these outer membrane-anchored proteins are in fact exquisitely specific for the highly branched xyloglucan (XyG) polysaccharide. The crystal structure of SGBP-A, a SusD homolog, with a bound XyG tetradecasaccharide reveals an extended carbohydrate-binding platform that primarily relies on recognition of the β-glucan backbone. The unique, tetra-modular structure of SGBP-B is comprised of tandem Ig-like folds, with XyG binding mediated at the distal C-terminal domain. Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Finally, together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture byB. ovatusand elaborate a model of how vegetable xyloglucans are accessed by theBacteroidetes.« less

Molecular Dissection of Xyloglucan Recognition in a Prominent Human Gut Symbiont

PubMed Central

Tauzin, Alexandra S.; Kwiatkowski, Kurt J.; Orlovsky, Nicole I.; Smith, Christopher J.; Creagh, A. Louise; Haynes, Charles A.; Wawrzak, Zdzislaw

2016-01-01

ABSTRACT Polysaccharide utilization loci (PUL) within the genomes of resident human gut Bacteroidetes are central to the metabolism of the otherwise indigestible complex carbohydrates known as “dietary fiber.” However, functional characterization of PUL lags significantly behind sequencing efforts, which limits physiological understanding of the human-bacterial symbiosis. In particular, the molecular basis of complex polysaccharide recognition, an essential prerequisite to hydrolysis by cell surface glycosidases and subsequent metabolism, is generally poorly understood. Here, we present the biochemical, structural, and reverse genetic characterization of two unique cell surface glycan-binding proteins (SGBPs) encoded by a xyloglucan utilization locus (XyGUL) from Bacteroides ovatus, which are integral to growth on this key dietary vegetable polysaccharide. Biochemical analysis reveals that these outer membrane-anchored proteins are in fact exquisitely specific for the highly branched xyloglucan (XyG) polysaccharide. The crystal structure of SGBP-A, a SusD homolog, with a bound XyG tetradecasaccharide reveals an extended carbohydrate-binding platform that primarily relies on recognition of the β-glucan backbone. The unique, tetra-modular structure of SGBP-B is comprised of tandem Ig-like folds, with XyG binding mediated at the distal C-terminal domain. Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes. PMID:27118585

An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells.

PubMed

Guo, Jianying; Ma, Dacheng; Huang, Rujin; Ming, Jia; Ye, Min; Kee, Kehkooi; Xie, Zhen; Na, Jie

2017-05-01

Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naïve pluripotent gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass (ICM) of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.

Genetic diversity analysis of sugarcane germplasm based on fluorescence-labeled simple sequence repeat markers and a capillary electrophoresis-based genotyping platform

USDA-ARS?s Scientific Manuscript database

Genetic diversity analysis, which refers to the elaboration of total extent of genetic characteristics in the genetic makeup of a certain species, constitutes a classical strategy for the study of diversity, population genetic structure, and breeding practices. In this study, fluorescence-labeled se...

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.

A halochromic stimuli-responsive reversible fluorescence switching 3, 4, 9, 10-perylene tetracarboxylic acid dye for fabricating rewritable platform

NASA Astrophysics Data System (ADS)

Hariharan, P. S.; Pitchaimani, J.; Madhu, Vedichi; Anthony, Savarimuthu Philip

2017-02-01

3, 4, 9, 10-perylene tetracarboxylic acid (PTCA), a strongly fluorescent water soluble dye with halochromic functionality showed pH dependent reversible fluorescence switching. The strong fluorescence of PTCA (Φf = 0.67) in basic medium was completely quenched upon acidification. The fluorescent PTCA has been transferred on to a solid substrate (filter paper and glass plate) that also showed reversible off-on fluorescence switching by acid/base and drying/water vapor exposure. The reversible fluorescence switching of PTCA could be of potential interest for fabricating rewritable fluorescent medium.

Seven R's for the Student Teacher.

ERIC Educational Resources Information Center

Newson, Graham

1983-01-01

Briefly discussed are seven qualities which provide a significant basic platform for teaching mathematics effectively: reinforcement, responsiveness, reliability, realism, reversibility, range, and radicalism. (MNS)

Development of a phenotyping platform for high throughput screening of nodal root angle in sorghum.

PubMed

Joshi, Dinesh C; Singh, Vijaya; Hunt, Colleen; Mace, Emma; van Oosterom, Erik; Sulman, Richard; Jordan, David; Hammer, Graeme

2017-01-01

In sorghum, the growth angle of nodal roots is a major component of root system architecture. It strongly influences the spatial distribution of roots of mature plants in the soil profile, which can impact drought adaptation. However, selection for nodal root angle in sorghum breeding programs has been restricted by the absence of a suitable high throughput phenotyping platform. The aim of this study was to develop a phenotyping platform for the rapid, non-destructive and digital measurement of nodal root angle of sorghum at the seedling stage. The phenotyping platform comprises of 500 soil filled root chambers (50 × 45 × 0.3 cm in size), made of transparent perspex sheets that were placed in metal tubs and covered with polycarbonate sheets. Around 3 weeks after sowing, once the first flush of nodal roots was visible, roots were imaged in situ using an imaging box that included two digital cameras that were remotely controlled by two android tablets. Free software ( openGelPhoto.tcl ) allowed precise measurement of nodal root angle from the digital images. The reliability and efficiency of the platform was evaluated by screening a large nested association mapping population of sorghum and a set of hybrids in six independent experimental runs that included up to 500 plants each. The platform revealed extensive genetic variation and high heritability (repeatability) for nodal root angle. High genetic correlations and consistent ranking of genotypes across experimental runs confirmed the reproducibility of the platform. This low cost, high throughput root phenotyping platform requires no sophisticated equipment, is adaptable to most glasshouse environments and is well suited to dissect the genetic control of nodal root angle of sorghum. The platform is suitable for use in sorghum breeding programs aiming to improve drought adaptation through root system architecture manipulation.

Applying a Genetic Algorithm to Reconfigurable Hardware

NASA Technical Reports Server (NTRS)

Wells, B. Earl; Weir, John; Trevino, Luis; Patrick, Clint; Steincamp, Jim

2004-01-01

This paper investigates the feasibility of applying genetic algorithms to solve optimization problems that are implemented entirely in reconfgurable hardware. The paper highlights the pe$ormance/design space trade-offs that must be understood to effectively implement a standard genetic algorithm within a modem Field Programmable Gate Array, FPGA, reconfgurable hardware environment and presents a case-study where this stochastic search technique is applied to standard test-case problems taken from the technical literature. In this research, the targeted FPGA-based platform and high-level design environment was the Starbridge Hypercomputing platform, which incorporates multiple Xilinx Virtex II FPGAs, and the Viva TM graphical hardware description language.

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

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

Algorithms and Architectures for Elastic-Wave Inversion Final Report CRADA No. TC02144.0

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

Larsen, S.; Lindtjorn, O.

2017-08-15

This was a collaborative effort between Lawrence Livermore National Security, LLC as manager and operator of Lawrence Livermore National Laboratory (LLNL) and Schlumberger Technology Corporation (STC), to perform a computational feasibility study that investigates hardware platforms and software algorithms applicable to STC for Reverse Time Migration (RTM) / Reverse Time Inversion (RTI) of 3-D seismic data.

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.

A single-layer platform for Boolean logic and arithmetic through DNA excision in mammalian cells

PubMed Central

Weinberg, Benjamin H.; Hang Pham, N. T.; Caraballo, Leidy D.; Lozanoski, Thomas; Engel, Adrien; Bhatia, Swapnil; Wong, Wilson W.

2017-01-01

Genetic circuits engineered for mammalian cells often require extensive fine-tuning to perform their intended functions. To overcome this problem, we present a generalizable biocomputing platform that can engineer genetic circuits which function in human cells with minimal optimization. We used our Boolean Logic and Arithmetic through DNA Excision (BLADE) platform to build more than 100 multi-input-multi-output circuits. We devised a quantitative metric to evaluate the performance of the circuits in human embryonic kidney and Jurkat T cells. Of 113 circuits analysed, 109 functioned (96.5%) with the correct specified behavior without any optimization. We used our platform to build a three-input, two-output Full Adder and six-input, one-output Boolean Logic Look Up Table. We also used BLADE to design circuits with temporal small molecule-mediated inducible control and circuits that incorporate CRISPR/Cas9 to regulate endogenous mammalian genes. PMID:28346402

Structural insights into the adaptation of proliferating cell nuclear antigen (PCNA) from Haloferax volcanii to a high-salt environment

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

Morgunova, Ekaterina, E-mail: ekaterina.morgunova@ki.se; Gray, Fiona C.; MacNeill, Stuart A.

2009-10-01

The crystal structure of PCNA from the halophilic archaeon H. volcanii reveals specific features of the charge distribution on the protein surface that reflect adaptation to a high-salt environment and suggests a different type of interaction with DNA in halophilic PCNAs. The sliding clamp proliferating cell nuclear antigen (PCNA) plays vital roles in many aspects of DNA replication and repair in eukaryotic cells and in archaea. Realising the full potential of archaea as a model for PCNA function requires a combination of biochemical and genetic approaches. In order to provide a platform for subsequent reverse genetic analysis, PCNA from themore » halophilic archaeon Haloferax volcanii was subjected to crystallographic analysis. The gene was cloned and expressed in Escherichia coli and the protein was purified by affinity chromatography and crystallized by the vapour-diffusion technique. The structure was determined by molecular replacement and refined at 3.5 Å resolution to a final R factor of 23.7% (R{sub free} = 25%). PCNA from H. volcanii was found to be homotrimeric and to resemble other homotrimeric PCNA clamps but with several differences that appear to be associated with adaptation of the protein to the high intracellular salt concentrations found in H. volcanii cells.« less

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.

Reverse-engineering the genetic circuitry of a cancer cell with predicted intervention in chronic lymphocytic leukemia.

PubMed

Vallat, Laurent; Kemper, Corey A; Jung, Nicolas; Maumy-Bertrand, Myriam; Bertrand, Frédéric; Meyer, Nicolas; Pocheville, Arnaud; Fisher, John W; Gribben, John G; Bahram, Seiamak

2013-01-08

Cellular behavior is sustained by genetic programs that are progressively disrupted in pathological conditions--notably, cancer. High-throughput gene expression profiling has been used to infer statistical models describing these cellular programs, and development is now needed to guide orientated modulation of these systems. Here we develop a regression-based model to reverse-engineer a temporal genetic program, based on relevant patterns of gene expression after cell stimulation. This method integrates the temporal dimension of biological rewiring of genetic programs and enables the prediction of the effect of targeted gene disruption at the system level. We tested the performance accuracy of this model on synthetic data before reverse-engineering the response of primary cancer cells to a proliferative (protumorigenic) stimulation in a multistate leukemia biological model (i.e., chronic lymphocytic leukemia). To validate the ability of our method to predict the effects of gene modulation on the global program, we performed an intervention experiment on a targeted gene. Comparison of the predicted and observed gene expression changes demonstrates the possibility of predicting the effects of a perturbation in a gene regulatory network, a first step toward an orientated intervention in a cancer cell genetic program.

Osmosis process for leachate treatment in industrial platform: Economic and performances evaluations to zero liquid discharge.

PubMed

Cingolani, Diego; Eusebi, Anna Laura; Battistoni, Paolo

2017-12-01

The industrial processes require large quantities of water. The presence of discharges results not only in significant environmental impact but implies wastage of water resources. This problem could be solved treating and reusing the produced wastewaters and applying the new zero liquid discharge approach. This paper discusses the design and the performances of reverse osmosis membranes for the upgrading of full scale platform for industrial liquid wastes. The final effluent from the ultrafiltration unit of the full scale plant was monitored to design the reverse osmosis unit. Previous modelling phase was used to evaluate the specific ordinary and maintenance costs and the final effluent quality (2.7 €/m 3 ). The system was designed in triple stages at different operative pressures. The economic feasibility and the payback period of the technology at different percentages of produced permeate were determined. The recovery of 90% was identified as profitable for the reverse osmosis application. One experimental pilot plant applying the reverse osmosis was used to test the final effluent. Moreover, the same flow was treated with second pilot system based on the forward osmosis process. The final efficiencies were compared. Removals higher than 95% using the reverse system were obtained for the main macropollutants and ions. No sustainable applicability of the forward osmosis was determined. Copyright © 2016 Elsevier Ltd. All rights reserved.

SMART – Sunflower Mutant population And Reverse genetic Tool for crop improvement

PubMed Central

2013-01-01

Background Sunflower (Helianthus annuus L.) is an important oilseed crop grown widely in various areas of the world. Classical genetic studies have been extensively undertaken for the improvement of this particular oilseed crop. Pertaining to this endeavor, we developed a “chemically induced mutated genetic resource for detecting SNP by TILLING” in sunflower to create new traits. Results To optimize the EMS mutagenesis, we first conducted a “kill curve” analysis with a range of EMS dose from 0.5% to 3%. Based on the observed germination rate, a 50% survival rate i.e. LD50, treatment with 0.6% EMS for 8 hours was chosen to generate 5,000 M2 populations, out of which, 4,763 M3 plants with fertile seed set. Phenotypic characterization of the 5,000 M2 mutagenised lines were undertaken to assess the mutagenesis quality and to identify traits of interest. In the M2 population, about 1.1% of the plants showed phenotypic variations. The sunflower TILLING platform was setup using Endo-1-nuclease as mismatch detection system coupled with an eight fold DNA pooling strategy. As proof-of-concept, we screened the M2 population for induced mutations in two genes related to fatty acid biosynthesis, FatA an acyl-ACP thioesterase and SAD the stearoyl-ACP desaturase and identified a total of 26 mutations. Conclusion Based on the TILLING of FatA and SAD genes, we calculated the overall mutation rate to one mutation every 480 kb, similar to other report for this crop so far. As sunflower is a plant model for seed oil biosynthesis, we anticipate that the developed genetic resource will be a useful tool to identify novel traits for sunflower crop improvement. PMID:23496999

Application of Genomic Technologies to the Breeding of Trees

PubMed Central

Badenes, Maria L.; Fernández i Martí, Angel; Ríos, Gabino; Rubio-Cabetas, María J.

2016-01-01

The recent introduction of next generation sequencing (NGS) technologies represents a major revolution in providing new tools for identifying the genes and/or genomic intervals controlling important traits for selection in breeding programs. In perennial fruit trees with long generation times and large sizes of adult plants, the impact of these techniques is even more important. High-throughput DNA sequencing technologies have provided complete annotated sequences in many important tree species. Most of the high-throughput genotyping platforms described are being used for studies of genetic diversity and population structure. Dissection of complex traits became possible through the availability of genome sequences along with phenotypic variation data, which allow to elucidate the causative genetic differences that give rise to observed phenotypic variation. Association mapping facilitates the association between genetic markers and phenotype in unstructured and complex populations, identifying molecular markers for assisted selection and breeding. Also, genomic data provide in silico identification and characterization of genes and gene families related to important traits, enabling new tools for molecular marker assisted selection in tree breeding. Deep sequencing of transcriptomes is also a powerful tool for the analysis of precise expression levels of each gene in a sample. It consists in quantifying short cDNA reads, obtained by NGS technologies, in order to compare the entire transcriptomes between genotypes and environmental conditions. The miRNAs are non-coding short RNAs involved in the regulation of different physiological processes, which can be identified by high-throughput sequencing of RNA libraries obtained by reverse transcription of purified short RNAs, and by in silico comparison with known miRNAs from other species. All together, NGS techniques and their applications have increased the resources for plant breeding in tree species, closing the former gap of genetic tools between trees and annual species. PMID:27895664

Application of Genomic Technologies to the Breeding of Trees.

PubMed

Badenes, Maria L; Fernández I Martí, Angel; Ríos, Gabino; Rubio-Cabetas, María J

2016-01-01

The recent introduction of next generation sequencing (NGS) technologies represents a major revolution in providing new tools for identifying the genes and/or genomic intervals controlling important traits for selection in breeding programs. In perennial fruit trees with long generation times and large sizes of adult plants, the impact of these techniques is even more important. High-throughput DNA sequencing technologies have provided complete annotated sequences in many important tree species. Most of the high-throughput genotyping platforms described are being used for studies of genetic diversity and population structure. Dissection of complex traits became possible through the availability of genome sequences along with phenotypic variation data, which allow to elucidate the causative genetic differences that give rise to observed phenotypic variation. Association mapping facilitates the association between genetic markers and phenotype in unstructured and complex populations, identifying molecular markers for assisted selection and breeding. Also, genomic data provide in silico identification and characterization of genes and gene families related to important traits, enabling new tools for molecular marker assisted selection in tree breeding. Deep sequencing of transcriptomes is also a powerful tool for the analysis of precise expression levels of each gene in a sample. It consists in quantifying short cDNA reads, obtained by NGS technologies, in order to compare the entire transcriptomes between genotypes and environmental conditions. The miRNAs are non-coding short RNAs involved in the regulation of different physiological processes, which can be identified by high-throughput sequencing of RNA libraries obtained by reverse transcription of purified short RNAs, and by in silico comparison with known miRNAs from other species. All together, NGS techniques and their applications have increased the resources for plant breeding in tree species, closing the former gap of genetic tools between trees and annual species.

Novel predictive models for metabolic syndrome risk: a "big data" analytic approach.

PubMed

Steinberg, Gregory B; Church, Bruce W; McCall, Carol J; Scott, Adam B; Kalis, Brian P

2014-06-01

We applied a proprietary "big data" analytic platform--Reverse Engineering and Forward Simulation (REFS)--to dimensions of metabolic syndrome extracted from a large data set compiled from Aetna's databases for 1 large national customer. Our goals were to accurately predict subsequent risk of metabolic syndrome and its various factors on both a population and individual level. The study data set included demographic, medical claim, pharmacy claim, laboratory test, and biometric screening results for 36,944 individuals. The platform reverse-engineered functional models of systems from diverse and large data sources and provided a simulation framework for insight generation. The platform interrogated data sets from the results of 2 Comprehensive Metabolic Syndrome Screenings (CMSSs) as well as complete coverage records; complete data from medical claims, pharmacy claims, and lab results for 2010 and 2011; and responses to health risk assessment questions. The platform predicted subsequent risk of metabolic syndrome, both overall and by risk factor, on population and individual levels, with ROC/AUC varying from 0.80 to 0.88. We demonstrated that improving waist circumference and blood glucose yielded the largest benefits on subsequent risk and medical costs. We also showed that adherence to prescribed medications and, particularly, adherence to routine scheduled outpatient doctor visits, reduced subsequent risk. The platform generated individualized insights using available heterogeneous data within 3 months. The accuracy and short speed to insight with this type of analytic platform allowed Aetna to develop targeted cost-effective care management programs for individuals with or at risk for metabolic syndrome.

Oncotargeting by Vesicular Stomatitis Virus (VSV): Advances in Cancer Therapy.

PubMed

Bishnoi, Suman; Tiwari, Ritudhwaj; Gupta, Sharad; Byrareddy, Siddappa N; Nayak, Debasis

2018-02-23

Modern oncotherapy approaches are based on inducing controlled apoptosis in tumor cells. Although a number of apoptosis-induction approaches are available, site-specific delivery of therapeutic agents still remain the biggest hurdle in achieving the desired cancer treatment benefit. Additionally, systemic treatment-induced toxicity remains a major limiting factor in chemotherapy. To specifically address drug-accessibility and chemotherapy side effects, oncolytic virotherapy (OV) has emerged as a novel cancer treatment alternative. In OV, recombinant viruses with higher replication capacity and stronger lytic properties are being considered for tumor cell-targeting and subsequent cell lysing. Successful application of OVs lies in achieving strict tumor-specific tropism called oncotropism, which is contingent upon the biophysical interactions of tumor cell surface receptors with viral receptors and subsequent replication of oncolytic viruses in cancer cells. In this direction, few viral vector platforms have been developed and some of these have entered pre-clinical/clinical trials. Among these, the Vesicular stomatitis virus (VSV)-based platform shows high promise, as it is not pathogenic to humans. Further, modern molecular biology techniques such as reverse genetics tools have favorably advanced this field by creating efficient recombinant VSVs for OV; some have entered into clinical trials. In this review, we discuss the current status of VSV based oncotherapy, challenges, and future perspectives regarding its therapeutic applications in the cancer treatment.

Oncotargeting by Vesicular Stomatitis Virus (VSV): Advances in Cancer Therapy

PubMed Central

Bishnoi, Suman; Tiwari, Ritudhwaj; Gupta, Sharad; Byrareddy, Siddappa N.; Nayak, Debasis

2018-01-01

Modern oncotherapy approaches are based on inducing controlled apoptosis in tumor cells. Although a number of apoptosis-induction approaches are available, site-specific delivery of therapeutic agents still remain the biggest hurdle in achieving the desired cancer treatment benefit. Additionally, systemic treatment-induced toxicity remains a major limiting factor in chemotherapy. To specifically address drug-accessibility and chemotherapy side effects, oncolytic virotherapy (OV) has emerged as a novel cancer treatment alternative. In OV, recombinant viruses with higher replication capacity and stronger lytic properties are being considered for tumor cell-targeting and subsequent cell lysing. Successful application of OVs lies in achieving strict tumor-specific tropism called oncotropism, which is contingent upon the biophysical interactions of tumor cell surface receptors with viral receptors and subsequent replication of oncolytic viruses in cancer cells. In this direction, few viral vector platforms have been developed and some of these have entered pre-clinical/clinical trials. Among these, the Vesicular stomatitis virus (VSV)-based platform shows high promise, as it is not pathogenic to humans. Further, modern molecular biology techniques such as reverse genetics tools have favorably advanced this field by creating efficient recombinant VSVs for OV; some have entered into clinical trials. In this review, we discuss the current status of VSV based oncotherapy, challenges, and future perspectives regarding its therapeutic applications in the cancer treatment. PMID:29473868

A truly Lego®-like modular microfluidics platform

NASA Astrophysics Data System (ADS)

Vittayarukskul, Kevin; Lee, Abraham Phillip

2017-03-01

Ideally, a modular microfluidics platform should be simple to assemble and support 3D configurations for increased versatility. The modular building blocks should also be mass producible like electrical components. These are fundamental features of world-renowned Legos® and why Legos® inspire many existing modular microfluidics platforms. In this paper, a truly Lego®-like microfluidics platform is introduced, and its basic feasibility is demonstrated. Here, PDMS building blocks resembling 2  ×  2 Lego® bricks are cast from 3D-printed master molds. The blocks are pegged and stacked on a traditional Lego® plate to create simple, 3D microfluidic networks, such as a single basket weave. Characteristics of the platform, including reversible sealing and automatic alignment of channels, are also analyzed and discussed in detail.

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.

An innovative SNP genotyping method adapting to multiple platforms and throughputs.

PubMed

Long, Y M; Chao, W S; Ma, G J; Xu, S S; Qi, L L

2017-03-01

An innovative genotyping method designated as semi-thermal asymmetric reverse PCR (STARP) was developed for genotyping individual SNPs with improved accuracy, flexible throughputs, low operational costs, and high platform compatibility. Multiplex chip-based technology for genome-scale genotyping of single nucleotide polymorphisms (SNPs) has made great progress in the past two decades. However, PCR-based genotyping of individual SNPs still remains problematic in accuracy, throughput, simplicity, and/or operational costs as well as the compatibility with multiple platforms. Here, we report a novel SNP genotyping method designated semi-thermal asymmetric reverse PCR (STARP). In this method, genotyping assay was performed under unique PCR conditions using two universal priming element-adjustable primers (PEA-primers) and one group of three locus-specific primers: two asymmetrically modified allele-specific primers (AMAS-primers) and their common reverse primer. The two AMAS-primers each were substituted one base in different positions at their 3' regions to significantly increase the amplification specificity of the two alleles and tailed at 5' ends to provide priming sites for PEA-primers. The two PEA-primers were developed for common use in all genotyping assays to stringently target the PCR fragments generated by the two AMAS-primers with similar PCR efficiencies and for flexible detection using either gel-free fluorescence signals or gel-based size separation. The state-of-the-art primer design and unique PCR conditions endowed STARP with all the major advantages of high accuracy, flexible throughputs, simple assay design, low operational costs, and platform compatibility. In addition to SNPs, STARP can also be employed in genotyping of indels (insertion-deletion polymorphisms). As vast variations in DNA sequences are being unearthed by many genome sequencing projects and genotyping by sequencing, STARP will have wide applications across all biological organisms in agriculture, medicine, and forensics.

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.

In situ monitoring of intracellular controlled drug release from mesoporous silica nanoparticles coated with pH-responsive charge-reversal polymer.

PubMed

Zhang, Peng; Wu, Tong; Kong, Ji-Lie

2014-10-22

Therapeutic platforms such as chemotherapy that respond to physical and biological stimuli are highly desirable for effective cancer therapy. In this study, pH-responsive charge-reversal, polymer-coated mesoporous silica nanoparticles [PAH-cit/APTES-MSNs; PAH-cit refers to poly(allylamine)-citraconic anhydride; APTES refers to (3-aminopropyl)triethoxysilane] were synthesized for application as drug-delivery systems for the treatment of malignant cells. Confocal laser scanning microscopy (CLSM) revealed that the PAH-cit/APTES-MSNs nanocomposite effectively delivered and released doxorubicin hydrochloride to the nucleus of HeLa (human cervical carcinoma) cells. Additionally, the real-time dynamic drug-release process was monitored by CLSM. The current pH-controlled-smart-release platform holds promise in drug-delivery and cancer therapy-related applications.

Current status of flavivirus vaccines.

PubMed

Barrett, A D

2001-12-01

Although there are approximately 68 flaviviruses recognized, vaccines have been developed to control very few human flavivirus diseases. Licensed live attenuated vaccines have been developed for yellow fever (strain 17D) and Japanese encephalitis (strain SA14-14-2) viruses, and inactivated vaccines have been developed for Japanese encephalitis and tick-borne encephalitis viruses. The yellow fever live attenuated 17D vaccine is one of the most efficacious and safe vaccines developed to date and has been used to immunize more than 300 million people. A number of experimental vaccines are being developed, most notably for dengue. Candidate tetravalent live attenuated dengue vaccines are undergoing clinical trials. Other vaccines are being developed using reverse genetics, DNA vaccines, and recombinant immunogens. In addition, the yellow fever 17D vaccine has been used as a backbone to generate chimeric viruses containing the premembrane and envelope protein genes from other flaviviruses. The "Chimerivax" platform has been used to construct chimeric Japanese encephalitis and dengue viruses that are in different phases of development. Similar strategies are being used by other laboratories.

SARS-like WIV1-CoV poised for human emergence

PubMed Central

Menachery, Vineet D.; Yount, Boyd L.; Sims, Amy C.; Debbink, Kari; Agnihothram, Sudhakar S.; Gralinski, Lisa E.; Graham, Rachel L.; Scobey, Trevor; Plante, Jessica A.; Royal, Scott R.; Swanstrom, Jesica; Sheahan, Timothy P.; Pickles, Raymond J.; Corti, Davide; Randell, Scott H.; Lanzavecchia, Antonio; Marasco, Wayne A.; Baric, Ralph S.

2016-01-01

Outbreaks from zoonotic sources represent a threat to both human disease as well as the global economy. Despite a wealth of metagenomics studies, methods to leverage these datasets to identify future threats are underdeveloped. In this study, we describe an approach that combines existing metagenomics data with reverse genetics to engineer reagents to evaluate emergence and pathogenic potential of circulating zoonotic viruses. Focusing on the severe acute respiratory syndrome (SARS)-like viruses, the results indicate that the WIV1-coronavirus (CoV) cluster has the ability to directly infect and may undergo limited transmission in human populations. However, in vivo attenuation suggests additional adaptation is required for epidemic disease. Importantly, available SARS monoclonal antibodies offered success in limiting viral infection absent from available vaccine approaches. Together, the data highlight the utility of a platform to identify and prioritize prepandemic strains harbored in animal reservoirs and document the threat posed by WIV1-CoV for emergence in human populations. PMID:26976607

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.

Functional integrity of flexible n-channel metal-oxide-semiconductor field-effect transistors on a reversibly bistable platform

NASA Astrophysics Data System (ADS)

Alfaraj, Nasir; Hussain, Aftab M.; Torres Sevilla, Galo A.; Ghoneim, Mohamed T.; Rojas, Jhonathan P.; Aljedaani, Abdulrahman B.; Hussain, Muhammad M.

2015-10-01

Flexibility can bring a new dimension to state-of-the-art electronics, such as rollable displays and integrated circuit systems being transformed into more powerful resources. Flexible electronics are typically hosted on polymeric substrates. Such substrates can be bent and rolled up, but cannot be independently fixed at the rigid perpendicular position necessary to realize rollable display-integrated gadgets and electronics. A reversibly bistable material can assume two stable states in a reversible way: flexibly rolled state and independently unbent state. Such materials are used in cycling and biking safety wristbands and a variety of ankle bracelets for orthopedic healthcare. They are often wrapped around an object with high impulsive force loading. Here, we study the effects of cumulative impulsive force loading on thinned (25 μm) flexible silicon-based n-channel metal-oxide-semiconductor field-effect transistor devices housed on a reversibly bistable flexible platform. We found that the transistors have maintained their high performance level up to an accumulated 180 kN of impact force loading. The gate dielectric layers have maintained their reliability, which is evidenced by the low leakage current densities. Also, we observed low variation in the effective electron mobility values, which manifests that the device channels have maintained their carrier transport properties.

A reverse engineering algorithm for neural networks, applied to the subthalamopallidal network of basal ganglia.

PubMed

Floares, Alexandru George

2008-01-01

Modeling neural networks with ordinary differential equations systems is a sensible approach, but also very difficult. This paper describes a new algorithm based on linear genetic programming which can be used to reverse engineer neural networks. The RODES algorithm automatically discovers the structure of the network, including neural connections, their signs and strengths, estimates its parameters, and can even be used to identify the biophysical mechanisms involved. The algorithm is tested on simulated time series data, generated using a realistic model of the subthalamopallidal network of basal ganglia. The resulting ODE system is highly accurate, and results are obtained in a matter of minutes. This is because the problem of reverse engineering a system of coupled differential equations is reduced to one of reverse engineering individual algebraic equations. The algorithm allows the incorporation of common domain knowledge to restrict the solution space. To our knowledge, this is the first time a realistic reverse engineering algorithm based on linear genetic programming has been applied to neural networks.

YouGenMap: a web platform for dynamic multi-comparative mapping and visualization of genetic maps

Treesearch

Keith Batesole; Kokulapalan Wimalanathan; Lin Liu; Fan Zhang; Craig S. Echt; Chun Liang

2014-01-01

Comparative genetic maps are used in examination of genome organization, detection of conserved gene order, and exploration of marker order variations. YouGenMap is an open-source web tool that offers dynamic comparative mapping capability of users' own genetic mapping between 2 or more map sets. Users' genetic map data and optional gene annotations are...

Web Platform vs In-Person Genetic Counselor for Return of Carrier Results From Exome Sequencing: A Randomized Clinical Trial.

PubMed

Biesecker, Barbara B; Lewis, Katie L; Umstead, Kendall L; Johnston, Jennifer J; Turbitt, Erin; Fishler, Kristen P; Patton, John H; Miller, Ilana M; Heidlebaugh, Alexis R; Biesecker, Leslie G

2018-03-01

A critical bottleneck in clinical genomics is the mismatch between large volumes of results and the availability of knowledgeable professionals to return them. To test whether a web-based platform is noninferior to a genetic counselor for educating patients about their carrier results from exome sequencing. A randomized noninferiority trial conducted in a longitudinal sequencing cohort at the National Institutes of Health from February 5, 2014, to December 16, 2016, was used to compare the web-based platform with a genetic counselor. Among the 571 eligible participants, 1 to 7 heterozygous variants were identified in genes that cause a phenotype that is recessively inherited. Surveys were administered after cohort enrollment, immediately following trial education, and 1 month and 6 months later to primarily healthy postreproductive participants who expressed interest in learning their carrier results. Both intention-to-treat and per-protocol analyses were applied. A web-based platform that integrated education on carrier results with personal test results was designed to directly parallel disclosure education by a genetic counselor. The sessions took a mean (SD) time of 21 (10.6), and 27 (9.3) minutes, respectively. The primary outcomes and noninferiority margins (δNI) were knowledge (0 to 8, δNI = -1), test-specific distress (0 to 30, δNI = +1), and decisional conflict (15 to 75, δNI = +6). After 462 participants (80.9%) provided consent and were randomized, all but 3 participants (n = 459) completed surveys following education and counseling; 398 (86.1%) completed 1-month surveys and 392 (84.8%) completed 6-month surveys. Participants were predominantly well-educated, non-Hispanic white, married parents; mean (SD) age was 63 (63.1) years and 246 (53.6%) were men. The web platform was noninferior to the genetic counselor on outcomes assessed at 1 and 6 months: knowledge (mean group difference, -0.18; lower limit of 97.5% CI, -0.63; δNI = -1), test-specific distress (median group difference, 0; upper limit of 97.5% CI, 0; δNI = +1), and decisional conflict about choosing to learn results (mean group difference, 1.18; upper limit of 97.5% CI, 2.66; δNI = +6). There were no significant differences between the genetic counselors and web-based platform detected between modes of education delivery in disclosure rates to spouses (151 vs 159; relative risk [RR], 1.04; 95% CI, 0.64-1.69; P > .99), children (103 vs 117; RR, 1.07; 95% CI, 0.85-1.36; P = .59), or siblings (91 vs 78; RR, 1.17; 95% CI, 0.94-1.46; P = .18). This trial demonstrates noninferiority of web-based return of carrier results among postreproductive, mostly healthy adults. Replication studies among younger and more diverse populations are needed to establish generalizability. Yet return of results via a web-based platform may be sufficient for subsets of test results, reserving genetic counselors for return of results with a greater health threat. clinicaltrials.gov Identifier: NCT00410241.

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...

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.…

Rescue of ATP7B function in hepatocyte-like cells from Wilson's disease induced pluripotent stem cells using gene therapy or the chaperone drug curcumin.

PubMed

Zhang, Shiqiang; Chen, Shen; Li, Wen; Guo, Xiangpeng; Zhao, Ping; Xu, Jianyong; Chen, Yan; Pan, Qiong; Liu, Xiaorong; Zychlinski, Daniela; Lu, Hai; Tortorella, Micky D; Schambach, Axel; Wang, Yan; Pei, Duanqing; Esteban, Miguel A

2011-08-15

Directed hepatocyte differentiation from human induced pluripotent stem cells (iPSCs) potentially provides a unique platform for modeling liver genetic diseases and performing drug-toxicity screening in vitro. Wilson's disease is a genetic disease caused by mutations in the ATP7B gene, whose product is a liver transporter protein responsible for coordinated copper export into bile and blood. Interestingly, the spectrum of ATP7B mutations is vast and can influence clinical presentation (a variable spectrum of hepatic and neural manifestations), though the reason is not well understood. We describe the generation of iPSCs from a Chinese patient with Wilson's disease that bears the R778L Chinese hotspot mutation in the ATP7B gene. These iPSCs were pluripotent and could be readily differentiated into hepatocyte-like cells that displayed abnormal cytoplasmic localization of mutated ATP7B and defective copper transport. Moreover, gene correction using a self-inactivating lentiviral vector that expresses codon optimized-ATP7B or treatment with the chaperone drug curcumin could reverse the functional defect in vitro. Hence, our work describes an attractive model for studying the pathogenesis of Wilson's disease that is valuable for screening compounds or gene therapy approaches aimed to correct the abnormality. In the future, once relevant safety concerns (including the stability of the mature liver-like phenotype) and technical issues for the transplantation procedure are solved, hepatocyte-like cells from similarly genetically corrected iPSCs could be an option for autologous transplantation in Wilson's disease.

Novel One-step Immunoassays to Quantify α-Synuclein

PubMed Central

Bidinosti, Michael; Shimshek, Derya R.; Mollenhauer, Brit; Marcellin, David; Schweizer, Tatjana; Lotz, Gregor P.; Schlossmacher, Michael G.; Weiss, Andreas

2012-01-01

Familial Parkinson disease (PD) can result from α-synuclein gene multiplication, implicating the reduction of neuronal α-synuclein as a therapeutic target. Moreover, α-synuclein content in human cerebrospinal fluid (CSF) represents a PD biomarker candidate. However, capture-based assays for α-synuclein quantification in CSF (such as by ELISA) have shown discrepancies and have limited suitability for high-throughput screening. Here, we describe two sensitive, in-solution, time-resolved Förster's resonance energy transfer (TR-FRET)-based immunoassays for total and oligomeric α-synuclein quantification. CSF analysis showed strong concordance for total α-synuclein content between two TR-FRET assays and, in agreement with a previously characterized 36 h protocol-based ELISA, demonstrated lower α-synuclein levels in PD donors. Critically, the assay suitability for high-throughput screening of siRNA constructs and small molecules aimed at reducing endogenous α-synuclein levels was established and validated. In a small-scale proof of concept compound screen using 384 well plates, signals ranged from <30 to >120% of the mean of vehicle-treated cells for molecules known to lower and increase cellular α-synuclein, respectively. Furthermore, a reverse genetic screen of a kinase-directed siRNA library identified seven genes that modulated α-synuclein protein levels (five whose knockdown increased and two that decreased cellular α-synuclein protein). This provides critical new biological insight into cellular pathways regulating α-synuclein steady-state expression that may help guide further drug discovery efforts. Moreover, we describe an inherent limitation in current α-synuclein oligomer detection methodology, a finding that will direct improvement of future assay design. Our one-step TR-FRET-based platform for α-synuclein quantification provides a novel platform with superior performance parameters for the rapid screening of large biomarker cohorts and of compound and genetic libraries, both of which are essential to the development of PD therapies. PMID:22843695

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.

Neuroimaging, Genetics, and Clinical Data Sharing in Python Using the CubicWeb Framework

PubMed Central

Grigis, Antoine; Goyard, David; Cherbonnier, Robin; Gareau, Thomas; Papadopoulos Orfanos, Dimitri; Chauvat, Nicolas; Di Mascio, Adrien; Schumann, Gunter; Spooren, Will; Murphy, Declan; Frouin, Vincent

2017-01-01

In neurosciences or psychiatry, the emergence of large multi-center population imaging studies raises numerous technological challenges. From distributed data collection, across different institutions and countries, to final data publication service, one must handle the massive, heterogeneous, and complex data from genetics, imaging, demographics, or clinical scores. These data must be both efficiently obtained and downloadable. We present a Python solution, based on the CubicWeb open-source semantic framework, aimed at building population imaging study repositories. In addition, we focus on the tools developed around this framework to overcome the challenges associated with data sharing and collaborative requirements. We describe a set of three highly adaptive web services that transform the CubicWeb framework into a (1) multi-center upload platform, (2) collaborative quality assessment platform, and (3) publication platform endowed with massive-download capabilities. Two major European projects, IMAGEN and EU-AIMS, are currently supported by the described framework. We also present a Python package that enables end users to remotely query neuroimaging, genetics, and clinical data from scripts. PMID:28360851

Neuroimaging, Genetics, and Clinical Data Sharing in Python Using the CubicWeb Framework.

PubMed

Grigis, Antoine; Goyard, David; Cherbonnier, Robin; Gareau, Thomas; Papadopoulos Orfanos, Dimitri; Chauvat, Nicolas; Di Mascio, Adrien; Schumann, Gunter; Spooren, Will; Murphy, Declan; Frouin, Vincent

2017-01-01

In neurosciences or psychiatry, the emergence of large multi-center population imaging studies raises numerous technological challenges. From distributed data collection, across different institutions and countries, to final data publication service, one must handle the massive, heterogeneous, and complex data from genetics, imaging, demographics, or clinical scores. These data must be both efficiently obtained and downloadable. We present a Python solution, based on the CubicWeb open-source semantic framework, aimed at building population imaging study repositories. In addition, we focus on the tools developed around this framework to overcome the challenges associated with data sharing and collaborative requirements. We describe a set of three highly adaptive web services that transform the CubicWeb framework into a (1) multi-center upload platform, (2) collaborative quality assessment platform, and (3) publication platform endowed with massive-download capabilities. Two major European projects, IMAGEN and EU-AIMS, are currently supported by the described framework. We also present a Python package that enables end users to remotely query neuroimaging, genetics, and clinical data from scripts.

Diversity, genetic mapping, and signatures of domestication in the carrot (Daucus carota L.) genome, as revealed by Diversity Arrays Technology (DArT) markers

USDA-ARS?s Scientific Manuscript database

Carrot is one of the most economically important vegetables worldwide, however, genetic and genomic resources supporting carrot breeding remain limited. We developed a Diversity Arrays Technology (DArT) platform for wild and cultivated carrot and used it to investigate genetic diversity and to devel...

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 ...

Knowledge discovery through games and game theory

NASA Astrophysics Data System (ADS)

Smith, James F., III; Rhyne, Robert D.

2001-03-01

A fuzzy logic based expert system has been developed that automatically allocates electronic attack (EA) resources in real-time over many dissimilar platforms. The platforms can be very general, e.g., ships, planes, robots, land based facilities, etc. Potential foes the platforms deal with can also be general. The initial version of the algorithm was optimized using a genetic algorithm employing fitness functions constructed based on expertise. A new approach is being explored that involves embedding the resource manager in a electronic game environment. The game allows a human expert to play against the resource manager in a simulated battlespace with each of the defending platforms being exclusively directed by the fuzzy resource manager and the attacking platforms being controlled by the human expert or operating autonomously under their own logic. This approach automates the data mining problem. The game automatically creates a database reflecting the domain expert's knowledge, it calls a data mining function, a genetic algorithm, for data mining of the database as required. The game allows easy evaluation of the information mined in the second step. The measure of effectiveness (MOE) for re-optimization is discussed. The mined information is extremely valuable as shown through demanding scenarios.

Genetic and Computational Approaches for Studying Plant Development and Abiotic Stress Responses Using Image-Based Phenotyping

NASA Astrophysics Data System (ADS)

Campbell, M. T.; Walia, H.; Grondin, A.; Knecht, A.

2017-12-01

The development of abiotic stress tolerant crops (i.e. drought, salinity, or heat stress) requires the discovery of DNA sequence variants associated with stress tolerance-related traits. However, many traits underlying adaptation to abiotic stress involve a suite of physiological pathways that may be induced at different times throughout the duration of stress. Conventional single-point phenotyping approaches fail to fully capture these temporal responses, and thus downstream genetic analysis may only identify a subset of the genetic variants that are important for adaptation to sub-optimal environments. Although genomic resources for crops have advanced tremendously, the collection of phenotypic data for morphological and physiological traits is laborious and remains a significant bottleneck in bridging the phenotype-genotype gap. In recent years, the availability of automated, image-based phenotyping platforms has provided researchers with an opportunity to collect morphological and physiological traits non-destructively in a highly controlled environment. Moreover, these platforms allow abiotic stress responses to be recorded throughout the duration of the experiment, and have facilitated the use of function-valued traits for genetic analyses in major crops. We will present our approaches for addressing abiotic stress tolerance in cereals. This talk will focus on novel open-source software to process and extract biological meaningful data from images generated from these phenomics platforms. In addition, we will discuss the statistical approaches to model longitudinal phenotypes and dissect the genetic basis of dynamic responses to these abiotic stresses throughout development.

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.

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.

Pathway Profiling and Rational Trial Design for Studies in Advanced Stage Cervical Carcinoma: A Review and a Perspective

PubMed Central

Scholl, Susy M. E.; Kenter, Gemma; Kurzeder, Christian; Beuzeboc, Philippe

2011-01-01

Multiple genetic abnormalities will have occurred in advanced cervical cancer and multiple targeting is likely to be needed to control tumor growth. To date, dominant therapeutic targets under scrutiny for cervical cancer treatment have been EGFR pathway and angiogenesis inhibition as well as anti-HPV vaccines. The potentially most effective targets to be blocked may be downstream from the membrane receptor or at the level of the nucleus. Alterations of the pathways involved in DNA repair and in checkpoint activations, as well as the specific site of HPV genome integration, appear worth assessing. For genetic mutational analysis, complete exon sequencing may become the norm in the future but at this stage frequent mutations (that matter) can be verified by PCR analysis. A precise documentation of relevant alterations of a large spectrum of protein biomarkers can be carried out by reverse phase protein array (RPPA) or by multiplex analysis. Clinical decision-making on the drug(s) of choice as a function of the biological alteration will need input from bio-informatics platforms as well as novel statistical designs. Endpoints are yet to be defined such as the loss (or reappearance) of a predictive biomarker. Single or dual targeting needs to be explored first in relevant preclinical animal and in xenograft models prior to clinical deployment. PMID:22091418

Use of whole exome sequencing for the identification of Ito-based arrhythmia mechanism and therapy.

PubMed

Sturm, Amy C; Kline, Crystal F; Glynn, Patric; Johnson, Benjamin L; Curran, Jerry; Kilic, Ahmet; Higgins, Robert S D; Binkley, Philip F; Janssen, Paul M L; Weiss, Raul; Raman, Subha V; Fowler, Steven J; Priori, Silvia G; Hund, Thomas J; Carnes, Cynthia A; Mohler, Peter J

2015-05-26

Identified genetic variants are insufficient to explain all cases of inherited arrhythmia. We tested whether the integration of whole exome sequencing with well-established clinical, translational, and basic science platforms could provide rapid and novel insight into human arrhythmia pathophysiology and disease treatment. We report a proband with recurrent ventricular fibrillation, resistant to standard therapeutic interventions. Using whole-exome sequencing, we identified a variant in a previously unidentified exon of the dipeptidyl aminopeptidase-like protein-6 (DPP6) gene. This variant is the first identified coding mutation in DPP6 and augments cardiac repolarizing current (Ito) causing pathological changes in Ito and action potential morphology. We designed a therapeutic regimen incorporating dalfampridine to target Ito. Dalfampridine, approved for multiple sclerosis, normalized the ECG and reduced arrhythmia burden in the proband by >90-fold. This was combined with cilostazol to accelerate the heart rate to minimize the reverse-rate dependence of augmented Ito. We describe a novel arrhythmia mechanism and therapeutic approach to ameliorate the disease. Specifically, we identify the first coding variant of DPP6 in human ventricular fibrillation. These findings illustrate the power of genetic approaches for the elucidation and treatment of disease when carefully integrated with clinical and basic/translational research teams. © 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Rhabdoviruses as vaccine platforms for infectious disease and cancer.

PubMed

Zemp, Franz; Rajwani, Jahanara; Mahoney, Douglas J

2018-05-21

The family Rhabdoviridae (RV) comprises a large, genetically diverse collection of single-stranded, negative sense RNA viruses from the order Mononegavirales. Several RV members are being developed as live-attenuated vaccine vectors for the prevention or treatment of infectious disease and cancer. These include the prototype recombinant Vesicular Stomatitis Virus (rVSV) and the more recently developed recombinant Maraba Virus, both species within the genus Vesiculoviridae. A relatively strong safety profile in humans, robust immunogenicity and genetic malleability are key features that make the RV family attractive vaccine platforms. Currently, the rVSV vector is in preclinical development for vaccination against numerous high-priority infectious diseases, with clinical evaluation underway for HIV/AIDS and Ebola virus disease. Indeed, the success of the rVSV-ZEBOV vaccine during the 2014-15 Ebola virus outbreak in West Africa highlights the therapeutic potential of rVSV as a vaccine vector for acute, life-threatening viral illnesses. The rVSV and rMaraba platforms are also being tested as 'oncolytic' cancer vaccines in a series of phase 1-2 clinical trials, after being proven effective at eliciting immune-mediated tumour regression in preclinical mouse models. In this review, we discuss the biological and genetic features that make RVs attractive vaccine platforms and the development and ongoing testing of rVSV and rMaraba strains as vaccine vectors for infectious disease and cancer.

Mastering the intermediaries.

PubMed

Edelman, Benjamin

2014-06-01

Almost every retailer looks to Google to refer customers, and it's rare to find a manufacturer whose products aren't sold on Amazon. But these and other big platforms can capture a disproportionate share of the value a company creates: Buy an app on iTunes, and Apple takes 30%. The author presents four strategies to help businesses reduce their dependence on powerful platforms. Exploit the platform's need to be comprehensive. American Airlines' strong coverage of key routes made its presence on the travel website Kayak indispensable to Kayak's value proposition. As a result, AA negotiated a better deal. Identify and discredit discrimination. Public complaints that eBay was giving search prominence to suppliers who advertised on the site forced a reversal of the policy. Create an alternative platform. When MovieTickets was on the verge of dominating phone and online ticketing, Regal Entertainment and two other large theater chains formed Fandango. Deal more directly. People ordering takeout through online platforms like Foodler and GrubHub have often already chosen their restaurant. Restaurants that deal directly can exit the platform.

Functional integrity of flexible n-channel metal–oxide–semiconductor field-effect transistors on a reversibly bistable platform

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

Alfaraj, Nasir; Hussain, Aftab M.; Torres Sevilla, Galo A.

Flexibility can bring a new dimension to state-of-the-art electronics, such as rollable displays and integrated circuit systems being transformed into more powerful resources. Flexible electronics are typically hosted on polymeric substrates. Such substrates can be bent and rolled up, but cannot be independently fixed at the rigid perpendicular position necessary to realize rollable display-integrated gadgets and electronics. A reversibly bistable material can assume two stable states in a reversible way: flexibly rolled state and independently unbent state. Such materials are used in cycling and biking safety wristbands and a variety of ankle bracelets for orthopedic healthcare. They are often wrappedmore » around an object with high impulsive force loading. Here, we study the effects of cumulative impulsive force loading on thinned (25 μm) flexible silicon-based n-channel metal–oxide–semiconductor field-effect transistor devices housed on a reversibly bistable flexible platform. We found that the transistors have maintained their high performance level up to an accumulated 180 kN of impact force loading. The gate dielectric layers have maintained their reliability, which is evidenced by the low leakage current densities. Also, we observed low variation in the effective electron mobility values, which manifests that the device channels have maintained their carrier transport properties.« less

[Review of Second Generation Sequencing and Its Application in Forensic Genetics].

PubMed

Zhang, S H; Bian, Y N; Zhao, Q; Li, C T

2016-08-01

The rapid development of second generation sequencing （SGS） within the past few years has led to the increasement of data throughput and read length while at the same time brought down substantially the sequencing cost. This made new breakthrough in the area of biology and ushered the forensic genetics into a new era. Based on the history of sequencing application in forensic genetics, this paper reviews the importance of sequencing technologies for genetic marker detection. The application status and potential of SGS in forensic genetics are discussed based on the already explored SGS platforms of Roche, Illumina and Life Technologies. With these platforms, DNA markers （SNP, STR）, RNA markers （mRNA, microRNA） and whole mtDNA can be sequenced. However, development and validation of application kits, maturation of analysis software, connection to the existing databases and the possible ethical issues occurred with big data will be the key factors that determine whether this technology can substitute or supplement PCR-CE, the mature technology, and be widely used for cases detection. Copyright© by the Editorial Department of Journal of Forensic Medicine.

SimHap GUI: an intuitive graphical user interface for genetic association analysis.

PubMed

Carter, Kim W; McCaskie, Pamela A; Palmer, Lyle J

2008-12-25

Researchers wishing to conduct genetic association analysis involving single nucleotide polymorphisms (SNPs) or haplotypes are often confronted with the lack of user-friendly graphical analysis tools, requiring sophisticated statistical and informatics expertise to perform relatively straightforward tasks. Tools, such as the SimHap package for the R statistics language, provide the necessary statistical operations to conduct sophisticated genetic analysis, but lacks a graphical user interface that allows anyone but a professional statistician to effectively utilise the tool. We have developed SimHap GUI, a cross-platform integrated graphical analysis tool for conducting epidemiological, single SNP and haplotype-based association analysis. SimHap GUI features a novel workflow interface that guides the user through each logical step of the analysis process, making it accessible to both novice and advanced users. This tool provides a seamless interface to the SimHap R package, while providing enhanced functionality such as sophisticated data checking, automated data conversion, and real-time estimations of haplotype simulation progress. SimHap GUI provides a novel, easy-to-use, cross-platform solution for conducting a range of genetic and non-genetic association analyses. This provides a free alternative to commercial statistics packages that is specifically designed for genetic association analysis.

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

Two-step ion-exchange chromatographic purification combined with reversed-phase chromatography to isolate C-peptide for mass spectrometric analysis.

PubMed

Kabytaev, Kuanysh; Durairaj, Anita; Shin, Dmitriy; Rohlfing, Curt L; Connolly, Shawn; Little, Randie R; Stoyanov, Alexander V

2016-02-01

A liquid chromatography with mass spectrometry on-line platform that includes the orthogonal techniques of ion exchange and reversed phase chromatography is applied for C-peptide analysis. Additional improvement is achieved by the subsequent application of cation- and anion-exchange purification steps that allow for isolating components that have their isoelectric points in a narrow pH range before final reversed-phase mass spectrometry analysis. The utility of this approach for isolating fractions in the desired "pI window" for profiling complex mixtures is discussed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Use of Sequence-independent, single-primer amplification (SISPA) with NGS platform for detection of RNA viruses in clinical samples

USDA-ARS?s Scientific Manuscript database

Current technologies for next generation sequencing (NGS) have revolutionized metagenomics analysis of clinical samples. One advantage of the NGS platform is the possibility to sequence the genetic material in samples without any prior knowledge of the sequence contained within. Sequence-Independent...

HaloTag Technology: A Versatile Platform for Biomedical Applications

PubMed Central

2015-01-01

Exploration of protein function and interaction is critical for discovering links among genomics, proteomics, and disease state; yet, the immense complexity of proteomics found in biological systems currently limits our investigational capacity. Although affinity and autofluorescent tags are widely employed for protein analysis, these methods have been met with limited success because they lack specificity and require multiple fusion tags and genetic constructs. As an alternative approach, the innovative HaloTag protein fusion platform allows protein function and interaction to be comprehensively analyzed using a single genetic construct with multiple capabilities. This is accomplished using a simplified process, in which a variable HaloTag ligand binds rapidly to the HaloTag protein (usually linked to the protein of interest) with high affinity and specificity. In this review, we examine all current applications of the HaloTag technology platform for biomedical applications, such as the study of protein isolation and purification, protein function, protein–protein and protein–DNA interactions, biological assays, in vitro cellular imaging, and in vivo molecular imaging. In addition, novel uses of the HaloTag platform are briefly discussed along with potential future applications. PMID:25974629

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.

Characterization of Tn6238 with a New Allele of aac(6′)-Ib-cr

PubMed Central

Quiroga, María P.; Orman, Betina; Errecalde, Laura; Kaufman, Sara

2015-01-01

Here, we report that the genetic structure of Tn1331 remained conserved in Argentina from 1989 to 2013 (72 of 73 isolates), with the exception being the plasmid-borne Tn1331-like transposon Tn6238 containing a new aac(6′)-Ib-cr allele recovered from a colistin-resistant Klebsiella pneumoniae clinical isolate. A bioinformatic analysis of aac(6′)-Ib-like gene cassettes suggests that this new aac(6′)-Ib-cr allele emerged through mutation or homologous recombination in the Tn1331 genetic platform. Tn6238 is a novel platform for the dissemination of aminoglycoside and fluoroquinolone resistance determinants. PMID:25691640

Supercontinuum optimization for dual-soliton based light sources using genetic algorithms in a grid platform.

PubMed

Arteaga-Sierra, F R; Milián, C; Torres-Gómez, I; Torres-Cisneros, M; Moltó, G; Ferrando, A

2014-09-22

We present a numerical strategy to design fiber based dual pulse light sources exhibiting two predefined spectral peaks in the anomalous group velocity dispersion regime. The frequency conversion is based on the soliton fission and soliton self-frequency shift occurring during supercontinuum generation. The optimization process is carried out by a genetic algorithm that provides the optimum input pulse parameters: wavelength, temporal width and peak power. This algorithm is implemented in a Grid platform in order to take advantage of distributed computing. These results are useful for optical coherence tomography applications where bell-shaped pulses located in the second near-infrared window are needed.

HpQTL: a geometric morphometric platform to compute the genetic architecture of heterophylly.

PubMed

Sun, Lidan; Wang, Jing; Zhu, Xuli; Jiang, Libo; Gosik, Kirk; Sang, Mengmeng; Sun, Fengsuo; Cheng, Tangren; Zhang, Qixiang; Wu, Rongling

2017-02-15

Heterophylly, i.e. morphological changes in leaves along the axis of an individual plant, is regarded as a strategy used by plants to cope with environmental change. However, little is known of the extent to which heterophylly is controlled by genes and how each underlying gene exerts its effect on heterophyllous variation. We described a geometric morphometric model that can quantify heterophylly in plants and further constructed an R-based computing platform by integrating this model into a genetic mapping and association setting. The platform, named HpQTL, allows specific quantitative trait loci mediating heterophyllous variation to be mapped throughout the genome. The statistical properties of HpQTL were examined and validated via computer simulation. Its biological relevance was demonstrated by results from a real data analysis of heterophylly in a wood plant, mei (Prunus mume). HpQTL provides a powerful tool to analyze heterophylly and its underlying genetic architecture in a quantitative manner. It also contributes a new approach for genome-wide association studies aimed to dissect the programmed regulation of plant development and evolution. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Data mining for multiagent rules, strategies, and fuzzy decision tree structure

NASA Astrophysics Data System (ADS)

Smith, James F., III; Rhyne, Robert D., II; Fisher, Kristin

2002-03-01

A fuzzy logic based resource manager (RM) has been developed that automatically allocates electronic attack resources in real-time over many dissimilar platforms. Two different data mining algorithms have been developed to determine rules, strategies, and fuzzy decision tree structure. The first data mining algorithm uses a genetic algorithm as a data mining function and is called from an electronic game. The game allows a human expert to play against the resource manager in a simulated battlespace with each of the defending platforms being exclusively directed by the fuzzy resource manager and the attacking platforms being controlled by the human expert or operating autonomously under their own logic. This approach automates the data mining problem. The game automatically creates a database reflecting the domain expert's knowledge. It calls a data mining function, a genetic algorithm, for data mining of the database as required and allows easy evaluation of the information mined in the second step. The criterion for re- optimization is discussed as well as experimental results. Then a second data mining algorithm that uses a genetic program as a data mining function is introduced to automatically discover fuzzy decision tree structures. Finally, a fuzzy decision tree generated through this process is discussed.

An innovative SNP genotyping method adapting to multiple platforms and throughputs

USDA-ARS?s Scientific Manuscript database

Single nucleotide polymorphisms (SNPs) are highly abundant, distributed throughout the genome in various species, and therefore they are widely used as genetic markers. However, the usefulness of this genetic tool relies heavily on the availability of user-friendly SNP genotyping methods. We have d...

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

Behavioral, Neurophysiological, and Synaptic Impairment in a Transgenic Neuregulin1 (NRG1-IV) Murine Schizophrenia Model

PubMed Central

Papaleo, Francesco; Yang, Feng; Paterson, Clare; Palumbo, Sara; Carr, Gregory V.; Wang, Yanhong; Floyd, Kirsten; Huang, Wenwei; Thomas, Craig J.; Chen, Jingshan; Weinberger, Daniel R.

2016-01-01

Schizophrenia is a chronic, disabling neuropsychiatric disorder with complex genetic origins. The development of strategies for genome manipulation in rodents provides a platform for understanding the pathogenic role of genes and for testing novel therapeutic agents. Neuregulin 1 (NRG1), a critical developmental neurotrophin, is associated with schizophrenia. The NRG1 gene undergoes extensive alternative splicing and, to date, little is known about the neurobiology of a novel NRG1 isoform, NRG1-IV, which is increased in the brains of individuals with schizophrenia and associated with genetic risk variation. Here, we developed a transgenic mouse model (NRG1-IV/NSE-tTA) in which human NRG1-IV is selectively overexpressed in a neuronal specific manner. Using a combination of molecular, biochemical, electrophysiological, and behavioral analyses, we demonstrate that NRG1-IV/NSE-tTA mice exhibit abnormal behaviors relevant to schizophrenia, including impaired sensorimotor gating, discrimination memory, and social behaviors. These neurobehavioral phenotypes are accompanied by increases in cortical expression of the NRG1 receptor, ErbB4 and the downstream signaling target, PIK3-p110δ, along with disrupted dendritic development, synaptic pathology, and altered prefrontal cortical excitatory–inhibitory balance. Pharmacological inhibition of p110δ reversed sensorimotor gating and cognitive deficits. These data demonstrate a novel role for NRG1-IV in learning, memory, and neural circuit formation and a potential neurobiological mechanism for schizophrenia risk; show that deficits are pharmacologically reversible in adulthood; and further highlight p110δ as a target for antipsychotic drug development. SIGNIFICANCE STATEMENT Schizophrenia is a disabling psychiatric disorder with neurodevelopmental origins. Genes that increase risk for schizophrenia have been identified. Understanding how these genes affect brain development and function is necessary. This work is the first report of a newly generated humanized transgenic mouse model engineered to express human NRG1-IV, an isoform of the NRG1 (Neuregulin 1) gene that is increased in the brains of patients with schizophrenia in association with genetic risk. Using behavioral neuroscience, molecular biology, electrophysiology, and pharmacology, we identify a role for NRG1-IV in learning, memory, and cognition and determine that this relates to brain excitatory–inhibitory balance and changes in ErbB4/PI3K/AKT signaling. Moreover, the study further highlights the potential of targeting the PI3K pathway for the treatment of schizophrenia. PMID:27122041

Additive Manufacturing of Catalytically Active Living Materials.

PubMed

Saha, Abhijit; Johnston, Trevor G; Shafranek, Ryan T; Goodman, Cassandra J; Zalatan, Jesse G; Storti, Duane W; Ganter, Mark A; Nelson, Alshakim

2018-04-25

Living materials, which are composites of living cells residing in a polymeric matrix, are designed to utilize the innate functionalities of the cells to address a broad range of applications such as fermentation and biosensing. Herein, we demonstrate the additive manufacturing of catalytically active living materials (AMCALM) for continuous fermentation. A multi-stimuli-responsive yeast-laden hydrogel ink, based on F127-dimethacrylate, was developed and printed using a direct-write 3D printer. The reversible stimuli-responsive behaviors of the polymer hydrogel inks to temperature and pressure are critical, as they enabled the facile incorporation of yeast cells and subsequent fabrication of 3D lattice constructs. Subsequent photo-cross-linking of the printed polymer hydrogel afforded a robust elastic material. These yeast-laden living materials were metabolically active in the fermentation of glucose into ethanol for 2 weeks in a continuous batch process without significant reduction in efficiency (∼90% yield of ethanol). This cell immobilization platform may potentially be applicable toward other genetically modified yeast strains to produce other high-value chemicals in a continuous biofermentation process.

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

Agrobacterium-mediated genetic transformation of yam (Dioscorea rotundata): an important tool for functional study of genes and crop improvement

PubMed Central

Nyaboga, Evans; Tripathi, Jaindra N.; Manoharan, Rajesh; Tripathi, Leena

2014-01-01

Although genetic transformation of clonally propagated crops has been widely studied as a tool for crop improvement and as a vital part of the development of functional genomics resources, there has been no report of any existing Agrobacterium-mediated transformation of yam (Dioscorea spp.) with evidence of stable integration of T-DNA. Yam is an important crop in the tropics and subtropics providing food security and income to over 300 million people. However, yam production remains constrained by increasing levels of field and storage pests and diseases. A major constraint to the development of biotechnological approaches for yam improvement has been the lack of an efficient and robust transformation and regeneration system. In this study, we developed an Agrobacterium-mediated transformation of Dioscorea rotundata using axillary buds as explants. Two cultivars of D. rotundata were transformed using Agrobacterium tumefaciens harboring the binary vectors containing selectable marker and reporter genes. After selection with appropriate concentrations of antibiotic, shoots were developed on shoot induction and elongation medium. The elongated antibiotic-resistant shoots were subsequently rooted on medium supplemented with selection agent. Successful transformation was confirmed by polymerase chain reaction, Southern blot analysis, and reporter genes assay. Expression of gusA gene in transgenic plants was also verified by reverse transcription polymerase chain reaction analysis. Transformation efficiency varied from 9.4 to 18.2% depending on the cultivars, selectable marker genes, and the Agrobacterium strain used for transformation. It took 3–4 months from Agro-infection to regeneration of complete transgenic plant. Here we report an efficient, fast and reproducible protocol for Agrobacterium-mediated transformation of D. rotundata using axillary buds as explants, which provides a useful platform for future genetic engineering studies in this economically important crop. PMID:25309562

Qatari intersections with global genetics research and discourse.

PubMed

Kilshaw, Susie

2018-04-01

Genetic discourses have taken a predominant role in approaches to combating a number of conditions that affect Qataris. This paper is derived from an exploration of Qatari encounters with globalizing discourses of genetics, particularly as they relate to notions of risk. It explores Qataris negotiations of global interactions and influences, including the discourses around genetic risk and cousin marriage. It suggests that family marriage can be seen as one of the main platforms of resistance and a means for modern, cosmopolitan and tradition to be negotiated.

Estimation of Genetic Relationships Between Individuals Across Cohorts and Platforms: Application to Childhood Height.

PubMed

Fedko, Iryna O; Hottenga, Jouke-Jan; Medina-Gomez, Carolina; Pappa, Irene; van Beijsterveldt, Catharina E M; Ehli, Erik A; Davies, Gareth E; Rivadeneira, Fernando; Tiemeier, Henning; Swertz, Morris A; Middeldorp, Christel M; Bartels, Meike; Boomsma, Dorret I

2015-09-01

Combining genotype data across cohorts increases power to estimate the heritability due to common single nucleotide polymorphisms (SNPs), based on analyzing a Genetic Relationship Matrix (GRM). However, the combination of SNP data across multiple cohorts may lead to stratification, when for example, different genotyping platforms are used. In the current study, we address issues of combining SNP data from different cohorts, the Netherlands Twin Register (NTR) and the Generation R (GENR) study. Both cohorts include children of Northern European Dutch background (N = 3102 + 2826, respectively) who were genotyped on different platforms. We explore imputation and phasing as a tool and compare three GRM-building strategies, when data from two cohorts are (1) just combined, (2) pre-combined and cross-platform imputed and (3) cross-platform imputed and post-combined. We test these three strategies with data on childhood height for unrelated individuals (N = 3124, average age 6.7 years) to explore their effect on SNP-heritability estimates and compare results to those obtained from the independent studies. All combination strategies result in SNP-heritability estimates with a standard error smaller than those of the independent studies. We did not observe significant difference in estimates of SNP-heritability based on various cross-platform imputed GRMs. SNP-heritability of childhood height was on average estimated as 0.50 (SE = 0.10). Introducing cohort as a covariate resulted in ≈2 % drop. Principal components (PCs) adjustment resulted in SNP-heritability estimates of about 0.39 (SE = 0.11). Strikingly, we did not find significant difference between cross-platform imputed and combined GRMs. All estimates were significant regardless the use of PCs adjustment. Based on these analyses we conclude that imputation with a reference set helps to increase power to estimate SNP-heritability by combining cohorts of the same ethnicity genotyped on different platforms. However, important factors should be taken into account such as remaining cohort stratification after imputation and/or phenotypic heterogeneity between and within cohorts. Whether one should use imputation, or just combine the genotype data, depends on the number of overlapping SNPs in relation to the total number of genotyped SNPs for both cohorts, and their ability to tag all the genetic variance related to the specific trait of interest.

SimHap GUI: An intuitive graphical user interface for genetic association analysis

PubMed Central

Carter, Kim W; McCaskie, Pamela A; Palmer, Lyle J

2008-01-01

Background Researchers wishing to conduct genetic association analysis involving single nucleotide polymorphisms (SNPs) or haplotypes are often confronted with the lack of user-friendly graphical analysis tools, requiring sophisticated statistical and informatics expertise to perform relatively straightforward tasks. Tools, such as the SimHap package for the R statistics language, provide the necessary statistical operations to conduct sophisticated genetic analysis, but lacks a graphical user interface that allows anyone but a professional statistician to effectively utilise the tool. Results We have developed SimHap GUI, a cross-platform integrated graphical analysis tool for conducting epidemiological, single SNP and haplotype-based association analysis. SimHap GUI features a novel workflow interface that guides the user through each logical step of the analysis process, making it accessible to both novice and advanced users. This tool provides a seamless interface to the SimHap R package, while providing enhanced functionality such as sophisticated data checking, automated data conversion, and real-time estimations of haplotype simulation progress. Conclusion SimHap GUI provides a novel, easy-to-use, cross-platform solution for conducting a range of genetic and non-genetic association analyses. This provides a free alternative to commercial statistics packages that is specifically designed for genetic association analysis. PMID:19109877

Genomic analysis of thermophilic Bacillus coagulans strains: efficient producers for platform bio-chemicals.

PubMed

Su, Fei; Xu, Ping

2014-01-29

Microbial strains with high substrate efficiency and excellent environmental tolerance are urgently needed for the production of platform bio-chemicals. Bacillus coagulans has these merits; however, little genetic information is available about this species. Here, we determined the genome sequences of five B. coagulans strains, and used a comparative genomic approach to reconstruct the central carbon metabolism of this species to explain their fermentation features. A novel xylose isomerase in the xylose utilization pathway was identified in these strains. Based on a genome-wide positive selection scan, the selection pressure on amino acid metabolism may have played a significant role in the thermal adaptation. We also researched the immune systems of B. coagulans strains, which provide them with acquired resistance to phages and mobile genetic elements. Our genomic analysis provides comprehensive insights into the genetic characteristics of B. coagulans and paves the way for improving and extending the uses of this species.

Genomic analysis of thermophilic Bacillus coagulans strains: efficient producers for platform bio-chemicals

PubMed Central

Su, Fei; Xu, Ping

2014-01-01

Microbial strains with high substrate efficiency and excellent environmental tolerance are urgently needed for the production of platform bio-chemicals. Bacillus coagulans has these merits; however, little genetic information is available about this species. Here, we determined the genome sequences of five B. coagulans strains, and used a comparative genomic approach to reconstruct the central carbon metabolism of this species to explain their fermentation features. A novel xylose isomerase in the xylose utilization pathway was identified in these strains. Based on a genome-wide positive selection scan, the selection pressure on amino acid metabolism may have played a significant role in the thermal adaptation. We also researched the immune systems of B. coagulans strains, which provide them with acquired resistance to phages and mobile genetic elements. Our genomic analysis provides comprehensive insights into the genetic characteristics of B. coagulans and paves the way for improving and extending the uses of this species. PMID:24473268

Salient Features of Endonuclease Platforms for Therapeutic Genome Editing.

PubMed

Certo, Michael T; Morgan, Richard A

2016-03-01

Emerging gene-editing technologies are nearing a revolutionary phase in genetic medicine: precisely modifying or repairing causal genetic defects. This may include any number of DNA sequence manipulations, such as knocking out a deleterious gene, introducing a particular mutation, or directly repairing a defective sequence by site-specific recombination. All of these edits can currently be achieved via programmable rare-cutting endonucleases to create targeted DNA breaks that can engage and exploit endogenous DNA repair pathways to impart site-specific genetic changes. Over the past decade, several distinct technologies for introducing site-specific DNA breaks have been developed, yet the different biological origins of these gene-editing technologies bring along inherent differences in parameters that impact clinical implementation. This review aims to provide an accessible overview of the various endonuclease-based gene-editing platforms, highlighting the strengths and weakness of each with respect to therapeutic applications.

Salient Features of Endonuclease Platforms for Therapeutic Genome Editing

PubMed Central

Certo, Michael T; Morgan, Richard A

2016-01-01

Emerging gene-editing technologies are nearing a revolutionary phase in genetic medicine: precisely modifying or repairing causal genetic defects. This may include any number of DNA sequence manipulations, such as knocking out a deleterious gene, introducing a particular mutation, or directly repairing a defective sequence by site-specific recombination. All of these edits can currently be achieved via programmable rare-cutting endonucleases to create targeted DNA breaks that can engage and exploit endogenous DNA repair pathways to impart site-specific genetic changes. Over the past decade, several distinct technologies for introducing site-specific DNA breaks have been developed, yet the different biological origins of these gene-editing technologies bring along inherent differences in parameters that impact clinical implementation. This review aims to provide an accessible overview of the various endonuclease-based gene-editing platforms, highlighting the strengths and weakness of each with respect to therapeutic applications. PMID:26796671

Electrically controlled microvalves to integrate microchip polymerase chain reaction and capillary electrophoresis.

PubMed

Kaigala, Govind V; Hoang, Viet N; Backhouse, Christopher J

2008-07-01

Microvalves are key in realizing portable miniaturized diagnostic platforms. We present a scalable microvalve that integrates well with standard lab on a chip (LOC) implementations, yet which requires essentially no external infrastructure for its operation. This electrically controlled, phase-change microvalve is used to integrate genetic amplification and analysis via capillary electrophoresis--the basis of many diagnostics. The microvalve is actuated using a polymer (polyethylene glycol, PEG) that exhibits a large volumetric change between its solid and liquid phases. Both the phase change of the PEG and the genetic amplification via polymerase chain reaction (PCR) are thermally controlled using thin film resistive elements that are patterned using standard microfabrication methods. By contrast with many other valve technologies, these microvalves and their control interface scale down in size readily. The novelty here lies in the use of fully integrated microvalves that require only electrical connections to realize a portable and inexpensive genetic analysis platform.

A Road Map for Precision Medicine in the Epilepsies

PubMed Central

2015-01-01

Summary Technological advances have paved the way for accelerated genomic discovery and are bringing precision medicine clearly into view. Epilepsy research in particular is well-suited to serve as a model for the development and deployment of targeted therapeutics in precision medicine because of the rapidly expanding genetic knowledge base in epilepsy, the availability of good in vitro and in vivo model systems to efficiently study the biological consequences of genetic mutations, the ability to turn these models into effective drug screening platforms, and the establishment of collaborative research groups. Moving forward, it is critical that we strengthen these collaborations, particularly through integrated research platforms to provide robust analyses both for accurate personal genome analysis and gene and drug discovery. Similarly, the implementation of clinical trial networks will allow the expansion of patient sample populations with genetically defined epilepsy so that drug discovery can be translated into clinical practice. PMID:26416172

Heating rates in furnace atomic absorption using the L'vov platform

USGS Publications Warehouse

Koirtyohann, S.R.; Giddings, R.C.; Taylor, Howard E.

1984-01-01

Heating rate profiles for the furnace tube wall, the furnace atmosphere, and a L'vov platform were established for a range of conditions in a cyclically heated graphite atomizer. The tube wall profile was made by direct observation with a recording optical pyrometer. The sodium line reversal method was used to establish the heating rate of the furnace atmosphere, and appearance temperatures for a series metals of differing volatility was used to establish platform profiles. The tube wall heating rate was nearly linear at 2240??C s- until the desired temperature was reached after which the temperature remained constant. The furnace atmosphere reached a given temperature 0.2-0.4 s later than the tube wall through most of the atomize cycle. The platform lagged the tube wall 0.5-0.8 s. Under typical operating conditions the furnace atmosphere was 100-200??C cooler than the tube wall and at nearly constant temperature when the analyte vaporized from the platform. The L'vov platform causes the cyclically heated commercial furnace to approximate the behavior of a constant temperature furnace during atomization. ?? 1984.

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

Optical and electrical interfacing technologies for living cell bio-chips.

PubMed

Shacham-Diamand, Y; Belkin, S; Rishpon, J; Elad, T; Melamed, S; Biran, A; Yagur-Kroll, S; Almog, R; Daniel, R; Ben-Yoav, H; Rabner, A; Vernick, S; Elman, N; Popovtzer, R

2010-06-01

Whole-cell bio-chips for functional sensing integrate living cells on miniaturized platforms made by micro-system-technologies (MST). The cells are integrated, deposited or immersed in a media which is in contact with the chip. The cells behavior is monitored via electrical, electrochemical or optical methods. In this paper we describe such whole-cell biochips where the signal is generated due to the genetic response of the cells. The solid-state platform hosts the biological component, i.e. the living cells, and integrates all the required micro-system technologies, i.e. the micro-electronics, micro-electro optics, micro-electro or magneto mechanics and micro-fluidics. The genetic response of the cells expresses proteins that generate: a. light by photo-luminescence or bioluminescence, b. electrochemical signal by interaction with a substrate, or c. change in the cell impedance. The cell response is detected by a front end unit that converts it to current or voltage amplifies and filters it. The resultant signal is analyzed and stored for further processing. In this paper we describe three examples of whole-cell bio chips, photo-luminescent, bioluminescent and electrochemical, which are based on the genetic response of genetically modified E. coli microbes integrated on a micro-fluidics MEMS platform. We describe the chip outline as well as the basic modeling scheme of such sensors. We discuss the highlights and problems of such system, from the point of view of micro-system-technology.

Construction of Escherichia Coli Cell Factories for Production of Organic Acids and Alcohols.

PubMed

Liu, Pingping; Zhu, Xinna; Tan, Zaigao; Zhang, Xueli; Ma, Yanhe

2016-01-01

Production of bulk chemicals from renewable biomass has been proved to be sustainable and environmentally friendly. Escherichia coli is the most commonly used host strain for constructing cell factories for production of bulk chemicals since it has clear physiological and genetic characteristics, grows fast in minimal salts medium, uses a wide range of substrates, and can be genetically modified easily. With the development of metabolic engineering, systems biology, and synthetic biology, a technology platform has been established to construct E. coli cell factories for bulk chemicals production. In this chapter, we will introduce this technology platform, as well as E. coli cell factories successfully constructed for production of organic acids and alcohols.

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.

The rare and undiagnosed diseases diagnostic service - application of massively parallel sequencing in a state-wide clinical service.

PubMed

Baynam, Gareth; Pachter, Nicholas; McKenzie, Fiona; Townshend, Sharon; Slee, Jennie; Kiraly-Borri, Cathy; Vasudevan, Anand; Hawkins, Anne; Broley, Stephanie; Schofield, Lyn; Verhoef, Hedwig; Walker, Caroline E; Molster, Caron; Blackwell, Jenefer M; Jamieson, Sarra; Tang, Dave; Lassmann, Timo; Mina, Kym; Beilby, John; Davis, Mark; Laing, Nigel; Murphy, Lesley; Weeramanthri, Tarun; Dawkins, Hugh; Goldblatt, Jack

2016-06-11

The Rare and Undiagnosed Diseases Diagnostic Service (RUDDS) refers to a genomic diagnostic platform operating within the Western Australian Government clinical services delivered through Genetic Services of Western Australia (GSWA). GSWA has provided a state-wide service for clinical genetic care for 28 years and it serves a population of 2.5 million people across a geographical area of 2.5milion Km(2). Within this context, GSWA has established a clinically integrated genomic diagnostic platform in partnership with other public health system managers and service providers, including but not limited to the Office of Population Health Genomics, Diagnostic Genomics (PathWest Laboratories) and with executive level support from the Department of Health. Herein we describe report presents the components of this service that are most relevant to the heterogeneity of paediatric clinical genetic care. Briefly the platform : i) offers multiple options including non-genetic testing; monogenic and genomic (targeted in silico filtered and whole exome) analysis; and matchmaking; ii) is delivered in a patient-centric manner that is resonant with the patient journey, it has multiple points for entry, exit and re-entry to allow people access to information they can use, when they want to receive it; iii) is synchronous with precision phenotyping methods; iv) captures new knowledge, including multiple expert review; v) is integrated with current translational genomic research activities and best practice; and vi) is designed for flexibility for interactive generation of, and integration with, clinical research for diagnostics, community engagement, policy and models of care. The RUDDS has been established as part of routine clinical genetic services and is thus sustainable, equitably managed and seeks to translate new knowledge into efficient diagnostics and improved health for the whole community.

Molecular genetic characterization of lasquerella new industrial crop using DArTseq markers

USDA-ARS?s Scientific Manuscript database

DArTseq, a new SNP-based marker platform, was developed and used to analyze the genetic diversity of the US germplasm collection of lesquerella. Lesquerella is a new oilseed crop in the Brassica family found native in the American Southwest. The potential of the species as a domestic source of indu...

Analysis of Human Plasma Metabolites across Different Liquid Chromatography - Mass Spectrometry Platforms: Cross-platform Transferable Chemical Signatures

PubMed Central

Telu, Kelly H.; Yan, Xinjian; Wallace, William E.; Stein, Stephen E.; Simón-Manso, Yamil

2016-01-01

RATIONALE The metabolite profiling of a NIST plasma Standard Reference Material (SRM 1950) on different LC-MS platforms showed significant differences. Although these findings suggest caution when interpreting metabolomics results, the degree of overlap of both profiles allowed us to use tandem mass spectral libraries of recurrent spectra to evaluate to what extent these results are transferable across platforms and to develop cross-platform chemical signatures. METHODS Non-targeted global metabolite profiles of SRM 1950 were obtained on different LC-MS platforms using reversed phase chromatography and different chromatographic scales (nano, conventional and UHPLC). The data processing and the metabolite differential analysis were carried out using publically available (XCMS), proprietary (Mass Profiler Professional) and in-house software (NIST pipeline). RESULTS Repeatability and intermediate precision showed that the non-targeted SRM 1950 profiling was highly reproducible when working on the same platform (RSD < 2%); however, substantial differences were found in the LC-MS patterns originating on different platforms or even using different chromatographic scales (conventional HPLC, UHPLC and nanoLC) on the same platform. A substantial degree of overlap (common molecular features) was also found. A procedure to generate consistent chemical signatures using tandem mass spectral libraries of recurrent spectra is proposed. CONLUSIONS Different platforms rendered significantly different metabolite profiles, but the results were highly reproducible when working within one platform. Tandem mass spectral libraries of recurrent spectra are proposed to evaluate the degree of transferability of chemical signatures generated on different platforms. Chemical signatures based on our procedure are most likely cross-platform transferable. PMID:26842580

Multi-stimuli-responsive biohybrid nanoparticles with cross-linked albumin coronae self-assembled by a polymer-protein biodynamer.

PubMed

Wang, Lin; Liu, Li; Dong, Bingyang; Zhao, Hanying; Zhang, Mingming; Chen, Wenjuan; Hong, Yanhang

2017-05-01

A thermoresponsive polymer-protein biodynamer was prepared via the bioconjugation of an aliphatic aldehyde-functionalized copolymer to hydrazine-modified bovine serum albumin (BSA) through reversible pyridylhydrazone linkages. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC) results indicated that the pyridylhydrazone linkages cleaved in an intracellular-mimicking acidic milieu, thus leading to the release of BSA. The dynamic character of the protein biodynamer was demonstrated by exchange reactions with aldehyde-containing molecules. The biodynamer self-assembled into spherical micelles at a temperature above its lower critical solution temperature (LCST). Subsequently, BSA molecules within the hydrophilic coronae of the micelles were readily cross-linked via reaction with cystamine at 45°C, and multi-stimuli-responsive nanoparticles were generated. The biohybrid nanoparticles reversibly swelled and shrank as the cores of the nanoparticles were solvated below the LCST and desolvated above the LCST. The accessible reversibility of the pyridylhydrazone bonds imparts pH-responsive and dynamic characteristics to the nanoparticles. The nanoparticles displayed glutathione (GSH) responsiveness, and the synergistic effects of pH and GSH resulted in complete disintegration of the nanoparticles under the intracellular-mimicking acidic and reductive conditions. The nanoparticles were also enzyme-responsive and disintegrated rapidly in the presence of protease. In vitro cytotoxicity and cell uptake assays demonstrated that the nanoparticles were highly biocompatible and effectively internalized by HepG2 cells, which make them interesting candidates as vehicles for drug delivery application and biomimetic platforms to investigate the biological process in nature. In this research, we report the synthesis of a temperature and pH dual-responsive polymer-protein biodynamer through reversible pyridylhydrazone formation. The prepared biodynamer can offer a potential platform for intracellular protein delivery. The multi-stimuli-responsive biohybrid nanoparticles containing disulfide functionalities are constructed by cross-linking albumin coronae of the biodynamer micelles. With the combination of a thermoresponsive polymer, protein and reversible covalent bonds, the biohybrid nanoparticles are endowed with highly biocompatible, environmentally responsive and adaptive features. These nanoparticles present the ability to undergo changes in their constitution, hydrodynamic size and nanostructure in response to physical, chemical and biological stimuli, which make them interesting candidates as vehicles for drug delivery application and a biomimetic platform to investigate the biological process in nature. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

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.

Automation of diagnostic genetic testing: mutation detection by cyclic minisequencing.

PubMed

Alagrund, Katariina; Orpana, Arto K

2014-01-01

The rising role of nucleic acid testing in clinical decision making is creating a need for efficient and automated diagnostic nucleic acid test platforms. Clinical use of nucleic acid testing sets demands for shorter turnaround times (TATs), lower production costs and robust, reliable methods that can easily adopt new test panels and is able to run rare tests in random access principle. Here we present a novel home-brew laboratory automation platform for diagnostic mutation testing. This platform is based on the cyclic minisequecing (cMS) and two color near-infrared (NIR) detection. Pipetting is automated using Tecan Freedom EVO pipetting robots and all assays are performed in 384-well micro plate format. The automation platform includes a data processing system, controlling all procedures, and automated patient result reporting to the hospital information system. We have found automated cMS a reliable, inexpensive and robust method for nucleic acid testing for a wide variety of diagnostic tests. The platform is currently in clinical use for over 80 mutations or polymorphisms. Additionally to tests performed from blood samples, the system performs also epigenetic test for the methylation of the MGMT gene promoter, and companion diagnostic tests for analysis of KRAS and BRAF gene mutations from formalin fixed and paraffin embedded tumor samples. Automation of genetic test reporting is found reliable and efficient decreasing the work load of academic personnel.

Three-Dimensional Root Phenotyping with a Novel Imaging and Software Platform1[C][W][OA

PubMed Central

Clark, Randy T.; MacCurdy, Robert B.; Jung, Janelle K.; Shaff, Jon E.; McCouch, Susan R.; Aneshansley, Daniel J.; Kochian, Leon V.

2011-01-01

A novel imaging and software platform was developed for the high-throughput phenotyping of three-dimensional root traits during seedling development. To demonstrate the platform’s capacity, plants of two rice (Oryza sativa) genotypes, Azucena and IR64, were grown in a transparent gellan gum system and imaged daily for 10 d. Rotational image sequences consisting of 40 two-dimensional images were captured using an optically corrected digital imaging system. Three-dimensional root reconstructions were generated and analyzed using a custom-designed software, RootReader3D. Using the automated and interactive capabilities of RootReader3D, five rice root types were classified and 27 phenotypic root traits were measured to characterize these two genotypes. Where possible, measurements from the three-dimensional platform were validated and were highly correlated with conventional two-dimensional measurements. When comparing gellan gum-grown plants with those grown under hydroponic and sand culture, significant differences were detected in morphological root traits (P < 0.05). This highly flexible platform provides the capacity to measure root traits with a high degree of spatial and temporal resolution and will facilitate novel investigations into the development of entire root systems or selected components of root systems. In combination with the extensive genetic resources that are now available, this platform will be a powerful resource to further explore the molecular and genetic determinants of root system architecture. PMID:21454799

Ubiquitin promoter-terminator cassette promotes genetically stable expression of the taste-modifying protein miraculin in transgenic lettuce.

PubMed

Hirai, Tadayoshi; Shohael, Abdullah Mohammad; Kim, You-Wang; Yano, Megumu; Ezura, Hiroshi

2011-12-01

Lettuce is a commercially important leafy vegetable that is cultivated worldwide, and it is also a target crop for plant factories. In this study, lettuce was selected as an alternative platform for recombinant miraculin production because of its fast growth, agronomic value, and wide availability. The taste-modifying protein miraculin is a glycoprotein extracted from the red berries of the West African native shrub Richadella dulcifica. Because of its limited natural availability, many attempts have been made to produce this protein in suitable alternative hosts. We produced transgenic lettuce with miraculin gene driven either by the ubiquitin promoter/terminator cassette from lettuce or a 35S promoter/nos terminator cassette. Miraculin gene expression and miraculin accumulation in both cassettes were compared by quantitative real-time PCR analysis, Western blotting, and enzyme-linked immunosorbent assay. The expression level of the miraculin gene and protein in transgenic lettuce was higher and more genetically stable in the ubiquitin promoter/terminator cassette than in the 35S promoter/nos terminator cassette. These results demonstrated that the ubiquitin promoter/terminator cassette is an efficient platform for the genetically stable expression of the miraculin protein in lettuce and hence this platform is of benefit for recombinant miraculin production on a commercial scale.

Multifunctional polymer-capped mesoporous silica nanoparticles for pH-responsive targeted drug delivery.

PubMed

Niedermayer, Stefan; Weiss, Veronika; Herrmann, Annika; Schmidt, Alexandra; Datz, Stefan; Müller, Katharina; Wagner, Ernst; Bein, Thomas; Bräuchle, Christoph

2015-05-07

A highly stable modular platform, based on the sequential covalent attachment of different functionalities to the surface of core-shell mesoporous silica nanoparticles (MSNs) for targeted drug delivery is presented. A reversible pH-responsive cap system based on covalently attached poly(2-vinylpyridine) (PVP) was developed as drug release mechanism. Our platform offers (i) tuneable interactions and release kinetics with the cargo drug in the mesopores based on chemically orthogonal core-shell design, (ii) an extremely robust and reversible closure and release mechanism based on endosomal acidification of the covalently attached PVP polymer block, (iii) high colloidal stability due to a covalently coupled PEG shell, and (iv) the ability to covalently attach a wide variety of dyes, targeting ligands and other functionalities at the outer periphery of the PEG shell. The functionality of the system was demonstrated in several cell studies, showing pH-triggered release in the endosome, light-triggered endosomal escape with an on-board photosensitizer, and efficient folic acid-based cell targeting.

Clustering and Network Analysis of Reverse Phase Protein Array Data.

PubMed

Byron, Adam

2017-01-01

Molecular profiling of proteins and phosphoproteins using a reverse phase protein array (RPPA) platform, with a panel of target-specific antibodies, enables the parallel, quantitative proteomic analysis of many biological samples in a microarray format. Hence, RPPA analysis can generate a high volume of multidimensional data that must be effectively interrogated and interpreted. A range of computational techniques for data mining can be applied to detect and explore data structure and to form functional predictions from large datasets. Here, two approaches for the computational analysis of RPPA data are detailed: the identification of similar patterns of protein expression by hierarchical cluster analysis and the modeling of protein interactions and signaling relationships by network analysis. The protocols use freely available, cross-platform software, are easy to implement, and do not require any programming expertise. Serving as data-driven starting points for further in-depth analysis, validation, and biological experimentation, these and related bioinformatic approaches can accelerate the functional interpretation of RPPA data.

Rapid targeted mutational analysis of human tumours: a clinical platform to guide personalized cancer medicine

PubMed Central

Dias-Santagata, Dora; Akhavanfard, Sara; David, Serena S; Vernovsky, Kathy; Kuhlmann, Georgiana; Boisvert, Susan L; Stubbs, Hannah; McDermott, Ultan; Settleman, Jeffrey; Kwak, Eunice L; Clark, Jeffrey W; Isakoff, Steven J; Sequist, Lecia V; Engelman, Jeffrey A; Lynch, Thomas J; Haber, Daniel A; Louis, David N; Ellisen, Leif W; Borger, Darrell R; Iafrate, A John

2010-01-01

Targeted cancer therapy requires the rapid and accurate identification of genetic abnormalities predictive of therapeutic response. We sought to develop a high-throughput genotyping platform that would allow prospective patient selection to the best available therapies, and that could readily and inexpensively be adopted by most clinical laboratories. We developed a highly sensitive multiplexed clinical assay that performs very well with nucleic acid derived from formalin fixation and paraffin embedding (FFPE) tissue, and tests for 120 previously described mutations in 13 cancer genes. Genetic profiling of 250 primary tumours was consistent with the documented oncogene mutational spectrum and identified rare events in some cancer types. The assay is currently being used for clinical testing of tumour samples and contributing to cancer patient management. This work therefore establishes a platform for real-time targeted genotyping that can be widely adopted. We expect that efforts like this one will play an increasingly important role in cancer management. PMID:20432502

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.

The Generation Challenge Programme Platform: Semantic Standards and Workbench for Crop Science

PubMed Central

Bruskiewich, Richard; Senger, Martin; Davenport, Guy; Ruiz, Manuel; Rouard, Mathieu; Hazekamp, Tom; Takeya, Masaru; Doi, Koji; Satoh, Kouji; Costa, Marcos; Simon, Reinhard; Balaji, Jayashree; Akintunde, Akinnola; Mauleon, Ramil; Wanchana, Samart; Shah, Trushar; Anacleto, Mylah; Portugal, Arllet; Ulat, Victor Jun; Thongjuea, Supat; Braak, Kyle; Ritter, Sebastian; Dereeper, Alexis; Skofic, Milko; Rojas, Edwin; Martins, Natalia; Pappas, Georgios; Alamban, Ryan; Almodiel, Roque; Barboza, Lord Hendrix; Detras, Jeffrey; Manansala, Kevin; Mendoza, Michael Jonathan; Morales, Jeffrey; Peralta, Barry; Valerio, Rowena; Zhang, Yi; Gregorio, Sergio; Hermocilla, Joseph; Echavez, Michael; Yap, Jan Michael; Farmer, Andrew; Schiltz, Gary; Lee, Jennifer; Casstevens, Terry; Jaiswal, Pankaj; Meintjes, Ayton; Wilkinson, Mark; Good, Benjamin; Wagner, James; Morris, Jane; Marshall, David; Collins, Anthony; Kikuchi, Shoshi; Metz, Thomas; McLaren, Graham; van Hintum, Theo

2008-01-01

The Generation Challenge programme (GCP) is a global crop research consortium directed toward crop improvement through the application of comparative biology and genetic resources characterization to plant breeding. A key consortium research activity is the development of a GCP crop bioinformatics platform to support GCP research. This platform includes the following: (i) shared, public platform-independent domain models, ontology, and data formats to enable interoperability of data and analysis flows within the platform; (ii) web service and registry technologies to identify, share, and integrate information across diverse, globally dispersed data sources, as well as to access high-performance computational (HPC) facilities for computationally intensive, high-throughput analyses of project data; (iii) platform-specific middleware reference implementations of the domain model integrating a suite of public (largely open-access/-source) databases and software tools into a workbench to facilitate biodiversity analysis, comparative analysis of crop genomic data, and plant breeding decision making. PMID:18483570

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.

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

Kinetic limitations of the Mg(2)Si system for reversible hydrogen storage.

PubMed

Kelly, Stephen T; Van Atta, Sky L; Vajo, John J; Olson, Gregory L; Clemens, B M

2009-05-20

Despite the promising thermodynamics and storage capacities of many destabilized metal hydride hydrogen storage material systems, they are often kinetically limited from achieving practical and reversible behavior. Such is the case with the Mg2Si system. We investigated the kinetic mechanisms responsible for limiting the reversibility of the MgH2+Si system using thin films as a controlled research platform. We observed that the reaction MgH2 + 1/2Mg2Si + H2 is limited by the mass transport of Mg and Si into separate phases. Hydrogen readily diffuses through the Mg2Si material and nucleating MgH2 phase growth does not result in reaction completion. By depositing and characterizing multilayer films of Mg2Si and Mg with varying Mg2Si layer thicknesses, we conclude that the hydrogenation reaction consumes no more than 1 nm of Mg2Si, making this system impractical for reversible hydrogen storage.

Tools to Analyze Morphology and Spatially Mapped Molecular Data | Informatics Technology for Cancer Research (ITCR)

Cancer.gov

This project is to develop, deploy, and disseminate a suite of open source tools and integrated informatics platform that will facilitate multi-scale, correlative analyses of high resolution whole slide tissue image data, spatially mapped genetics and molecular data for cancer research. This platform will play an essential role in supporting studies of tumor initiation, development, heterogeneity, invasion, and metastasis.

Assembly/disassembly of a complex icosahedral virus to incorporate heterologous nucleic acids

NASA Astrophysics Data System (ADS)

Pascual, Elena; Mata, Carlos P.; Carrascosa, José L.; Castón, José R.

2017-12-01

Hollow protein containers are widespread in nature, and include virus capsids as well as eukaryotic and bacterial complexes. Protein cages are studied extensively for applications in nanotechnology, nanomedicine and materials science. Their inner and outer surfaces can be modified chemically or genetically, and the internal cavity can be used to template, store and/or arrange molecular cargos. Virus capsids and virus-like particles (VLP, noninfectious particles) provide versatile platforms for nanoscale bioengineering. Study of capsid protein self-assembly into monodispersed particles, and of VLP structure and biophysics is necessary not only to understand natural processes, but also to infer how these platforms can be redesigned to furnish novel functional VLP. Here we address the assembly dynamics of infectious bursal disease virus (IBDV), a complex icosahedral virus. IBDV has a ~70 nm-diameter T  =  13 capsid with VP2 trimers as the only structural subunits. During capsid assembly, VP2 is synthesized as a precursor (pVP2) whose C terminus is cleaved. The pVP2 C terminus has an amphipathic helix that controls VP2 polymorphism. In the absence of the VP3 scaffolding protein, necessary for control of assembly, 466/456-residue pVP2 intermediates bearing this helix assemble into VLP only when expressed with an N-terminal His6 tag (the HT-VP2-466 protein). HT-VP2-466 capsids are optimal for genetic insertion of proteins (cargo space ~78 000 nm3). We established an in vitro assembly/disassembly system of HT-VP2-466-based VLP for heterologous nucleic acid packaging and/or encapsulation of drugs and other molecules. HT-VP2-466 (empty) capsids were disassembled and reassembled by dialysis against low-salt/basic pH and high-salt/acid pH buffers, respectively, thus illustrating the reversibility in vitro of IBDV capsid assembly. HT-VP2-466 VLP also packed heterologous DNA by non-specific confinement during assembly. These and previous results establish the bases for biotechnological applications based on the IBDV capsid and its ability to incorporate exogenous proteins and nucleic acids.

Ligand interaction scan: a general method for engineering ligand-sensitive protein alleles.

PubMed

Erster, Oran; Eisenstein, Miriam; Liscovitch, Mordechai

2007-05-01

The ligand interaction scan (LIScan) method is a general procedure for engineering small molecule ligand-regulated forms of a protein that is complementary to other 'reverse' genetic and chemical-genetic methods for drug-target validation. It involves insertional mutagenesis by a chemical-genetic 'switch', comprising a genetically encoded peptide module that binds with high affinity to a small-molecule ligand. We demonstrated the method with TEM-1 beta-lactamase, using a tetracysteine hexapeptide insert and a biarsenical fluorescein ligand (FlAsH).

Development of a dynamic traffic assignment model to evaluate lane-reversal plans for I-65.

DOT National Transportation Integrated Search

2010-05-01

This report presents the methodology and results from a project that studied contra-flow operations in support of : hurricane evacuations in the state of Alabama. As part of this effort, a simulation model was developed using the : VISTA platform for...

Self-digitization microfluidic chip for absolute quantification of mRNA in single cells.

PubMed

Thompson, Alison M; Gansen, Alexander; Paguirigan, Amy L; Kreutz, Jason E; Radich, Jerald P; Chiu, Daniel T

2014-12-16

Quantification of mRNA in single cells provides direct insight into how intercellular heterogeneity plays a role in disease progression and outcomes. Quantitative polymerase chain reaction (qPCR), the current gold standard for evaluating gene expression, is insufficient for providing absolute measurement of single-cell mRNA transcript abundance. Challenges include difficulties in handling small sample volumes and the high variability in measurements. Microfluidic digital PCR provides far better sensitivity for minute quantities of genetic material, but the typical format of this assay does not allow for counting of the absolute number of mRNA transcripts samples taken from single cells. Furthermore, a large fraction of the sample is often lost during sample handling in microfluidic digital PCR. Here, we report the absolute quantification of single-cell mRNA transcripts by digital, one-step reverse transcription PCR in a simple microfluidic array device called the self-digitization (SD) chip. By performing the reverse transcription step in digitized volumes, we find that the assay exhibits a linear signal across a wide range of total RNA concentrations and agrees well with standard curve qPCR. The SD chip is found to digitize a high percentage (86.7%) of the sample for single-cell experiments. Moreover, quantification of transferrin receptor mRNA in single cells agrees well with single-molecule fluorescence in situ hybridization experiments. The SD platform for absolute quantification of single-cell mRNA can be optimized for other genes and may be useful as an independent control method for the validation of mRNA quantification techniques.

Desktop Genetics.

PubMed

Hough, Soren H; Ajetunmobi, Ayokunmi; Brody, Leigh; Humphryes-Kirilov, Neil; Perello, Edward

2016-11-01

Desktop Genetics is a bioinformatics company building a gene-editing platform for personalized medicine. The company works with scientists around the world to design and execute state-of-the-art clustered regularly interspaced short palindromic repeats (CRISPR) experiments. Desktop Genetics feeds the lessons learned about experimental intent, single-guide RNA design and data from international genomics projects into a novel CRISPR artificial intelligence system. We believe that machine learning techniques can transform this information into a cognitive therapeutic development tool that will revolutionize medicine.

Single-Cell Quantitative PCR: Advances and Potential in Cancer Diagnostics.

PubMed

Ok, Chi Young; Singh, Rajesh R; Salim, Alaa A

2016-01-01

Tissues are heterogeneous in their components. If cells of interest are a minor population of collected tissue, it would be difficult to obtain genetic or genomic information of the interested cell population with conventional genomic DNA extraction from the collected tissue. Single-cell DNA analysis is important in the analysis of genetics of cell clonality, genetic anticipation, and single-cell DNA polymorphisms. Single-cell PCR using Single Cell Ampligrid/GeXP platform is described in this chapter.

Ricebase: a breeding and genetics platform for rice, integrating individual molecular markers, pedigrees, and whole-genome-based data

USDA-ARS?s Scientific Manuscript database

Ricebase (http://ricebase.org) is an integrative genomic database for rice (Oryza sativa) with an emphasis on combining data sets in a way that maintains the key links between past and current genetic studies. Ricebase includes DNA sequence data, gene annotations, nucleotide variation data, and mol...

Development of DArT marker platforms and genetic diversity assessment of the U.S. collection of new oilseed crop lesquerella and relative species

USDA-ARS?s Scientific Manuscript database

The advantages of using molecular markers in modern genebanks are well documented. They are commonly used to understand the distribution of genetic diversity in populations and among species which is crucial for efficient management and effective utilization of the collections. We describe the devel...

Development of a 690K SNP array in catfish and its application for genetic mapping and validation of the reference genome sequence

USDA-ARS?s Scientific Manuscript database

Single nucleotide polymorphisms (SNPs) are capable of providing the highest level of genome coverage for genomic and genetic analysis because of their abundance and relatively even distribution in the genome. Such a capacity, however, cannot be achieved without an efficient genotyping platform such ...

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.

Recent advances in the molecular genetics of epilepsy.

PubMed

Hildebrand, Michael S; Dahl, Hans-Henrik M; Damiano, John Anthony; Smith, Richard J H; Scheffer, Ingrid E; Berkovic, Samuel F

2013-05-01

Recent advances in molecular genetics have translated into the increasing utilisation of genetic testing in the routine clinical practice of neurologists. There has been a steady, incremental increase in understanding the genetic variation associated with epilepsies. Genetic testing in the epilepsies is not yet widely practiced, but the advent of new screening technologies promises to exponentially expand both knowledge and clinical utility. To maximise the value of this new genetic insight we need to rapidly extrapolate genetic findings to inform patients of their diagnosis, prognosis, recurrence risk and the clinical management options available for their specific genetic condition. Comprehensive, highly specific and sensitive genetic test results improve the management of patients by neurologists and clinical geneticists. Here we discuss the latest developments in clinical genetic testing for epilepsy and describe new molecular genetics platforms that will transform both genetic screening and novel gene discovery.

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).

Avoiding pitfalls in molecular genetic testing: case studies of high-resolution array comparative genomic hybridization testing in the definitive diagnosis of Mowat-Wilson syndrome.

PubMed

Kluk, Michael Joseph; An, Yu; James, Philip; Coulter, David; Harris, David; Wu, Bai-Lin; Shen, Yiping

2011-05-01

The molecular testing options available for the diagnosis of genetic disorders are numerous and include a variety of different assay platforms. The consultative input of molecular pathologists and cytogeneticists, working closely with the ordering clinicians, is often important for definitive diagnosis. Herein, we describe two patients who had long histories of unexplained signs and symptoms with a high clinical suspicion of an underlying genetic etiology. Initial molecular testing in both cases was negative, but the application of high-resolution array comparative genomic hybridization technology lead to definitive diagnosis in both cases. We summarize the clinical findings and molecular testing in each case, discuss the differential diagnoses, and review the clinical and pathological findings of Mowat-Wilson syndrome. This report highlights the importance for those involved in molecular testing to know the nature of the underlying genetic abnormalities associated with the suspected diagnosis, to recognize the limitations of each testing platform, and to persistently pursue repeat testing using high-resolution technologies when indicated. This concept is applicable to both germline and somatic molecular genetic testing. Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Analysis of human plasma metabolites across different liquid chromatography/mass spectrometry platforms: Cross-platform transferable chemical signatures.

PubMed

Telu, Kelly H; Yan, Xinjian; Wallace, William E; Stein, Stephen E; Simón-Manso, Yamil

2016-03-15

The metabolite profiling of a NIST plasma Standard Reference Material (SRM 1950) on different liquid chromatography/mass spectrometry (LC/MS) platforms showed significant differences. Although these findings suggest caution when interpreting metabolomics results, the degree of overlap of both profiles allowed us to use tandem mass spectral libraries of recurrent spectra to evaluate to what extent these results are transferable across platforms and to develop cross-platform chemical signatures. Non-targeted global metabolite profiles of SRM 1950 were obtained on different LC/MS platforms using reversed-phase chromatography and different chromatographic scales (conventional HPLC, UHPLC and nanoLC). The data processing and the metabolite differential analysis were carried out using publically available (XCMS), proprietary (Mass Profiler Professional) and in-house software (NIST pipeline). Repeatability and intermediate precision showed that the non-targeted SRM 1950 profiling was highly reproducible when working on the same platform (relative standard deviation (RSD) <2%); however, substantial differences were found in the LC/MS patterns originating on different platforms or even using different chromatographic scales (conventional HPLC, UHPLC and nanoLC) on the same platform. A substantial degree of overlap (common molecular features) was also found. A procedure to generate consistent chemical signatures using tandem mass spectral libraries of recurrent spectra is proposed. Different platforms rendered significantly different metabolite profiles, but the results were highly reproducible when working within one platform. Tandem mass spectral libraries of recurrent spectra are proposed to evaluate the degree of transferability of chemical signatures generated on different platforms. Chemical signatures based on our procedure are most likely cross-platform transferable. Published in 2016. This article is a U.S. Government work and is in the public domain in the USA.

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

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.

Droplet based microfluidics for highthroughput screening of antibody secreting cells

NASA Astrophysics Data System (ADS)

Cai, Liheng; Heyman, John; Mazutis, Linas; Ung, Lloyd; Guerra, Rodrigo; Aubrecht, Donald; Weitz, David

2014-03-01

We present a droplet based microfluidic platform that allows highthroughput screening of antibody secreting cells. We coencapsulate single cells, fluorescent probes, and detection beads into emulsion droplets with diameter of 40 micron. The beads capture antibodies secreted by cells, resulting in a pronounced fluorescent signal that activates dielectrophoresis sorting at rate about 500 droplets per second. Moreover, we demonstrate that Reverse Transcription Polymerase Chain Reaction (RT-PCR) can be successfully applied to the cell encapsulated in a single sorted droplet. Our work highlights the potential of droplet based microfluidics as a platform to generate recombinant antibodies.

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

How Surrogate and Chemical Genetics in Model Organisms Can Suggest Therapies for Human Genetic Diseases.

PubMed

Strynatka, Katherine A; Gurrola-Gal, Michelle C; Berman, Jason N; McMaster, Christopher R

2018-03-01

Genetic diseases are both inherited and acquired. Many genetic diseases fall under the paradigm of orphan diseases, a disease found in < 1 in 2000 persons. With rapid and cost-effective genome sequencing becoming the norm, many causal mutations for genetic diseases are being rapidly determined. In this regard, model organisms are playing an important role in validating if specific mutations identified in patients drive the observed phenotype. An emerging challenge for model organism researchers is the application of genetic and chemical genetic platforms to discover drug targets and drugs/drug-like molecules for potential treatment options for patients with genetic disease. This review provides an overview of how model organisms have contributed to our understanding of genetic disease, with a focus on the roles of yeast and zebrafish in gene discovery and the identification of compounds that could potentially treat human genetic diseases. Copyright © 2018 by the Genetics Society of America.

Refactored M13 Bacteriophage as a Platform for Tumor Cell Imaging and Drug Delivery

PubMed Central

MOSER, FELIX; ENDY, DREW; BELCHER, ANGELA M.

2014-01-01

M13 bacteriophage is a well-characterized platform for peptide display. The utility of the M13 display platform is derived from the ability to encode phage protein fusions with display peptides at the genomic level. However, the genome of the phage is complicated by overlaps of key genetic elements. These overlaps directly couple the coding sequence of one gene to the coding or regulatory sequence of another, making it difficult to alter one gene without disrupting the other. Specifically, overlap of the end of gene VII and the beginning of gene IX has prevented the functional genomic modification of the N-terminus of p9. By redesigning the M13 genome to physically separate these overlapping genetic elements, a process known as “refactoring,” we enabled independent manipulation of gene VII and gene IX and the construction of the first N-terminal genomic modification of p9 for peptide display. We demonstrate the utility of this refactored genome by developing an M13 bacteriophage-based platform for targeted imaging of and drug delivery to prostate cancer cells in vitro. This successful use of refactoring principles to reengineer a natural biological system strengthens the suggestion that natural genomes can be rationally designed for a number of applications. PMID:23656279

Refactored M13 bacteriophage as a platform for tumor cell imaging and drug delivery.

PubMed

Ghosh, Debadyuti; Kohli, Aditya G; Moser, Felix; Endy, Drew; Belcher, Angela M

2012-12-21

M13 bacteriophage is a well-characterized platform for peptide display. The utility of the M13 display platform is derived from the ability to encode phage protein fusions with display peptides at the genomic level. However, the genome of the phage is complicated by overlaps of key genetic elements. These overlaps directly couple the coding sequence of one gene to the coding or regulatory sequence of another, making it difficult to alter one gene without disrupting the other. Specifically, overlap of the end of gene VII and the beginning of gene IX has prevented the functional genomic modification of the N-terminus of p9. By redesigning the M13 genome to physically separate these overlapping genetic elements, a process known as "refactoring," we enabled independent manipulation of gene VII and gene IX and the construction of the first N-terminal genomic modification of p9 for peptide display. We demonstrate the utility of this refactored genome by developing an M13 bacteriophage-based platform for targeted imaging of and drug delivery to prostate cancer cells in vitro. This successful use of refactoring principles to re-engineer a natural biological system strengthens the suggestion that natural genomes can be rationally designed for a number of applications.

Autonomous self-organizing resource manager for multiple networked platforms

NASA Astrophysics Data System (ADS)

Smith, James F., III

2002-08-01

A fuzzy logic based expert system for resource management has been developed that automatically allocates electronic attack (EA) resources in real-time over many dissimilar autonomous naval platforms defending their group against attackers. The platforms can be very general, e.g., ships, planes, robots, land based facilities, etc. Potential foes the platforms deal with can also be general. This paper provides an overview of the resource manager including the four fuzzy decision trees that make up the resource manager; the fuzzy EA model; genetic algorithm based optimization; co-evolutionary data mining through gaming; and mathematical, computational and hardware based validation. Methods of automatically designing new multi-platform EA techniques are considered. The expert system runs on each defending platform rendering it an autonomous system requiring no human intervention. There is no commanding platform. Instead the platforms work cooperatively as a function of battlespace geometry; sensor data such as range, bearing, ID, uncertainty measures for sensor output; intelligence reports; etc. Computational experiments will show the defending networked platform's ability to self- organize. The platforms' ability to self-organize is illustrated through the output of the scenario generator, a software package that automates the underlying data mining problem and creates a computer movie of the platforms' interaction for evaluation.

Investigation of left and right lateral fluid percussion injury in C57BL6/J mice: In vivo functional consequences

PubMed Central

Schurman, Lesley D.; Smith, Terry L.; Morales, Anthony J.; Lee, Nancy N.; Reeves, Thomas M.; Phillips, Linda L.; Lichtman, Aron H.

2017-01-01

Although rodent models of traumatic brain injury (TBI) reliably produce cognitive and motor disturbances, behavioral characterization resulting from left and right hemisphere injuries remains unexplored. Here we examined the functional consequences of targeting the left versus right parietal cortex in lateral fluid percussion injury, on Morris water maze (MWM) spatial memory tasks (fixed platform and reversal) and neurological motor deficits (neurological severity score and rotarod). In the MWM fixed platform task, right lateral injury produced a small delay in acquisition rate compared to left. However, injury to either hemisphere resulted in probe trial deficits. In the MWM reversal task, left-right performance deficits were not evident, though left lateral injury produced mild acquisition and probe trial deficits compared to sham controls. Additionally, left and right injury produced similar neurological motor task deficits, impaired righting times, and lesion volumes. Injury to either hemisphere also produced robust ipsilateral, and modest contralateral, morphological changes in reactive microglia and astrocytes. In conclusion, left and right lateral TBI impaired MWM performance, with mild fixed platform acquisition rate differences, despite similar motor deficits, histological damage, and glial cell reactivity. Thus, while both left and right lateral TBI produce cognitive deficits, laterality in mouse MWM learning and memory merits consideration in the investigation of TBI-induced cognitive consequences. PMID:28527714

Investigation of left and right lateral fluid percussion injury in C57BL6/J mice: In vivo functional consequences.

PubMed

Schurman, Lesley D; Smith, Terry L; Morales, Anthony J; Lee, Nancy N; Reeves, Thomas M; Phillips, Linda L; Lichtman, Aron H

2017-07-13

Although rodent models of traumatic brain injury (TBI) reliably produce cognitive and motor disturbances, behavioral characterization resulting from left and right hemisphere injuries remains unexplored. Here we examined the functional consequences of targeting the left versus right parietal cortex in lateral fluid percussion injury, on Morris water maze (MWM) spatial memory tasks (fixed platform and reversal) and neurological motor deficits (neurological severity score and rotarod). In the MWM fixed platform task, right lateral injury produced a small delay in acquisition rate compared to left. However, injury to either hemisphere resulted in probe trial deficits. In the MWM reversal task, left-right performance deficits were not evident, though left lateral injury produced mild acquisition and probe trial deficits compared to sham controls. Additionally, left and right injury produced similar neurological motor task deficits, impaired righting times, and lesion volumes. Injury to either hemisphere also produced robust ipsilateral, and modest contralateral, morphological changes in reactive microglia and astrocytes. In conclusion, left and right lateral TBI impaired MWM performance, with mild fixed platform acquisition rate differences, despite similar motor deficits, histological damage, and glial cell reactivity. Thus, while both left and right lateral TBI produce cognitive deficits, laterality in mouse MWM learning and memory merits consideration in the investigation of TBI-induced cognitive consequences. Copyright © 2017. Published by Elsevier B.V.

Killing cancer cells by targeted drug-carrying phage nanomedicines

PubMed Central

Bar, Hagit; Yacoby, Iftach; Benhar, Itai

2008-01-01

Background Systemic administration of chemotherapeutic agents, in addition to its anti-tumor benefits, results in indiscriminate drug distribution and severe toxicity. This shortcoming may be overcome by targeted drug-carrying platforms that ferry the drug to the tumor site while limiting exposure to non-target tissues and organs. Results We present a new form of targeted anti-cancer therapy in the form of targeted drug-carrying phage nanoparticles. Our approach is based on genetically-modified and chemically manipulated filamentous bacteriophages. The genetic manipulation endows the phages with the ability to display a host-specificity-conferring ligand. The phages are loaded with a large payload of a cytotoxic drug by chemical conjugation. In the presented examples we used anti ErbB2 and anti ERGR antibodies as targeting moieties, the drug hygromycin conjugated to the phages by a covalent amide bond, or the drug doxorubicin conjugated to genetically-engineered cathepsin-B sites on the phage coat. We show that targeting of phage nanomedicines via specific antibodies to receptors on cancer cell membranes results in endocytosis, intracellular degradation, and drug release, resulting in growth inhibition of the target cells in vitro with a potentiation factor of >1000 over the corresponding free drugs. Conclusion The results of the proof-of concept study presented here reveal important features regarding the potential of filamentous phages to serve as drug-delivery platform, on the affect of drug solubility or hydrophobicity on the target specificity of the platform and on the effect of drug release mechanism on the potency of the platform. These results define targeted drug-carrying filamentous phage nanoparticles as a unique type of antibody-drug conjugates. PMID:18387177

Conversion to Reverse Total Shoulder Arthroplasty with and without Humeral Stem Retention: The Role of a Convertible-Platform Stem.

PubMed

Crosby, Lynn A; Wright, Thomas W; Yu, Stephen; Zuckerman, Joseph D

2017-05-03

Revision shoulder arthroplasty is a technically challenging procedure. It is associated with increased blood loss and operative time, and it frequently necessitates revision implants, augments, and bone-grafting. Shoulder arthroplasty systems with a convertible-platform humeral stem have been developed to reduce the complexity of revision procedures by eliminating the need for humeral component explantation when converting from anatomic shoulder arthroplasty (hemiarthroplasty or total shoulder arthroplasty) to reverse total shoulder arthroplasty (rTSA). A multicenter, retrospective analysis involving 102 consecutive shoulders (102 patients) that underwent revision of an anatomic shoulder arthroplasty to an rTSA was conducted. During the revision, 73 of the shoulders needed exchange of the humeral stem (the exchange group) and 29 had retention of a convertible-platform humeral component (the retention group). Patient demographics, operative time, blood management, range of motion, complications, and patient-reported outcomes were compared between the 2 groups. Patients with retention had significantly shorter operative time (mean and standard deviation, 130 ± 48 versus 195 ± 58 minutes; p < 0.001) and lower estimated blood loss (292 ± 118 versus 492 ± 334 mL; p = 0.034). The rate of intraoperative complications was lower in the retention group (0% versus 15%; p = 0.027). Patients with retention had slightly better postoperative range of motion (active external rotation, 26° ± 23° versus 11° ± 23° [p = 0.006]; active forward elevation, 112° ± 37° versus 96° ± 33° [p = 0.055]). Shoulder arthroplasty systems that utilize a convertible-platform humeral stem offer an advantage for rTSA conversion in that a well-fixed, well-positioned humeral stem can be retained. There were significantly fewer complications as well as significantly decreased blood loss and operative time when a convertible-platform stem was utilized (p < 0.050). Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

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 Tool Development for a New Host for Biotechnology, the Thermotolerant Bacterium Bacillus coagulans▿ †

PubMed Central

Kovács, Ákos T.; van Hartskamp, Mariska; Kuipers, Oscar P.; van Kranenburg, Richard

2010-01-01

Bacillus coagulans has good potential as an industrial production organism for platform chemicals from renewable resources but has limited genetic tools available. Here, we present a targeted gene disruption system using the Cre-lox system, development of a LacZ reporter assay for monitoring gene transcription, and heterologous d-lactate dehydrogenase expression. PMID:20400555

Reverse genetics of measles virus and resulting multivalent recombinant vaccines: applications of recombinant measles viruses.

PubMed

Billeter, M A; Naim, H Y; Udem, S A

2009-01-01

An overview is given on the development of technologies to allow reverse genetics of RNA viruses, i.e., the rescue of viruses from cDNA, with emphasis on nonsegmented negative-strand RNA viruses (Mononegavirales), as exemplified for measles virus (MV). Primarily, these technologies allowed site-directed mutagenesis, enabling important insights into a variety of aspects of the biology of these viruses. Concomitantly, foreign coding sequences were inserted to (a) allow localization of virus replication in vivo through marker gene expression, (b) develop candidate multivalent vaccines against measles and other pathogens, and (c) create candidate oncolytic viruses. The vector use of these viruses was experimentally encouraged by the pronounced genetic stability of the recombinants unexpected for RNA viruses, and by the high load of insertable genetic material, in excess of 6 kb. The known assets, such as the small genome size of the vector in comparison to DNA viruses proposed as vectors, the extensive clinical experience of attenuated MV as vaccine with a proven record of high safety and efficacy, and the low production cost per vaccination dose are thus favorably complemented.

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

How Surrogate and Chemical Genetics in Model Organisms Can Suggest Therapies for Human Genetic Diseases

PubMed Central

Strynatka, Katherine A.; Gurrola-Gal, Michelle C.; Berman, Jason N.; McMaster, Christopher R.

2018-01-01

Genetic diseases are both inherited and acquired. Many genetic diseases fall under the paradigm of orphan diseases, a disease found in < 1 in 2000 persons. With rapid and cost-effective genome sequencing becoming the norm, many causal mutations for genetic diseases are being rapidly determined. In this regard, model organisms are playing an important role in validating if specific mutations identified in patients drive the observed phenotype. An emerging challenge for model organism researchers is the application of genetic and chemical genetic platforms to discover drug targets and drugs/drug-like molecules for potential treatment options for patients with genetic disease. This review provides an overview of how model organisms have contributed to our understanding of genetic disease, with a focus on the roles of yeast and zebrafish in gene discovery and the identification of compounds that could potentially treat human genetic diseases. PMID:29487144

A Food Chain Algorithm for Capacitated Vehicle Routing Problem with Recycling in Reverse Logistics

NASA Astrophysics Data System (ADS)

Song, Qiang; Gao, Xuexia; Santos, Emmanuel T.

2015-12-01

This paper introduces the capacitated vehicle routing problem with recycling in reverse logistics, and designs a food chain algorithm for it. Some illustrative examples are selected to conduct simulation and comparison. Numerical results show that the performance of the food chain algorithm is better than the genetic algorithm, particle swarm optimization as well as quantum evolutionary algorithm.

Chemical screening platforms for autophagy drug discovery to identify therapeutic candidates for Huntington's disease and other neurodegenerative disorders.

PubMed

Sarkar, Sovan

2013-01-01

Autophagy is a cellular degradation process involved in the clearance of aggregate-prone proteins associated with neurodegenerative diseases. While the mTOR pathway has been known to be the major regulator of autophagy, recent advancements into the regulation of autophagy have identified mTOR-independent autophagy pathways that are amenable to chemical perturbations. Several chemical and genetic screens have been undertaken to identify small molecule and genetic regulators of autophagy, respectively. The small molecule autophagy enhancers offer great potential as therapeutic candidates not only for neurodegenerative diseases, but also for diverse human diseases where autophagy acts as a protective pathway. This review highlights the various chemical screening platforms for autophagy drug discovery pertinent for the treatment of neurodegenerative diseases.

A Neuron-Based Screening Platform for Optimizing Genetically-Encoded Calcium Indicators

PubMed Central

Schreiter, Eric R.; Hasseman, Jeremy P.; Tsegaye, Getahun; Fosque, Benjamin F.; Behnam, Reza; Shields, Brenda C.; Ramirez, Melissa; Kimmel, Bruce E.; Kerr, Rex A.; Jayaraman, Vivek; Looger, Loren L.; Svoboda, Karel; Kim, Douglas S.

2013-01-01

Fluorescent protein-based sensors for detecting neuronal activity have been developed largely based on non-neuronal screening systems. However, the dynamics of neuronal state variables (e.g., voltage, calcium, etc.) are typically very rapid compared to those of non-excitable cells. We developed an electrical stimulation and fluorescence imaging platform based on dissociated rat primary neuronal cultures. We describe its use in testing genetically-encoded calcium indicators (GECIs). Efficient neuronal GECI expression was achieved using lentiviruses containing a neuronal-selective gene promoter. Action potentials (APs) and thus neuronal calcium levels were quantitatively controlled by electrical field stimulation, and fluorescence images were recorded. Images were segmented to extract fluorescence signals corresponding to individual GECI-expressing neurons, which improved sensitivity over full-field measurements. We demonstrate the superiority of screening GECIs in neurons compared with solution measurements. Neuronal screening was useful for efficient identification of variants with both improved response kinetics and high signal amplitudes. This platform can be used to screen many types of sensors with cellular resolution under realistic conditions where neuronal state variables are in relevant ranges with respect to timing and amplitude. PMID:24155972

A reverse genetics system for enterovirus D68 using human RNA polymerase I.

PubMed

Pan, Minglei; Gao, Shuai; Zhou, Zhenwei; Zhang, Keke; Liu, Sihua; Wang, Zhiyun; Wang, Tao

2018-05-17

Human enterovirus D68 (EV-D68) is a highly contagious virus, which causes respiratory tract infections. However, no effective vaccines are currently available for controlling EV-D68 infection. Here, we developed a reverse genetics system to recover EV-D68 minireplicons and infectious EV-D68 from transfected plasmids using the RNA polymerase I (Pol I) promoter. The EV-D68 minireplicons contained the luciferase reporter gene, which flanked by the non-coding regions of the EV-D68 RNA. The luciferase signals could be detected in cells after transfection and Pol I promoter-mediated luciferase signal was significantly stronger than that mediated by the T7 promoter. Furthermore, recombinant viruses were generated by transfecting plasmids that contained the genomic RNA segments of EV-D68, under the control of Pol I promoter into 293T cells or RD cells. On plaque morphology and growth kinetics, the rescued virus and parental virus were indistinguishable. In addition, we showed that the G394C mutation disrupts the viral 5'-UTR structure and suppresses the viral cap-independent translation. This reverse genetics system for EV-D68 recovery can greatly facilitate research into EV-D68 biology. Moreover, this system could accelerate the development of EV-D68 vaccines and anti-EV-D68 drugs.

Arenavirus reverse genetics for vaccine development

PubMed Central

Ortiz-Riaño, Emilio; Cheng, Benson Yee Hin; Carlos de la Torre, Juan

2013-01-01

Arenaviruses are important human pathogens with no Food and Drug Administration (FDA)-licensed vaccines available and current antiviral therapy being limited to an off-label use of the nucleoside analogue ribavirin of limited prophylactic efficacy. The development of reverse genetics systems represented a major breakthrough in arenavirus research. However, rescue of recombinant arenaviruses using current reverse genetics systems has been restricted to rodent cells. In this study, we describe the rescue of recombinant arenaviruses from human 293T cells and Vero cells, an FDA-approved line for vaccine development. We also describe the generation of novel vectors that mediate synthesis of both negative-sense genome RNA and positive-sense mRNA species of lymphocytic choriomeningitis virus (LCMV) directed by the human RNA polymerases I and II, respectively, within the same plasmid. This approach reduces by half the number of vectors required for arenavirus rescue, which could facilitate virus rescue in cell lines approved for human vaccine production but that cannot be transfected at high efficiencies. We have shown the feasibility of this approach by rescuing both the Old World prototypic arenavirus LCMV and the live-attenuated vaccine Candid#1 strain of the New World arenavirus Junín. Moreover, we show the feasibility of using these novel strategies for efficient rescue of recombinant tri-segmented both LCMV and Candid#1. PMID:23364194

MINIGENOMES, TRANSCRIPTION AND REPLICATION COMPETENT VIRUS-LIKE PARTICLES AND BEYOND: REVERSE GENETICS SYSTEMS FOR FILOVIRUSES AND OTHER NEGATIVE STRANDED HEMORRHAGIC FEVER VIRUSES

PubMed Central

Hoenen, Thomas; Groseth, Allison; de Kok-Mercado, Fabian; Kuhn, Jens H.; Wahl-Jensen, Victoria

2012-01-01

Reverse-genetics systems are powerful tools enabling researchers to study the replication cycle of RNA viruses, including filoviruses and other hemorrhagic fever viruses, as well as to discover new antivirals. They include full-length clone systems as well as a number of life cycle modeling systems. Full-length clone systems allow for the generation of infectious, recombinant viruses, and thus are an important tool for studying the virus replication cycle in its entirety. In contrast, life cycle modeling systems such as minigenome and transcription and replication competent virus-like particle systems can be used to simulate and dissect parts of the virus life cycle outside of containment facilities. Minigenome systems are used to model viral genome replication and transcription, whereas transcription and replication competent virus-like particle systems also model morphogenesis and budding as well as infection of target cells. As such, these modeling systems have tremendous potential to further the discovery and screening of new antivirals targeting hemorrhagic fever viruses. This review provides an overview of currently established reverse genetics systems for hemorrhagic fever-causing negative-sense RNA viruses, with a particular emphasis on filoviruses, and the potential application of these systems for antiviral research. PMID:21699921

Novel droplet platforms for the detection of disease biomarkers.

PubMed

Zec, Helena; Shin, Dong Jin; Wang, Tza-Huei

2014-09-01

Personalized medicine - healthcare based on individual genetic variation - has the potential to transform the way healthcare is delivered to patients. The promise of personalized medicine has been predicated on the predictive and diagnostic power of genomic and proteomic biomarkers. Biomarker screening may help improve health outcomes, for example, by identifying individuals' susceptibility to diseases and predicting how patients will respond to drugs. Microfluidic droplet technology offers an exciting opportunity to revolutionize the accessibility of personalized medicine. A framework for the role of droplet microfluidics in biomarker detection can be based on two main themes. Emulsion-based microdroplet platforms can provide new ways to measure and detect biomolecules. In addition, microdroplet platforms facilitate high-throughput screening of biomarkers. Meanwhile, surface-based droplet platforms provide an opportunity to develop miniaturized diagnostic systems. These platforms may function as portable benchtop environments that dramatically shorten the transition of a benchtop assay into a point-of-care format.

Genetic diversity and structure of core collection of winter mushroom (Flammulina velutipes) developed by genomic SSR markers.

PubMed

Liu, Xiao Bin; Li, Jing; Yang, Zhu L

2018-01-01

A core collection is a subset of an entire collection that represents as much of the genetic diversity of the entire collection as possible. The establishment of a core collection for crops is practical for efficient management and use of germplasm. However, the establishment of a core collection of mushrooms is still in its infancy, and no established core collection of the economically important species Flammulina velutipes has been reported. We established the first core collection of F. velutipes , containing 32 strains based on 81 genetically different F. veltuipes strains. The allele retention proportion of the core collection for the entire collection was 100%. Moreover, the genetic diversity parameters (the effective number of alleles, Nei's expected heterozygosity, the number of observed heterozygosity, and Shannon's information index) of the core collection showed no significant differences from the entire collection ( p  > 0.01). Thus, the core collection is representative of the genetic diversity of the entire collection. Genetic structure analyses of the core collection revealed that the 32 strains could be clustered into 6 groups, among which groups 1 to 3 were cultivars and groups 4 to 6 were wild strains. The wild strains from different locations harbor their own specific alleles, and were clustered stringently in accordance with their geographic origins. Genetic diversity analyses of the core collection revealed that the wild strains possessed greater genetic diversity than the cultivars. We established the first core collection of F. velutipes in China, which is an important platform for efficient breeding of this mushroom in the future. In addition, the wild strains in the core collection possess favorable agronomic characters and produce unique bioactive compounds, adding value to the platform. More attention should be paid to wild strains in further strain breeding.

Magnetic nanoparticles: Applications in gene delivery and gene therapy.

PubMed

Majidi, Sima; Zeinali Sehrig, Fatemeh; Samiei, Mohammad; Milani, Morteza; Abbasi, Elham; Dadashzadeh, Kianoosh; Akbarzadeh, Abolfazl

2016-06-01

Gene therapy is defined as the direct transfer of genetic material to tissues or cells for the treatment of inherited disorders and acquired diseases. For gene delivery, magnetic nanoparticles (MNPs) are typically combined with a delivery platform to encapsulate the gene, and promote cell uptake. Delivery technologies that have been used with MNPs contain polymeric, viral, as well as non-viral platforms. In this review, we focus on targeted gene delivery using MNPs.

Development and Evaluation of a 9K SNP Array for Peach by Internationally Coordinated SNP Detection and Validation in Breeding Germplasm

PubMed Central

Scalabrin, Simone; Gilmore, Barbara; Lawley, Cynthia T.; Gasic, Ksenija; Micheletti, Diego; Rosyara, Umesh R.; Cattonaro, Federica; Vendramin, Elisa; Main, Dorrie; Aramini, Valeria; Blas, Andrea L.; Mockler, Todd C.; Bryant, Douglas W.; Wilhelm, Larry; Troggio, Michela; Sosinski, Bryon; Aranzana, Maria José; Arús, Pere; Iezzoni, Amy; Morgante, Michele; Peace, Cameron

2012-01-01

Although a large number of single nucleotide polymorphism (SNP) markers covering the entire genome are needed to enable molecular breeding efforts such as genome wide association studies, fine mapping, genomic selection and marker-assisted selection in peach [Prunus persica (L.) Batsch] and related Prunus species, only a limited number of genetic markers, including simple sequence repeats (SSRs), have been available to date. To address this need, an international consortium (The International Peach SNP Consortium; IPSC) has pursued a coordinated effort to perform genome-scale SNP discovery in peach using next generation sequencing platforms to develop and characterize a high-throughput Illumina Infinium® SNP genotyping array platform. We performed whole genome re-sequencing of 56 peach breeding accessions using the Illumina and Roche/454 sequencing technologies. Polymorphism detection algorithms identified a total of 1,022,354 SNPs. Validation with the Illumina GoldenGate® assay was performed on a subset of the predicted SNPs, verifying ∼75% of genic (exonic and intronic) SNPs, whereas only about a third of intergenic SNPs were verified. Conservative filtering was applied to arrive at a set of 8,144 SNPs that were included on the IPSC peach SNP array v1, distributed over all eight peach chromosomes with an average spacing of 26.7 kb between SNPs. Use of this platform to screen a total of 709 accessions of peach in two separate evaluation panels identified a total of 6,869 (84.3%) polymorphic SNPs. The almost 7,000 SNPs verified as polymorphic through extensive empirical evaluation represent an excellent source of markers for future studies in genetic relatedness, genetic mapping, and dissecting the genetic architecture of complex agricultural traits. The IPSC peach SNP array v1 is commercially available and we expect that it will be used worldwide for genetic studies in peach and related stone fruit and nut species. PMID:22536421

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.

Plastid, nuclear and reverse transcriptase sequences in the mitochondrial genome of Oenothera: is genetic information transferred between organelles via RNA?

PubMed Central

Schuster, W; Brennicke, A

1987-01-01

We describe an open reading frame (ORF) with high homology to reverse transcriptase in the mitochondrial genome of Oenothera. This ORF displays all the characteristics of an active plant mitochondrial gene with a possible ribosome binding site and 39% T in the third codon position. It is located between a sequence fragment from the plastid genome and one of nuclear origin downstream from the gene encoding subunit 5 of the NADH dehydrogenase. The nuclear derived sequence consists of 528 nucleotides from the small ribosomal RNA and contains an expansion segment unique to nuclear rRNAs. The plastid sequence contains part of the ribosomal protein S4 and the complete tRNA(Ser). The observation that only transcribed sequences have been found i more than one subcellular compartment in higher plants suggests that interorganellar transfer of genetic information may occur via RNA and subsequent local reverse transcription and genomic integration. PMID:14650433

Reverse transcription-polymerase chain reaction molecular testing of cytology specimens: Pre-analytic and analytic factors.

PubMed

Bridge, Julia A

2017-01-01

The introduction of molecular testing into cytopathology laboratory practice has expanded the types of samples considered feasible for identifying genetic alterations that play an essential role in cancer diagnosis and treatment. Reverse transcription-polymerase chain reaction (RT-PCR), a sensitive and specific technical approach for amplifying a defined segment of RNA after it has been reverse-transcribed into its DNA complement, is commonly used in clinical practice for the identification of recurrent or tumor-specific fusion gene events. Real-time RT-PCR (quantitative RT-PCR), a technical variation, also permits the quantitation of products generated during each cycle of the polymerase chain reaction process. This review addresses qualitative and quantitative pre-analytic and analytic considerations of RT-PCR as they relate to various cytologic specimens. An understanding of these aspects of genetic testing is central to attaining optimal results in the face of the challenges that cytology specimens may present. Cancer Cytopathol 2017;125:11-19. © 2016 American Cancer Society. © 2016 American Cancer Society.

Using exposure prediction tools to link exposure and dosimetry for risk based decisions: a case study with phthalates

EPA Science Inventory

The Population Life-course Exposure to Health Effects Modeling (PLETHEM) platform being developed provides a tool that links results from emerging toxicity testing tools to exposure estimates for humans as defined by the USEPA. A reverse dosimetry case study using phthalates was ...

Redox mediators modify end product distribution in biomass fermentations by mixed ruminal microbes in vitro

USDA-ARS?s Scientific Manuscript database

The fermentation system of mixed ruminal bacteria is capable of generating large amounts of short-chain volatile fatty acids (VFA) via the carboxylate platform in vitro. These VFAs are subject to elongation to larger, more energy-dense products through reverse beta-oxidation. This study examined the...

Evaluation of a reverse-hybridization StripAssay for the detection of genetic polymorphisms leading to acenocoumarol sensitivity.

PubMed

Gialeraki, Argyri; Markatos, Christos; Grouzi, Elisabeth; Merkouri, Efrosyni; Travlou, Anthi; Politou, Marianna

2010-04-01

Acenocoumarol is mainly catabolized by CYP2C9 isoform of cytochrome P450 (CYP) liver complex and exerts its anticoagulant effect through the inhibition of Vitamin K Epoxide Reductase (VKOR). The most important genetic polymorphisms which lead to an impaired enzymatic activity and therefore predispose to acenocoumarol sensitivity, are considered to be CYP2C9*2 (Arg144Cys), CYP2C9*3 (Ile359Leu) and VKORC1-1639G>A, respectively. In this study we compared the results of the PGXThrombo StripAssay kit (ViennaLab Diagnostics,Vienna, Austria) with direct DNA sequencing and in house Restriction Fragment Length Polymorphisms (RFLP) for the detection of the aforementioned Single Nucleotide Polymorphisms (SNPs). The reverse hybridization StripAssay was found to be equally effective with RFLP and direct DNA sequencing for the detection of CYP2C9*2 and CYP2C9*3 polymorphisms, respectively. The comparison of the RFLP reference method with the reverse hybridization StripAssay for the detection of VKORC1-1639 G>A polymorphism showed that the reverse hybridization StripAsssay might misclassify some A/A homozygotes as heterozygotes. Optimization of the hybridization procedures may eliminate the extra low signal band observed in some samples at the reverse hybridization StripAssay and improve its diagnostic value.

Fast imaging diagnostics on the C-2U advanced beam-driven field-reversed configuration device

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

Granstedt, E. M., E-mail: egranstedt@trialphaenergy.com; Petrov, P.; Knapp, K.

2016-11-15

The C-2U device employed neutral beam injection, end-biasing, and various particle fueling techniques to sustain a Field-Reversed Configuration (FRC) plasma. As part of the diagnostic suite, two fast imaging instruments with radial and nearly axial plasma views were developed using a common camera platform. To achieve the necessary viewing geometry, imaging lenses were mounted behind re-entrant viewports attached to welded bellows. During gettering, the vacuum optics were retracted and isolated behind a gate valve permitting their removal if cleaning was necessary. The axial view incorporated a stainless-steel mirror in a protective cap assembly attached to the vacuum-side of the viewport.more » For each system, a custom lens-based, high-throughput optical periscope was designed to relay the plasma image about half a meter to a high-speed camera. Each instrument also contained a remote-controlled filter wheel, set between shots to isolate a particular hydrogen or impurity emission line. The design of the camera platform, imaging performance, and sample data for each view is presented.« less

Fast imaging diagnostics on the C-2U advanced beam-driven field-reversed configuration device

NASA Astrophysics Data System (ADS)

Granstedt, E. M.; Petrov, P.; Knapp, K.; Cordero, M.; Patel, V.

2016-11-01

The C-2U device employed neutral beam injection, end-biasing, and various particle fueling techniques to sustain a Field-Reversed Configuration (FRC) plasma. As part of the diagnostic suite, two fast imaging instruments with radial and nearly axial plasma views were developed using a common camera platform. To achieve the necessary viewing geometry, imaging lenses were mounted behind re-entrant viewports attached to welded bellows. During gettering, the vacuum optics were retracted and isolated behind a gate valve permitting their removal if cleaning was necessary. The axial view incorporated a stainless-steel mirror in a protective cap assembly attached to the vacuum-side of the viewport. For each system, a custom lens-based, high-throughput optical periscope was designed to relay the plasma image about half a meter to a high-speed camera. Each instrument also contained a remote-controlled filter wheel, set between shots to isolate a particular hydrogen or impurity emission line. The design of the camera platform, imaging performance, and sample data for each view is presented.

Mendelian Genetics as a Platform for Teaching about Nature of Science and Scientific Inquiry: The Value of Textbooks

ERIC Educational Resources Information Center

Campanile, Megan F.; Lederman, Norman G.; Kampourakis, Kostas

2015-01-01

The purpose of this study was to analyze seven widely used high school biology textbooks in order to assess the nature of science knowledge (NOS) and scientific inquiry (SI) aspects they, explicitly or implicitly, conveyed in the Mendelian genetics sections. Textbook excerpts that directly and/or fully matched our statements about NOS and SI were…

Avidin-Based Targeting and Purification of a Protein IX-Modified, Metabolically Biotinylated Adenoviral Vector

PubMed Central

Campos, Samuel K.; Parrott, M. Brandon; Barry, Michael A.

2014-01-01

While genetic modification of adenoviral vectors can produce vectors with modified tropism, incorporation of targeting peptides/proteins into the structural context of the virion can also result in destruction of ligand targeting or virion integrity. To combat this problem, we have developed a versatile targeting system using metabolically biotinylated adenoviral vectors bearing biotinylated fiber proteins. These vectors have been demonstrated to be useful as a platform for avidin-based ligand screening and vector targeting by conjugating biotinylated ligands to the virus using high-affinity tetrameric avidin (Kd = 10−15 M). The biotinylated vector could also be purified by biotin-reversible binding on monomeric avidin (Kd = 10−7 M). In this report, a second metabolically biotinylated adenovirus vector, Ad-IX-BAP, has been engineered by fusing a biotin acceptor peptide (BAP) to the C-terminus of the adenovirus pIX protein. This biotinylated vector displays twice as many biotins and was markedly superior for single-step affinity purification on monomeric avidin resin. However, unlike the fiber-biotinylated vector, Ad-IX-BAP failed to retarget to cells with biotinylated antibodies including anti-CD71 against the transferrin receptor. In contrast, Ad-IX-BAP was retargeted if transferrin, the cognate ligand for CD71, was used as a ligand rather than the anti-CD71. This work demonstrates the utility of metabolic biotinylation as a molecular screening tool to assess the utility of different viral capsid proteins for ligand display and the biology and compatibility of different ligands and receptors for vector targeting applications. These results also demonstrate the utility of the pIX-biotinylated vector as a platform for gentle single-step affinity purification of adenoviral vectors. PMID:15194061

MONITORING MYCOTOXIN PRODUCTION AT THE GENETIC LEVEL ON VARIOUS GROWTH SUBSTRATES USING QUANTITATIVE REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION?EXPERIMENT DESIGN

EPA Science Inventory

The paper describes a method of analyzing the production of mycotoxins at the genetic level by monitoring the intracellular levels of messenger RNA (mRNA). Initial work will focus on threshing out the mycotoxin gene clusters in Stachybotrys chartarum followed by analysis of toxin...

Are we there yet? Tracking the development of new model systems

Treesearch

A. Abzhanov; C. Extavour; A. Groover; S. Hodges; H. Hoekstra; E. Kramer; A. Monteiro

2008-01-01

It is increasingly clear that additional ‘model’ systems are needed to elucidate the genetic and developmental basis of organismal diversity. Whereas model system development previously required enormous investment, recent advances including the decreasing cost of DNA sequencing and the power of reverse genetics to study gene function are greatly facilitating...

Comprehensive Study of the Flow Control Strategy in a Wirelessly Charged Centrifugal Microfluidic Platform with Two Rotation Axes.

PubMed

Zhu, Yunzeng; Chen, Yiqi; Meng, Xiangrui; Wang, Jing; Lu, Ying; Xu, Youchun; Cheng, Jing

2017-09-05

Centrifugal microfluidics has been widely applied in the sample-in-answer-out systems for the analyses of nucleic acids, proteins, and small molecules. However, the inherent characteristic of unidirectional fluid propulsion limits the flexibility of these fluidic chips. Providing an extra degree of freedom to allow the unconstrained and reversible pumping of liquid is an effective strategy to address this limitation. In this study, a wirelessly charged centrifugal microfluidic platform with two rotation axes has been constructed and the flow control strategy in such platform with two degrees of freedom was comprehensively studied for the first time. Inductively coupled coils are installed on the platform to achieve wireless power transfer to the spinning stage. A micro servo motor is mounted on both sides of the stage to alter the orientation of the device around a secondary rotation axis on demand during stage rotation. The basic liquid operations on this platform, including directional transport of liquid, valving, metering, and mixing, are comprehensively studied and realized. Finally, a chip for the simultaneous determination of hexavalent chromium [Cr(VI)] and methanal in water samples is designed and tested based on the strategy presented in this paper, demonstrating the potential use of this platform for on-site environmental monitoring, food safety testing, and other life science applications.

Genetic Instability at the Agouti Locus of the Mouse (Mus Musculus). I. Increased Reverse Mutation Frequency to the A(w) Allele in a/a Heterozygotes

PubMed Central

Sandulache, R.; Neuhauser-Klaus, A.; Favor, J.

1994-01-01

We have compiled the reverse mutation rate data to the white bellied agouti (A(w)) allele in heterozygous A/a mice and shown it to be increased by a factor of at least 350 in comparison to the reverse mutation rate in homozygous a/a mice. Employing tightly linked flanking restriction fragment length polymorphism DNA markers, we have shown that reversion to A(w) is associated with crossing over in the vicinity of the agouti locus. The non-agouti (a) allele has been recently shown to contain an 11-kb insert within the first intron of the agouti gene. Together with our present results, these observations suggest possible mechanisms to explain the reversion events. PMID:7982562

The iCRISPR platform for rapid genome editing in human pluripotent stem cells.

PubMed

Zhu, Zengrong; González, Federico; Huangfu, Danwei

2014-01-01

Human pluripotent stem cells (hPSCs) have the potential to generate all adult cell types, including rare or inaccessible human cell populations, thus providing a unique platform for disease studies. To realize this promise, it is essential to develop methods for efficient genetic manipulations in hPSCs. Established using TALEN (transcription activator-like effector nuclease) and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) systems, the iCRISPR platform supports a variety of genome-engineering approaches with high efficiencies. Here, we first describe the establishment of the iCRISPR platform through TALEN-mediated targeting of inducible Cas9 expression cassettes into the AAVS1 locus. Next, we provide a series of technical procedures for using iCRISPR to achieve one-step knockout of one or multiple gene(s), "scarless" introduction of precise nucleotide alterations, as well as inducible knockout during hPSC differentiation. We present an optimized workflow, as well as guidelines for the selection of CRISPR targeting sequences and the design of single-stranded DNA (ssDNA) homology-directed DNA repair templates for the introduction of specific nucleotide alterations. We have successfully used these protocols in four different hPSC lines, including human embryonic stem cells and induced pluripotent stem cells. Once the iCRISPR platform is established, clonal lines with desired genetic modifications can be established in as little as 1 month. The methods described here enable a wide range of genome-engineering applications in hPSCs, thus providing a valuable resource for the creation of diverse hPSC-based disease models with superior speed and ease.

The impact of sex-role reversal on the diversity of the major histocompatibility complex: insights from the seahorse (Hippocampus abdominalis).

PubMed

Bahr, Angela; Wilson, Anthony B

2011-05-10

Both natural and sexual selection are thought to influence genetic diversity, but the study of the relative importance of these two factors on ecologically-relevant traits has traditionally focused on species with conventional sex-roles, with male-male competition and female-based mate choice. With its high variability and significance in both immune function and olfactory-mediated mate choice, the major histocompatibility complex (MHC/MH) is an ideal system in which to evaluate the relative contributions of these two selective forces to genetic diversity. Intrasexual competition and mate choice are both reversed in sex-role reversed species, and sex-related differences in the detection and use of MH-odor cues are expected to influence the intensity of sexual selection in such species. The seahorse, Hippocampus abdominalis, has an exceptionally highly developed form of male parental care, with female-female competition and male mate choice. Here, we demonstrate that the sex-role reversed seahorse has a single MH class II beta-chain gene and that the diversity of the seahorse MHIIβ locus and its pattern of variation are comparable to those detected in species with conventional sex roles. Despite the presence of only a single gene copy, intralocus MHIIβ allelic diversity in this species exceeds that observed in species with multiple copies of this locus. The MHIIβ locus of the seahorse exhibits a novel expression domain in the male brood pouch. The high variation found at the seahorse MHIIβ gene indicates that sex-role reversed species are capable of maintaining the high MHC diversity typical in most vertebrates.Whether such species have evolved the capacity to use MH-odor cues during mate choice is presently being investigated using mate choice experiments. If this possibility can be rejected, such systems would offer an exceptional opportunity to study the effects of natural selection in isolation, providing powerful comparative models for understanding the relative importance of selective factors in shaping patterns of genetic variation.

Integration of Multiplexed Microfluidic Electrokinetic Concentrators with a Morpholino Microarray via Reversible Surface Bonding for Enhanced DNA Hybridization.

PubMed

Martins, Diogo; Wei, Xi; Levicky, Rastislav; Song, Yong-Ak

2016-04-05

We describe a microfluidic concentration device to accelerate the surface hybridization reaction between DNA and morpholinos (MOs) for enhanced detection. The microfluidic concentrator comprises a single polydimethylsiloxane (PDMS) microchannel onto which an ion-selective layer of conductive polymer poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) ( PSS) was directly printed and then reversibly surface bonded onto a morpholino microarray for hybridization. Using this electrokinetic trapping concentrator, we could achieve a maximum concentration factor of ∼800 for DNA and a limit of detection of 10 nM within 15 min. In terms of the detection speed, it enabled faster hybridization by around 10-fold when compared to conventional diffusion-based hybridization. A significant advantage of our approach is that the fabrication of the microfluidic concentrator is completely decoupled from the microarray; by eliminating the need to deposit an ion-selective layer on the microarray surface prior to device integration, interfacing between both modules, the PDMS chip for electrokinetic concentration and the substrate for DNA sensing are easier and applicable to any microarray platform. Furthermore, this fabrication strategy facilitates a multiplexing of concentrators. We have demonstrated the proof-of-concept for multiplexing by building a device with 5 parallel concentrators connected to a single inlet/outlet and applying it to parallel concentration and hybridization. Such device yielded similar concentration and hybridization efficiency compared to that of a single-channel device without adding any complexity to the fabrication and setup. These results demonstrate that our concentrator concept can be applied to the development of a highly multiplexed concentrator-enhanced microarray detection system for either genetic analysis or other diagnostic assays.

SysFinder: A customized platform for search, comparison and assisted design of appropriate animal models based on systematic similarity.

PubMed

Yang, Shuang; Zhang, Guoqing; Liu, Wan; Wang, Zhen; Zhang, Jifeng; Yang, Dongshan; Chen, Y Eugene; Sun, Hong; Li, Yixue

2017-05-20

Animal models are increasingly gaining values by cross-comparisons of response or resistance to clinical agents used for patients. However, many disease mechanisms and drug effects generated from animal models are not transferable to human. To address these issues, we developed SysFinder (http://lifecenter.sgst.cn/SysFinder), a platform for scientists to find appropriate animal models for translational research. SysFinder offers a "topic-centered" approach for systematic comparisons of human genes, whose functions are involved in a specific scientific topic, to the corresponding homologous genes of animal models. Scientific topic can be a certain disease, drug, gene function or biological pathway. SysFinder calculates multi-level similarity indexes to evaluate the similarities between human and animal models in specified scientific topics. Meanwhile, SysFinder offers species-specific information to investigate the differences in molecular mechanisms between humans and animal models. Furthermore, SysFinder provides a user-friendly platform for determination of short guide RNAs (sgRNAs) and homology arms to design a new animal model. Case studies illustrate the ability of SysFinder in helping experimental scientists. SysFinder is a useful platform for experimental scientists to carry out their research in the human molecular mechanisms. Copyright © 2017 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

The pINDUCER lentiviral toolkit for inducible RNA interference in vitro and in vivo

PubMed Central

Meerbrey, Kristen L.; Hu, Guang; Kessler, Jessica D.; Roarty, Kevin; Fang, Justin E.; Herschkowitz, Jason I.; Burrows, Anna E.; Ciccia, Alberto; Sun, Tingting; Schmitt, Earlene M.; Bernardi, Ronald J.; Fu, Xiaoyong; Bland, Christopher S.; Cooper, Thomas A.; Schiff, Rachel; Rosen, Jeffrey M.; Westbrook, Thomas F.; Elledge, Stephen J.

2011-01-01

The discovery of RNAi has revolutionized loss-of-function genetic studies in mammalian systems. However, significant challenges still remain to fully exploit RNAi for mammalian genetics. For instance, genetic screens and in vivo studies could be broadly improved by methods that allow inducible and uniform gene expression control. To achieve this, we built the lentiviral pINDUCER series of expression vehicles for inducible RNAi in vivo. Using a multicistronic design, pINDUCER vehicles enable tracking of viral transduction and shRNA or cDNA induction in a broad spectrum of mammalian cell types in vivo. They achieve this uniform temporal, dose-dependent, and reversible control of gene expression across heterogenous cell populations via fluorescence-based quantification of reverse tet-transactivator expression. This feature allows isolation of cell populations that exhibit a potent, inducible target knockdown in vitro and in vivo that can be used in human xenotransplantation models to examine cancer drug targets. PMID:21307310

A reverse genetics approach identifies novel mutants in light responses and anthocyanin metabolism in petunia.

PubMed

Berenschot, Amanda S; Quecini, Vera

2014-01-01

Flower color and plant architecture are important commercially valuable features for ornamental petunias (Petunia x hybrida Vilm.). Photoperception and light signaling are the major environmental factors controlling anthocyanin and chlorophyll biosynthesis and shade-avoidance responses in higher plants. The genetic regulators of these processes were investigated in petunia by in silico analyses and the sequence information was used to devise a reverse genetics approach to probe mutant populations. Petunia orthologs of photoreceptor, light-signaling components and anthocyanin metabolism genes were identified and investigated for functional conservation by phylogenetic and protein motif analyses. The expression profiles of photoreceptor gene families and of transcription factors regulating anthocyanin biosynthesis were obtained by bioinformatic tools. Two mutant populations, generated by an alkalyting agent and by gamma irradiation, were screened using a phenotype-independent, sequence-based method by high-throughput PCR-based assay. The strategy allowed the identification of novel mutant alleles for anthocyanin biosynthesis (CHALCONE SYNTHASE) and regulation (PH4), and for light signaling (CONSTANS) genes.

Molecular Cloning Designer Simulator (MCDS): All-in-one molecular cloning and genetic engineering design, simulation and management software for complex synthetic biology and metabolic engineering projects.

PubMed

Shi, Zhenyu; Vickers, Claudia E

2016-12-01

Molecular Cloning Designer Simulator (MCDS) is a powerful new all-in-one cloning and genetic engineering design, simulation and management software platform developed for complex synthetic biology and metabolic engineering projects. In addition to standard functions, it has a number of features that are either unique, or are not found in combination in any one software package: (1) it has a novel interactive flow-chart user interface for complex multi-step processes, allowing an integrated overview of the whole project; (2) it can perform a user-defined workflow of cloning steps in a single execution of the software; (3) it can handle multiple types of genetic recombineering, a technique that is rapidly replacing classical cloning for many applications; (4) it includes experimental information to conveniently guide wet lab work; and (5) it can store results and comments to allow the tracking and management of the whole project in one platform. MCDS is freely available from https://mcds.codeplex.com.

Real-time PCR array as a universal platform for the detection of genetically modified crops and its application in identifying unapproved genetically modified crops in Japan.

PubMed

Mano, Junichi; Shigemitsu, Natsuki; Futo, Satoshi; Akiyama, Hiroshi; Teshima, Reiko; Hino, Akihiro; Furui, Satoshi; Kitta, Kazumi

2009-01-14

We developed a novel type of real-time polymerase chain reaction (PCR) array with TaqMan chemistry as a platform for the comprehensive and semiquantitative detection of genetically modified (GM) crops. Thirty primer-probe sets for the specific detection of GM lines, recombinant DNA (r-DNA) segments, endogenous reference genes, and donor organisms were synthesized, and a 96-well PCR plate was prepared with a different primer-probe in each well as the real-time PCR array. The specificity and sensitivity of the array were evaluated. A comparative analysis with the data and publicly available information on GM crops approved in Japan allowed us to assume the possibility of unapproved GM crop contamination. Furthermore, we designed a Microsoft Excel spreadsheet application, Unapproved GMO Checker version 2.01, which helps process all the data of real-time PCR arrays for the easy assumption of unapproved GM crop contamination. The spreadsheet is available free of charge at http://cse.naro.affrc.go.jp/jmano/index.html .

The Microphenotron: a robotic miniaturized plant phenotyping platform with diverse applications in chemical biology.

PubMed

Burrell, Thomas; Fozard, Susan; Holroyd, Geoff H; French, Andrew P; Pound, Michael P; Bigley, Christopher J; James Taylor, C; Forde, Brian G

2017-01-01

Chemical genetics provides a powerful alternative to conventional genetics for understanding gene function. However, its application to plants has been limited by the lack of a technology that allows detailed phenotyping of whole-seedling development in the context of a high-throughput chemical screen. We have therefore sought to develop an automated micro-phenotyping platform that would allow both root and shoot development to be monitored under conditions where the phenotypic effects of large numbers of small molecules can be assessed. The 'Microphenotron' platform uses 96-well microtitre plates to deliver chemical treatments to seedlings of Arabidopsis thaliana L. and is based around four components: (a) the 'Phytostrip', a novel seedling growth device that enables chemical treatments to be combined with the automated capture of images of developing roots and shoots; (b) an illuminated robotic platform that uses a commercially available robotic manipulator to capture images of developing shoots and roots; (c) software to control the sequence of robotic movements and integrate these with the image capture process; (d) purpose-made image analysis software for automated extraction of quantitative phenotypic data. Imaging of each plate (representing 80 separate assays) takes 4 min and can easily be performed daily for time-course studies. As currently configured, the Microphenotron has a capacity of 54 microtitre plates in a growth room footprint of 2.1 m 2 , giving a potential throughput of up to 4320 chemical treatments in a typical 10 days experiment. The Microphenotron has been validated by using it to screen a collection of 800 natural compounds for qualitative effects on root development and to perform a quantitative analysis of the effects of a range of concentrations of nitrate and ammonium on seedling development. The Microphenotron is an automated screening platform that for the first time is able to combine large numbers of individual chemical treatments with a detailed analysis of whole-seedling development, and particularly root system development. The Microphenotron should provide a powerful new tool for chemical genetics and for wider chemical biology applications, including the development of natural and synthetic chemical products for improved agricultural sustainability.

An automated Genomes-to-Natural Products platform (GNP) for the discovery of modular natural products.

PubMed

Johnston, Chad W; Skinnider, Michael A; Wyatt, Morgan A; Li, Xiang; Ranieri, Michael R M; Yang, Lian; Zechel, David L; Ma, Bin; Magarvey, Nathan A

2015-09-28

Bacterial natural products are a diverse and valuable group of small molecules, and genome sequencing indicates that the vast majority remain undiscovered. The prediction of natural product structures from biosynthetic assembly lines can facilitate their discovery, but highly automated, accurate, and integrated systems are required to mine the broad spectrum of sequenced bacterial genomes. Here we present a genome-guided natural products discovery tool to automatically predict, combinatorialize and identify polyketides and nonribosomal peptides from biosynthetic assembly lines using LC-MS/MS data of crude extracts in a high-throughput manner. We detail the directed identification and isolation of six genetically predicted polyketides and nonribosomal peptides using our Genome-to-Natural Products platform. This highly automated, user-friendly programme provides a means of realizing the potential of genetically encoded natural products.

Comparative Genomics of Bacillus species and its Relevance in Industrial Microbiology.

PubMed

Sharma, Archana; Satyanarayana, T

2013-01-01

With the advent of high throughput sequencing platforms and relevant analytical tools, the rate of microbial genome sequencing has accelerated which has in turn led to better understanding of microbial molecular biology and genetics. The complete genome sequences of important industrial organisms provide opportunities for human health, industry, and the environment. Bacillus species are the dominant workhorses in industrial fermentations. Today, genome sequences of several Bacillus species are available, and comparative genomics of this genus helps in understanding their physiology, biochemistry, and genetics. The genomes of these bacterial species are the sources of many industrially important enzymes and antibiotics and, therefore, provide an opportunity to tailor enzymes with desired properties to suit a wide range of applications. A comparative account of strengths and weaknesses of the different sequencing platforms are also highlighted in the review.

Cocaine Analog Coupled to Disrupted Adenovirus: A Vaccine Strategy to Evoke High-titer Immunity Against Addictive Drugs

PubMed Central

Hicks, Martin J; De, Bishnu P; Rosenberg, Jonathan B; Davidson, Jesse T; Moreno, Amira Y; Janda, Kim D; Wee, Sunmee; Koob, George F; Hackett, Neil R; Kaminsky, Stephen M; Worgall, Stefan; Toth, Miklos; Mezey, Jason G; Crystal, Ronald G

2011-01-01

Based on the concept that anticocaine antibodies could prevent inhaled cocaine from reaching its target receptors in the brain, an effective anticocaine vaccine could help reverse cocaine addiction. Leveraging the knowledge that E1−E3− adenovirus (Ad) gene transfer vectors are potent immunogens, we have developed a novel vaccine platform for addictive drugs by covalently linking a cocaine analog to the capsid proteins of noninfectious, disrupted Ad vector. The Ad-based anticocaine vaccine evokes high-titer anticocaine antibodies in mice sufficient to completely reverse, on a persistent basis, the hyperlocomotor activity induced by intravenous administration of cocaine. PMID:21206484

FitSearch: a robust way to interpret a yeast fitness profile in terms of drug's mode-of-action.

PubMed

Lee, Minho; Han, Sangjo; Chang, Hyeshik; Kwak, Youn-Sig; Weller, David M; Kim, Dongsup

2013-01-01

Yeast deletion-mutant collections have been successfully used to infer the mode-of-action of drugs especially by profiling chemical-genetic and genetic-genetic interactions on a genome-wide scale. Although tens of thousands of those profiles are publicly available, a lack of an accurate method for mining such data has been a major bottleneck for more widespread use of these useful resources. For general usage of those public resources, we designed FitRankDB as a general repository of fitness profiles, and developed a new search algorithm, FitSearch, for identifying the profiles that have a high similarity score with statistical significance for a given fitness profile. We demonstrated that our new repository and algorithm are highly beneficial to researchers who attempting to make hypotheses based on unknown modes-of-action of bioactive compounds, regardless of the types of experiments that have been performed using yeast deletion-mutant collection in various types of different measurement platforms, especially non-chip-based platforms. We showed that our new database and algorithm are useful when attempting to construct a hypothesis regarding the unknown function of a bioactive compound through small-scale experiments with a yeast deletion collection in a platform independent manner. The FitRankDB and FitSearch enhance the ease of searching public yeast fitness profiles and obtaining insights into unknown mechanisms of action of drugs. FitSearch is freely available at http://fitsearch.kaist.ac.kr.

FitSearch: a robust way to interpret a yeast fitness profile in terms of drug's mode-of-action

PubMed Central

2013-01-01

Background Yeast deletion-mutant collections have been successfully used to infer the mode-of-action of drugs especially by profiling chemical-genetic and genetic-genetic interactions on a genome-wide scale. Although tens of thousands of those profiles are publicly available, a lack of an accurate method for mining such data has been a major bottleneck for more widespread use of these useful resources. Results For general usage of those public resources, we designed FitRankDB as a general repository of fitness profiles, and developed a new search algorithm, FitSearch, for identifying the profiles that have a high similarity score with statistical significance for a given fitness profile. We demonstrated that our new repository and algorithm are highly beneficial to researchers who attempting to make hypotheses based on unknown modes-of-action of bioactive compounds, regardless of the types of experiments that have been performed using yeast deletion-mutant collection in various types of different measurement platforms, especially non-chip-based platforms. Conclusions We showed that our new database and algorithm are useful when attempting to construct a hypothesis regarding the unknown function of a bioactive compound through small-scale experiments with a yeast deletion collection in a platform independent manner. The FitRankDB and FitSearch enhance the ease of searching public yeast fitness profiles and obtaining insights into unknown mechanisms of action of drugs. FitSearch is freely available at http://fitsearch.kaist.ac.kr. PMID:23368702

Comparison of reverse transcription-quantitative polymerase chain reaction methods and platforms for single cell gene expression analysis.

PubMed

Fox, Bridget C; Devonshire, Alison S; Baradez, Marc-Olivier; Marshall, Damian; Foy, Carole A

2012-08-15

Single cell gene expression analysis can provide insights into development and disease progression by profiling individual cellular responses as opposed to reporting the global average of a population. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the "gold standard" for the quantification of gene expression levels; however, the technical performance of kits and platforms aimed at single cell analysis has not been fully defined in terms of sensitivity and assay comparability. We compared three kits using purification columns (PicoPure) or direct lysis (CellsDirect and Cells-to-CT) combined with a one- or two-step RT-qPCR approach using dilutions of cells and RNA standards to the single cell level. Single cell-level messenger RNA (mRNA) analysis was possible using all three methods, although the precision, linearity, and effect of lysis buffer and cell background differed depending on the approach used. The impact of using a microfluidic qPCR platform versus a standard instrument was investigated for potential variability introduced by preamplification of template or scaling down of the qPCR to nanoliter volumes using laser-dissected single cell samples. The two approaches were found to be comparable. These studies show that accurate gene expression analysis is achievable at the single cell level and highlight the importance of well-validated experimental procedures for low-level mRNA analysis. Copyright © 2012 Elsevier Inc. All rights reserved.

Reverse phase protein arrays in signaling pathways: a data integration perspective

PubMed Central

Creighton, Chad J; Huang, Shixia

2015-01-01

The reverse phase protein array (RPPA) data platform provides expression data for a prespecified set of proteins, across a set of tissue or cell line samples. Being able to measure either total proteins or posttranslationally modified proteins, even ones present at lower abundances, RPPA represents an excellent way to capture the state of key signaling transduction pathways in normal or diseased cells. RPPA data can be combined with those of other molecular profiling platforms, in order to obtain a more complete molecular picture of the cell. This review offers perspective on the use of RPPA as a component of integrative molecular analysis, using recent case examples from The Cancer Genome Altas consortium, showing how RPPA may provide additional insight into cancer besides what other data platforms may provide. There also exists a clear need for effective visualization approaches to RPPA-based proteomic results; this was highlighted by the recent challenge, put forth by the HPN-DREAM consortium, to develop visualization methods for a highly complex RPPA dataset involving many cancer cell lines, stimuli, and inhibitors applied over time course. In this review, we put forth a number of general guidelines for effective visualization of complex molecular datasets, namely, showing the data, ordering data elements deliberately, enabling generalization, focusing on relevant specifics, and putting things into context. We give examples of how these principles can be utilized in visualizing the intrinsic subtypes of breast cancer and in meaningfully displaying the entire HPN-DREAM RPPA dataset within a single page. PMID:26185419

Microstructured Optical Fiber-based Biosensors: Reversible and Nanoliter-Scale Measurement of Zinc Ions.

PubMed

Heng, Sabrina; McDevitt, Christopher A; Kostecki, Roman; Morey, Jacqueline R; Eijkelkamp, Bart A; Ebendorff-Heidepriem, Heike; Monro, Tanya M; Abell, Andrew D

2016-05-25

Sensing platforms that allow rapid and efficient detection of metal ions would have applications in disease diagnosis and study, as well as environmental sensing. Here, we report the first microstructured optical fiber-based biosensor for the reversible and nanoliter-scale measurement of metal ions. Specifically, a photoswitchable spiropyran Zn(2+) sensor is incorporated within the microenvironment of a liposome attached to microstructured optical fibers (exposed-core and suspended-core microstructured optical fibers). Both fiber-based platforms retains high selectivity of ion binding associated with a small molecule sensor, while also allowing nanoliter volume sampling and on/off switching. We have demonstrated that multiple measurements can be made on a single sample without the need to change the sensor. The ability of the new sensing platform to sense Zn(2+) in pleural lavage and nasopharynx of mice was compared to that of established ion sensing methodologies such as inductively coupled plasma mass spectrometry (ICP-MS) and a commercially available fluorophore (Fluozin-3), where the optical-fiber-based sensor provides a significant advantage in that it allows the use of nanoliter (nL) sampling when compared to ICP-MS (mL) and FluoZin-3 (μL). This work paves the way to a generic approach for developing surface-based ion sensors using a range of sensor molecules, which can be attached to a surface without the need for its chemical modification and presents an opportunity for the development of new and highly specific ion sensors for real time sensing applications.

Development of High Data Rate Acoustic Multiple-Input/Multiple-Output Modems

DTIC Science & Technology

2015-09-30

communication capabilities of underwater platforms and facilitate real-time adaptive operations in the ocean. OBJECTIVES The ...signaling at the transmitter and low-complexity time reversal processing at the receiver. APPROACH Underwater acoustic (UWA) communication is useful...digital communications in shallow water environments. The advancement has direct impacts on defense appliations since underwater acoustic modems

Viral metagenomics, protein structure, and reverse genetics: Key strategies for investigating coronaviruses.

PubMed

Johnson, Bryan A; Graham, Rachel L; Menachery, Vineet D

2018-04-01

Viral metagenomics, modeling of protein structure, and manipulation of viral genetics are key approaches that have laid the foundations of our understanding of coronavirus biology. In this review, we discuss the major advances each method has provided and discuss how future studies should leverage these strategies synergistically to answer novel questions. Copyright © 2017 Elsevier Inc. All rights reserved.

Genetic Detection and Isolation of Crimean-Congo hemorrhagic fever virus, Kosovo, Yugoslavia

PubMed Central

Boźović, Bojana; Pavlidou, Vassiliki; Papadimitriou, Evangelia; Pelemis, Mijomir; Antoniadis, Aantonis

2002-01-01

Crimean-Congo hemorrhagic fever virus (C-CHFV) strains were isolated from a fatal case and the attending physician in Kosovo, Yugoslavia. Early, rapid diagnosis of the disease was achieved by reverse transcription-polymerase chain reaction. The physician was successfully treated with oral ribavirin. These cases yielded the first genetically studied C-CHFV human isolates in the Balkans. PMID:12141973

An Integrated Systems Genetics and Omics Toolkit to Probe Gene Function.

PubMed

Li, Hao; Wang, Xu; Rukina, Daria; Huang, Qingyao; Lin, Tao; Sorrentino, Vincenzo; Zhang, Hongbo; Bou Sleiman, Maroun; Arends, Danny; McDaid, Aaron; Luan, Peiling; Ziari, Naveed; Velázquez-Villegas, Laura A; Gariani, Karim; Kutalik, Zoltan; Schoonjans, Kristina; Radcliffe, Richard A; Prins, Pjotr; Morgenthaler, Stephan; Williams, Robert W; Auwerx, Johan

2018-01-24

Identifying genetic and environmental factors that impact complex traits and common diseases is a high biomedical priority. Here, we developed, validated, and implemented a series of multi-layered systems approaches, including (expression-based) phenome-wide association, transcriptome-/proteome-wide association, and (reverse-) mediation analysis, in an open-access web server (systems-genetics.org) to expedite the systems dissection of gene function. We applied these approaches to multi-omics datasets from the BXD mouse genetic reference population, and identified and validated associations between genes and clinical and molecular phenotypes, including previously unreported links between Rpl26 and body weight, and Cpt1a and lipid metabolism. Furthermore, through mediation and reverse-mediation analysis we established regulatory relations between genes, such as the co-regulation of BCKDHA and BCKDHB protein levels, and identified targets of transcription factors E2F6, ZFP277, and ZKSCAN1. Our multifaceted toolkit enabled the identification of gene-gene and gene-phenotype links that are robust and that translate well across populations and species, and can be universally applied to any populations with multi-omics datasets. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Genetically attenuated Trypanosoma cruzi parasites as a potential vaccination tool

PubMed Central

Brandan, Cecilia Pérez; Basombrío, Miguel Ángel

2012-01-01

Chagas disease is the clinical manifestation of the infection produced by the parasite Trypanosoma cruzi. Currently there is no vaccine to prevent this disease and the protection attained with vaccines containing non-replicating parasites is limited. Genetically attenuated trypanosomatid parasites can be obtained by deletion of selected genes. Gene deletion takes advantage of the fact that this parasite can undergo homologous recombination between endogenous and foreign DNA sequences artificially introduced in the cells. This approach facilitated the discovery of several unknown gene functions, as well as allowing us to speculate about the potential for genetically attenuated live organisms as experimental immunogens. Vaccination with live attenuated parasites has been used effectively in mice to reduce parasitemia and histological damage, and in dogs, to prevent vector-delivered infection in the field. However, the use of live parasites as immunogens is controversial due to the risk of reversion to a virulent phenotype. Herein, we present our results from experiments on genetic manipulation of two T. cruzi strains to produce parasites with impaired replication and infectivity, and using the mutation of the dhfr-ts gene as a safety device against reversion to virulence. PMID:22705838

A Novel ‘Gene Insertion/Marker Out’ (GIMO) Method for Transgene Expression and Gene Complementation in Rodent Malaria Parasites

PubMed Central

Sajid, Mohammed; Chevalley-Maurel, Séverine; Ramesar, Jai; Klop, Onny; Franke-Fayard, Blandine M. D.; Janse, Chris J.; Khan, Shahid M.

2011-01-01

Research on the biology of malaria parasites has greatly benefited from the application of reverse genetic technologies, in particular through the analysis of gene deletion mutants and studies on transgenic parasites that express heterologous or mutated proteins. However, transfection in Plasmodium is limited by the paucity of drug-selectable markers that hampers subsequent genetic modification of the same mutant. We report the development of a novel ‘gene insertion/marker out’ (GIMO) method for two rodent malaria parasites, which uses negative selection to rapidly generate transgenic mutants ready for subsequent modifications. We have created reference mother lines for both P. berghei ANKA and P. yoelii 17XNL that serve as recipient parasites for GIMO-transfection. Compared to existing protocols GIMO-transfection greatly simplifies and speeds up the generation of mutants expressing heterologous proteins, free of drug-resistance genes, and requires far fewer laboratory animals. In addition we demonstrate that GIMO-transfection is also a simple and fast method for genetic complementation of mutants with a gene deletion or mutation. The implementation of GIMO-transfection procedures should greatly enhance Plasmodium reverse-genetic research. PMID:22216235

Comprehensive comparison of three commercial human whole-exome capture platforms.

PubMed

Asan; Xu, Yu; Jiang, Hui; Tyler-Smith, Chris; Xue, Yali; Jiang, Tao; Wang, Jiawei; Wu, Mingzhi; Liu, Xiao; Tian, Geng; Wang, Jun; Wang, Jian; Yang, Huangming; Zhang, Xiuqing

2011-09-28

Exome sequencing, which allows the global analysis of protein coding sequences in the human genome, has become an effective and affordable approach to detecting causative genetic mutations in diseases. Currently, there are several commercial human exome capture platforms; however, the relative performances of these have not been characterized sufficiently to know which is best for a particular study. We comprehensively compared three platforms: NimbleGen's Sequence Capture Array and SeqCap EZ, and Agilent's SureSelect. We assessed their performance in a variety of ways, including number of genes covered and capture efficacy. Differences that may impact on the choice of platform were that Agilent SureSelect covered approximately 1,100 more genes, while NimbleGen provided better flanking sequence capture. Although all three platforms achieved similar capture specificity of targeted regions, the NimbleGen platforms showed better uniformity of coverage and greater genotype sensitivity at 30- to 100-fold sequencing depth. All three platforms showed similar power in exome SNP calling, including medically relevant SNPs. Compared with genotyping and whole-genome sequencing data, the three platforms achieved a similar accuracy of genotype assignment and SNP detection. Importantly, all three platforms showed similar levels of reproducibility, GC bias and reference allele bias. We demonstrate key differences between the three platforms, particularly advantages of solutions over array capture and the importance of a large gene target set.

Quinolinic acid injection in mouse medial prefrontal cortex affects reversal learning abilities, cortical connectivity and hippocampal synaptic plasticity

PubMed Central

Latif-Hernandez, Amira; Shah, Disha; Ahmed, Tariq; Lo, Adrian C.; Callaerts-Vegh, Zsuzsanna; Van der Linden, Annemie; Balschun, Detlef; D’Hooge, Rudi

2016-01-01

Intracerebral injection of the excitotoxic, endogenous tryptophan metabolite, quinolinic acid (QA), constitutes a chemical model of neurodegenerative brain disease. Complementary techniques were combined to examine the consequences of QA injection into medial prefrontal cortex (mPFC) of C57BL6 mice. In accordance with the NMDAR-mediated synapto- and neurotoxic action of QA, we found an initial increase in excitability and an augmentation of hippocampal long-term potentiation, converting within two weeks into a reduction and impairment, respectively, of these processes. QA-induced mPFC excitotoxicity impaired behavioral flexibility in a reversal variant of the hidden-platform Morris water maze (MWM), whereas regular, extended MWM training was unaffected. QA-induced mPFC damage specifically affected the spatial-cognitive strategies that mice use to locate the platform during reversal learning. These behavioral and cognitive defects coincided with changes in cortical functional connectivity (FC) and hippocampal neuroplasticity. FC between various cortical regions was assessed by resting-state fMRI (rsfMRI) methodology, and mice that had received QA injection into mPFC showed increased FC between various cortical regions. mPFC and hippocampus (HC) are anatomically as well as functionally linked as part of a cortical network that controls higher-order cognitive functions. Together, these observations demonstrate the central functional importance of rodent mPFC as well as the validity of QA-induced mPFC damage as a preclinical rodent model of the early stages of neurodegeneration. PMID:27819338

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

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

Linshiz, Gregory; Jensen, Erik; Stawski, Nina

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

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

DOE PAGES

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

2016-02-02

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

A genetic platform to model sarcomagenesis from primary adult mesenchymal stem cells

PubMed Central

Guarnerio, Jlenia; Riccardi, Luisa; Taulli, Riccardo; Maeda, Takahiro; Wang, Guocan; Hobbs, Robin M.; Song, Min Sup; Sportoletti, Paolo; Bernardi, Rosa; Bronson, Roderick T.; Castillo-Martin, Mireia; Cordon-Cardo, Carlos; Lunardi, Andrea; Pandolfi, Pier Paolo

2015-01-01

The regulatory factors governing adult mesenchymal stem cells (MSCs) physiology and their tumorigenic potential are still largely unknown, which substantially delays the identification of effective therapeutic approaches for the treatment of aggressive and lethal form of MSC-derived mesenchymal tumors, such as undifferentiated sarcomas. Here we have developed a novel platform to screen and quickly identify genes and pathways responsible for adult MSCs transformation, modeled undifferentiated sarcoma in vivo, and, ultimately, tested the efficacy of targeting the identified oncopathways. Importantly, by taking advantage of this new platform, we demonstrate the key role of an aberrant LRF-DLK1-SOX9 pathway in the pathogenesis of undifferentiated sarcoma with important therapeutic implications. PMID:25614485

[Orange Platform].

PubMed

Toba, Kenji

2017-07-01

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

Adenovirus-based genetic vaccines for biodefense.

PubMed

Boyer, Julie L; Kobinger, Gary; Wilson, James M; Crystal, Ronald G

2005-02-01

The robust host responses elicited against transgenes encoded by (E1-)(E3-) adenovirus (Ad) gene transfer vectors can be used to develop Ad-based vectors as platform technologies for vaccines against potential bioterror pathogens. This review focuses on pathogens of major concern as bioterror agents and why Ad vectors are ideal as anti-bioterror vaccine platforms, providing examples from our laboratories of using Ad vectors as vaccines against potential bioterror pathogens and how Ad vectors can be developed to enhance vaccine efficacy in the bioterror war.

Display of HIV-1 Envelope Protein on Lambda Phage Scaffold as a Vaccine Platform.

PubMed

Mattiacio, Jonelle L; Brewer, Matt; Dewhurst, Stephen

2017-01-01

The generation of a strong antibody response to target antigens is a major goal for vaccine development. Here we describe the display of the human immunodeficiency virus (HIV) envelope spike protein (Env) on a virus-like scaffold provided by the lambda phage capsid. Phage vectors, in general, have advantages over mammalian virus vectors due to their genetic tractability, inexpensive production, suitability for scale-up, as well as their physical stability, making them an attractive vaccine platform.

Regional Crustal Velocity Models for Northern Arabian Platform and Turkish-Iranian Plateau

NASA Astrophysics Data System (ADS)

Aleqabi, G.; Wysession, M.; Ghalib, H.

2008-12-01

The geological structure of the Northern Arabian platform and surrounding mountains is dominated by the collision and suturing of the Arabian plate with the Eurasian plate and the formation of the Turkish-Iranian plateau. The structure of the Northern Arabian platform and surrounding region is poorly constrained. A recent deployment of 10 broadband seismometers in northern and central Iraq provides an opportunity to refine velocity models of the region. We have applied the Niching Genetic Algorithm waveform inversion technique to Rayleigh and Love waves traversing the Northern Arabian platform, the Zagros fold belt, the southern Turkish Plateau, the Iranian Plateau. Results show variations in crustal thickness and shear wave velocity between the Northern Arabian platform and the Turkish-Iranian plateau. In general the shear wave velocities are higher in the Northern Arabian platform than in the Plateaus. Variation of shear velocities within each of the provinces reflects the diversity in tectonic environment across the Zagros fold belt and the complex tectonic history of the region. Crustal thickness results show little crustal thickening has occurred due to collision.

Task and Participant Scheduling of Trading Platforms in Vehicular Participatory Sensing Networks

PubMed Central

Shi, Heyuan; Song, Xiaoyu; Gu, Ming; Sun, Jiaguang

2016-01-01

The vehicular participatory sensing network (VPSN) is now becoming more and more prevalent, and additionally has shown its great potential in various applications. A general VPSN consists of many tasks from task, publishers, trading platforms and a crowd of participants. Some literature treats publishers and the trading platform as a whole, which is impractical since they are two independent economic entities with respective purposes. For a trading platform in markets, its purpose is to maximize the profit by selecting tasks and recruiting participants who satisfy the requirements of accepted tasks, rather than to improve the quality of each task. This scheduling problem for a trading platform consists of two parts: which tasks should be selected and which participants to be recruited? In this paper, we investigate the scheduling problem in vehicular participatory sensing with the predictable mobility of each vehicle. A genetic-based trading scheduling algorithm (GTSA) is proposed to solve the scheduling problem. Experiments with a realistic dataset of taxi trajectories demonstrate that GTSA algorithm is efficient for trading platforms to gain considerable profit in VPSN. PMID:27916807

Task and Participant Scheduling of Trading Platforms in Vehicular Participatory Sensing Networks.

PubMed

Shi, Heyuan; Song, Xiaoyu; Gu, Ming; Sun, Jiaguang

2016-11-28

The vehicular participatory sensing network (VPSN) is now becoming more and more prevalent, and additionally has shown its great potential in various applications. A general VPSN consists of many tasks from task, publishers, trading platforms and a crowd of participants. Some literature treats publishers and the trading platform as a whole, which is impractical since they are two independent economic entities with respective purposes. For a trading platform in markets, its purpose is to maximize the profit by selecting tasks and recruiting participants who satisfy the requirements of accepted tasks, rather than to improve the quality of each task. This scheduling problem for a trading platform consists of two parts: which tasks should be selected and which participants to be recruited? In this paper, we investigate the scheduling problem in vehicular participatory sensing with the predictable mobility of each vehicle. A genetic-based trading scheduling algorithm (GTSA) is proposed to solve the scheduling problem. Experiments with a realistic dataset of taxi trajectories demonstrate that GTSA algorithm is efficient for trading platforms to gain considerable profit in VPSN.

Linear phase conjugation for atmospheric aberration compensation

NASA Astrophysics Data System (ADS)

Grasso, Robert J.; Stappaerts, Eddy A.

1998-01-01

Atmospheric induced aberrations can seriously degrade laser performance, greatly affecting the beam that finally reaches the target. Lasers propagated over any distance in the atmosphere suffer from a significant decrease in fluence at the target due to these aberrations. This is especially so for propagation over long distances. It is due primarily to fluctuations in the atmosphere over the propagation path, and from platform motion relative to the intended aimpoint. Also, delivery of high fluence to the target typically requires low beam divergence, thus, atmospheric turbulence, platform motion, or both results in a lack of fine aimpoint control to keep the beam directed at the target. To improve both the beam quality and amount of laser energy delivered to the target, Northrop Grumman has developed the Active Tracking System (ATS); a novel linear phase conjugation aberration compensation technique. Utilizing a silicon spatial light modulator (SLM) as a dynamic wavefront reversing element, ATS undoes aberrations induced by the atmosphere, platform motion or both. ATS continually tracks the target as well as compensates for atmospheric and platform motion induced aberrations. This results in a high fidelity, near-diffraction limited beam delivered to the target.

Pancreatic cancer: Are "liquid biopsies" ready for prime-time?

PubMed Central

Lewis, Alexandra R; Valle, Juan W; McNamara, Mairead G

2016-01-01

Pancreatic cancer is a disease that carries a poor prognosis. Accurate tissue diagnosis is required. Tumours contain a high content of stromal tissue and therefore biopsies may be inconclusive. Circulating tumour cells (CTCs) have been investigated as a potential “liquid biopsy” in several malignancies and have proven to be of prognostic value in breast, prostate and colorectal cancers. They have been detected in patients with localised and metastatic pancreatic cancer with sensitivities ranging from 38%-100% using a variety of platforms. Circulating tumour DNA (ctDNA) has also been detected in pancreas cancer with a sensitivity ranging from 26%-100% in studies across different platforms and using different genetic markers. However, there is no clear consensus on which platform is the most effective for detection, nor which genetic markers are the most useful to use. Potential roles of liquid biopsies include diagnosis, screening, guiding therapies and prognosis. The presence of CTCs or ctDNA has been shown to be of prognostic value both at diagnosis and after treatment in patients with pancreatic cancer. However, more prospective studies are required before this promising technology is ready for adoption into routine clinical practice. PMID:27621566

Designing multilayered nanoplatforms for SERS-based detection of genetically modified organisms

NASA Astrophysics Data System (ADS)

Uluok, Saadet; Guven, Burcu; Eksi, Haslet; Ustundag, Zafer; Tamer, Ugur; Boyaci, Ismail Hakki

2015-01-01

In this study, the multilayered surface-enhanced Raman spectroscopy (SERS) platforms were developed for the analysis of genetically modified organisms (GMOs). For this purpose, two molecules [11-mercaptoundecanoic acid (11-MUA) and 2-mercaptoethylamine (2-MEA)] were attached with Aurod and Auspherical nanoparticles to form multilayered constructions on the gold (Au)slide surface. The best multilayered platform structure was chosen depending on SERS enhancement, and this surface was characterised with atomic force microscopy (AFM) and attenuated total reflectance Fourier transform infrared spectroscopy. After the optimum multilayered SERS platform and nanoparticle interaction was identified, the oligonucleotides on the Aurod nanoparticles and Auslide were combined to determine target concentrations from the 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) signals using SERS. The correlation between the SERS intensities for DTNB and target concentrations was found to be linear within a range of 10 pM to 1 µM, and with a detection limit of 34 fM. The selectivity and specificity of the developed sandwich assay were tested using negative and positive controls, and nonsense and real sample studies. The obtained results showed that the multilayered SERS sandwich method allows for sensitive, selective, and specific detection of oligonucleotide sequences.

Intracellular Protein Delivery System Using a Target-Specific Repebody and Translocation Domain of Bacterial Exotoxin.

PubMed

Kim, Hee-Yeon; Kang, Jung Ae; Ryou, Jeong-Hyun; Lee, Gyeong Hee; Choi, Dae Seong; Lee, Dong Eun; Kim, Hak-Sung

2017-11-17

With the high efficacy of protein-based therapeutics and plenty of intracellular drug targets, cytosolic protein delivery in a cell-specific manner has attracted considerable attention in the field of precision medicine. Herein, we present an intracellular protein delivery system based on a target-specific repebody and the translocation domain of Pseudomonas aeruginosa exotoxin A. The delivery platform was constructed by genetically fusing an EGFR-specific repebody as a targeting moiety to the translocation domain, while a protein cargo was fused to the C-terminal end of the delivery platform. The delivery platform was revealed to efficiently translocate a protein cargo to the cytosol in a target-specific manner. We demonstrate the utility and potential of the delivery platform by showing a remarkable tumor regression with negligible toxicity in a xenograft mice model when gelonin was used as the cytotoxic protein cargo. The present platform can find wide applications to the cell-selective cytosolic delivery of diverse proteins in many areas.

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.

Optical Time Reversal from Time-Dependent Epsilon-Near-Zero Media

NASA Astrophysics Data System (ADS)

Vezzoli, Stefano; Bruno, Vincenzo; DeVault, Clayton; Roger, Thomas; Shalaev, Vladimir M.; Boltasseva, Alexandra; Ferrera, Marcello; Clerici, Matteo; Dubietis, Audrius; Faccio, Daniele

2018-01-01

Materials with a spatially uniform but temporally varying optical response have applications ranging from magnetic field-free optical isolators to fundamental studies of quantum field theories. However, these effects typically become relevant only for time variations oscillating at optical frequencies, thus presenting a significant hurdle that severely limits the realization of such conditions. Here we present a thin-film material with a permittivity that pulsates (uniformly in space) at optical frequencies and realizes a time-reversing medium of the form originally proposed by Pendry [Science 322, 71 (2008), 10.1126/science.1162087]. We use an optically pumped, 500 nm thick film of epsilon-near-zero (ENZ) material based on Al-doped zinc oxide. An incident probe beam is both negatively refracted and time reversed through a reflected phase-conjugated beam. As a result of the high nonlinearity and the refractive index that is close to zero, the ENZ film leads to time reversed beams (simultaneous negative refraction and phase conjugation) with near-unit efficiency and greater-than-unit internal conversion efficiency. The ENZ platform therefore presents the time-reversal features required, e.g., for efficient subwavelength imaging, all-optical isolators and fundamental quantum field theory studies.

Coastal Marine Terraces Define Late Quaternary Fault Activity and Deformation Within Northern East Bay Hills, San Francisco Bay Region

NASA Astrophysics Data System (ADS)

Kelson, K. I.

2004-12-01

Detailed mapping of uplifted marine platforms bordering the Carquinez Strait between Benicia and Pinole, California, provides data on the pattern and rate of late Quaternary deformation across the northern East Bay Hills. Field mapping, interpretation of early 20th-century topographic data, analysis of aerial photography, and compilation of onshore borehole data show the presence of remnants of three platforms, with back-edge elevations of about 4 m, 12 m, and 18 m. Based on U-series dates (Helley et al., 1993) and comparison of platform elevations to published sea-level curves, the 12-m-high and 18-m-high platforms correlate with substage 5e (ca. 120 ka) and stage 9 (ca. 330 ka) sea-level high stands, respectively. West of the Southhampton fault, longitudinal profiles of platform back-edges suggest that the East Bay Hills between Pinole and Vallejo have undergone block uplift at a rate of 0.05 +/- 0.01 m/ka without substantial tilting or warping. With uncertainty of <3 m, the 120 ka and 330 ka platforms are at the same elevations across the NW-striking Franklin fault. This west-vergent reverse fault previously was interpreted to have had late Pleistocene activity and to accommodate crustal shortening in the East Bay Hills. Our data indicate an absence of vertical displacement across the Franklin fault within at least the past 120ka and perhaps 330ka. In contrast, the stage 5e and 9 have up-on-the-east vertical displacement and gentle westward tilting across the N-striking Southhampton fault, with a late Pleistocene vertical slip rate of >0.02 m/ka. The northerly strike and prominent geomorphic expression of this potentially active fault differs from the Franklin fault. Our mapping of the Southhampton fault suggests that it accommodates dextral shear in the East Bay Hills, and is one of several left-stepping, en echelon N-striking faults (collectively, the "Contra Costa shear zone", CCSZ) in the East Bay Hills. Faults within this zone coincide with geomorphic features suggestive of late Quaternary dextral strike slip and appear to truncate or displace NW-striking reverse faults (e.g., Franklin fault) that do not displace the late Quaternary marine platform sequence. These data support an interpretation that the CCSZ accommodates regional dextral shear, and possibly represents the northern extension of the Calaveras fault. Overall, the marine terraces provide excellent strain gauges from which to evaluate the pattern and rate of late Quaternary deformation throughout the northern East Bay Hills.

Simulated breeding with QU-GENE graphical user interface.

PubMed

Hathorn, Adrian; Chapman, Scott; Dieters, Mark

2014-01-01

Comparing the efficiencies of breeding methods with field experiments is a costly, long-term process. QU-GENE is a highly flexible genetic and breeding simulation platform capable of simulating the performance of a range of different breeding strategies and for a continuum of genetic models ranging from simple to complex. In this chapter we describe some of the basic mechanics behind the QU-GENE user interface and give a simplified example of how it works.

Diversity Arrays Technology (DArT) Marker Platforms for Diversity Analysis and Linkage Mapping in a Complex Crop, the Octoploid Cultivated Strawberry (Fragaria × ananassa)

PubMed Central

Sánchez-Sevilla, José F.; Horvath, Aniko; Botella, Miguel A.; Gaston, Amèlia; Folta, Kevin; Kilian, Andrzej; Denoyes, Beatrice; Amaya, Iraida

2015-01-01

Cultivated strawberry (Fragaria × ananassa) is a genetically complex allo-octoploid crop with 28 pairs of chromosomes (2n = 8x = 56) for which a genome sequence is not yet available. The diploid Fragaria vesca is considered the donor species of one of the octoploid sub-genomes and its available genome sequence can be used as a reference for genomic studies. A wide number of strawberry cultivars are stored in ex situ germplasm collections world-wide but a number of previous studies have addressed the genetic diversity present within a limited number of these collections. Here, we report the development and application of two platforms based on the implementation of Diversity Array Technology (DArT) markers for high-throughput genotyping in strawberry. The first DArT microarray was used to evaluate the genetic diversity of 62 strawberry cultivars that represent a wide range of variation based on phenotype, geographical and temporal origin and pedigrees. A total of 603 DArT markers were used to evaluate the diversity and structure of the population and their cluster analyses revealed that these markers were highly efficient in classifying the accessions in groups based on historical, geographical and pedigree-based cues. The second DArTseq platform took benefit of the complexity reduction method optimized for strawberry and the development of next generation sequencing technologies. The strawberry DArTseq was used to generate a total of 9,386 SNP markers in the previously developed ‘232’ × ‘1392’ mapping population, of which, 4,242 high quality markers were further selected to saturate this map after several filtering steps. The high-throughput platforms here developed for genotyping strawberry will facilitate genome-wide characterizations of large accessions sets and complement other available options. PMID:26675207

The Application of a Homologous Recombination Assay Revealed Amino Acid Residues in an LTR-Retrotransposon That Were Critical for Integration

PubMed Central

Atwood, Angela; Choi, Jeannie; Levin, Henry L.

1998-01-01

Retroviruses and their relatives, the LTR-retrotransposons, possess an integrase protein (IN) that is required for the insertion of reverse transcripts into the genome of host cells. Schizosaccharomyces pombe is the host of Tf1, an LTR-retrotransposon with integration activity that can be studied by using techniques of yeast genetics. In this study, we sought to identify amino acid substitutions in Tf1 that specifically affected the integration step of transposition. In addition to seeking amino acid substitutions in IN, we also explored the possibility that other Tf1 proteins contributed to integration. By comparing the results of genetic assays that monitored both transposition and reverse transcription, we were able to seek point mutations throughout Tf1 that blocked transposition but not the synthesis of reverse transcripts. These mutant versions of Tf1 were candidates of elements that possessed defects in the integration step of transposition. Five mutations in Tf1 that resulted in low levels of integration were found to be located in the IN protein: two substitutions in the N-terminal Zn domain, two in the catalytic core, and one in the C-terminal domain. These results suggested that each of the three IN domains was required for Tf1 transposition. The potential role of these five amino acid residues in the function of IN is discussed. Two of the mutations that reduced integration mapped to the RNase H (RH) domain of Tf1 reverse transcriptase. The Tf1 elements with the RH mutations produced high levels of reverse transcripts, as determined by recombination and DNA blot analysis. These results indicated that the RH of Tf1 possesses a function critical for transposition that is independent of the accumulation of reverse transcripts. PMID:9445033

A detrital sediment budget of a Maldivian reef platform

NASA Astrophysics Data System (ADS)

Morgan, K. M.; Kench, P. S.

2014-10-01

Sediment dynamics are an important control on the morphology and development of reef systems by actively removing and redistributing excess detrital sediment. This study presents quantitative data from direct point measurements of sediment transport on the platform surface and fore-reef slope of Vabbinfaru reef, North Malé Atoll, Maldives. A suite of sediment traps were used to construct actual rates of platform sediment fluxes and off-reef export over different spatial and temporal (seasonal) scales to establish key sediment transport pathways. Findings showed that high sediment fluxes occur on Vabbinfaru platform in the absence of major storm activity (up to 1905 g m- 1 d- 1), with 95% of annual transport occurring during the southwest monsoon as a result of increased wave energy. Climate-driven changes in the platform process regime caused a reversal of net sediment transport pathways between each monsoon season. Off-reef export rates were high, reaching a maximum of 12.58 kg m- 1 y- 1 for gravel and 407 g m- 1 d- 1 for sand-sized sediment. An estimated 127,120 kg is exported from the platform annually equating to a significant loss from the reef sediment budget and contributing to the long-term geomorphic development of the fore-reef slope and atoll basin. Detrital sediment reservoirs on Vabbinfaru are not purely depositional carbonate sinks, but rather temporary stores that are important in the transfer of sediment between reef zones.

Implementation of a Multiplex and Quantitative Proteomics Platform for Assessing Protein Lysates Using DNA-Barcoded Antibodies.

PubMed

Lee, Jinho; Geiss, Gary K; Demirkan, Gokhan; Vellano, Christopher P; Filanoski, Brian; Lu, Yiling; Ju, Zhenlin; Yu, Shuangxing; Guo, Huifang; Bogatzki, Lisa Y; Carter, Warren; Meredith, Rhonda K; Krishnamurthy, Savitri; Ding, Zhiyong; Beechem, Joseph M; Mills, Gordon B

2018-06-01

Molecular analysis of tumors forms the basis for personalized cancer medicine and increasingly guides patient selection for targeted therapy. Future opportunities for personalized medicine are highlighted by the measurement of protein expression levels via immunohistochemistry, protein arrays, and other approaches; however, sample type, sample quantity, batch effects, and "time to result" are limiting factors for clinical application. Here, we present a development pipeline for a novel multiplexed DNA-labeled antibody platform which digitally quantifies protein expression from lysate samples. We implemented a rigorous validation process for each antibody and show that the platform is amenable to multiple protocols covering nitrocellulose and plate-based methods. Results are highly reproducible across technical and biological replicates, and there are no observed "batch effects" which are common for most multiplex molecular assays. Tests from basal and perturbed cancer cell lines indicate that this platform is comparable to orthogonal proteomic assays such as Reverse-Phase Protein Array, and applicable to measuring the pharmacodynamic effects of clinically-relevant cancer therapeutics. Furthermore, we demonstrate the potential clinical utility of the platform with protein profiling from breast cancer patient samples to identify molecular subtypes. Together, these findings highlight the potential of this platform for enhancing our understanding of cancer biology in a clinical translation setting. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Next-generation sequencing library preparation method for identification of RNA viruses on the Ion Torrent Sequencing Platform.

PubMed

Chen, Guiqian; Qiu, Yuan; Zhuang, Qingye; Wang, Suchun; Wang, Tong; Chen, Jiming; Wang, Kaicheng

2018-05-09

Next generation sequencing (NGS) is a powerful tool for the characterization, discovery, and molecular identification of RNA viruses. There were multiple NGS library preparation methods published for strand-specific RNA-seq, but some methods are not suitable for identifying and characterizing RNA viruses. In this study, we report a NGS library preparation method to identify RNA viruses using the Ion Torrent PGM platform. The NGS sequencing adapters were directly inserted into the sequencing library through reverse transcription and polymerase chain reaction, without fragmentation and ligation of nucleic acids. The results show that this method is simple to perform, able to identify multiple species of RNA viruses in clinical samples.

Yeast as a tool to identify anti-aging compounds

PubMed Central

Zimmermann, Andreas; Hofer, Sebastian; Pendl, Tobias; Kainz, Katharina; Madeo, Frank; Carmona-Gutierrez, Didac

2018-01-01

Abstract In the search for interventions against aging and age-related diseases, biological screening platforms are indispensable tools to identify anti-aging compounds among large substance libraries. The budding yeast, Saccharomyces cerevisiae, has emerged as a powerful chemical and genetic screening platform, as it combines a rapid workflow with experimental amenability and the availability of a wide range of genetic mutant libraries. Given the amount of conserved genes and aging mechanisms between yeast and human, testing candidate anti-aging substances in yeast gene-deletion or overexpression collections, or de novo derived mutants, has proven highly successful in finding potential molecular targets. Yeast-based studies, for example, have led to the discovery of the polyphenol resveratrol and the natural polyamine spermidine as potential anti-aging agents. Here, we present strategies for pharmacological anti-aging screens in yeast, discuss common pitfalls and summarize studies that have used yeast for drug discovery and target identification. PMID:29905792

Clinical analysis of genome next-generation sequencing data using the Omicia platform

PubMed Central

Coonrod, Emily M; Margraf, Rebecca L; Russell, Archie; Voelkerding, Karl V; Reese, Martin G

2013-01-01

Aims Next-generation sequencing is being implemented in the clinical laboratory environment for the purposes of candidate causal variant discovery in patients affected with a variety of genetic disorders. The successful implementation of this technology for diagnosing genetic disorders requires a rapid, user-friendly method to annotate variants and generate short lists of clinically relevant variants of interest. This report describes Omicia’s Opal platform, a new software tool designed for variant discovery and interpretation in a clinical laboratory environment. The software allows clinical scientists to process, analyze, interpret and report on personal genome files. Materials & Methods To demonstrate the software, the authors describe the interactive use of the system for the rapid discovery of disease-causing variants using three cases. Results & Conclusion Here, the authors show the features of the Opal system and their use in uncovering variants of clinical significance. PMID:23895124

Modeling Human Cancers in Drosophila.

PubMed

Sonoshita, M; Cagan, R L

2017-01-01

Cancer is a complex disease that affects multiple organs. Whole-body animal models provide important insights into oncology that can lead to clinical impact. Here, we review novel concepts that Drosophila studies have established for cancer biology, drug discovery, and patient therapy. Genetic studies using Drosophila have explored the roles of oncogenes and tumor-suppressor genes that when dysregulated promote cancer formation, making Drosophila a useful model to study multiple aspects of transformation. Not limited to mechanism analyses, Drosophila has recently been showing its value in facilitating drug development. Flies offer rapid, efficient platforms by which novel classes of drugs can be identified as candidate anticancer leads. Further, we discuss the use of Drosophila as a platform to develop therapies for individual patients by modeling the tumor's genetic complexity. Drosophila provides both a classical and a novel tool to identify new therapeutics, complementing other more traditional cancer tools. © 2017 Elsevier Inc. All rights reserved.

Toward a new paradigm of DNA writing using a massively parallel sequencing platform and degenerate oligonucleotide

PubMed Central

Hwang, Byungjin; Bang, Duhee

2016-01-01

All synthetic DNA materials require prior programming of the building blocks of the oligonucleotide sequences. The development of a programmable microarray platform provides cost-effective and time-efficient solutions in the field of data storage using DNA. However, the scalability of the synthesis is not on par with the accelerating sequencing capacity. Here, we report on a new paradigm of generating genetic material (writing) using a degenerate oligonucleotide and optomechanical retrieval method that leverages sequencing (reading) throughput to generate the desired number of oligonucleotides. As a proof of concept, we demonstrate the feasibility of our concept in digital information storage in DNA. In simulation, the ability to store data is expected to exponentially increase with increase in degenerate space. The present study highlights the major framework change in conventional DNA writing paradigm as a sequencer itself can become a potential source of making genetic materials. PMID:27876825

The yellow fever 17D virus as a platform for new live attenuated vaccines.

PubMed

Bonaldo, Myrna C; Sequeira, Patrícia C; Galler, Ricardo

2014-01-01

The live-attenuated yellow fever 17D virus is one of the most outstanding human vaccines ever developed. It induces efficacious immune responses at a low production cost with a well-established manufacture process. These advantages make the YF17D virus attractive as a vector for the development of new vaccines. At the beginning of vector development studies, YF17D was genetically manipulated to express other flavivirus prM and E proteins, components of the viral envelope. While these 17D recombinants are based on the substitution of equivalent YF17D genes, other antigens from unrelated pathogens have also been successfully expressed and delivered by recombinant YF17D viruses employing alternative strategies for genetic manipulation of the YF17D genome. Herein, we discuss these strategies in terms of possibilities of single epitope or larger sequence expression and the main properties of these replication-competent viral platforms.

Toward a new paradigm of DNA writing using a massively parallel sequencing platform and degenerate oligonucleotide.

PubMed

Hwang, Byungjin; Bang, Duhee

2016-11-23

All synthetic DNA materials require prior programming of the building blocks of the oligonucleotide sequences. The development of a programmable microarray platform provides cost-effective and time-efficient solutions in the field of data storage using DNA. However, the scalability of the synthesis is not on par with the accelerating sequencing capacity. Here, we report on a new paradigm of generating genetic material (writing) using a degenerate oligonucleotide and optomechanical retrieval method that leverages sequencing (reading) throughput to generate the desired number of oligonucleotides. As a proof of concept, we demonstrate the feasibility of our concept in digital information storage in DNA. In simulation, the ability to store data is expected to exponentially increase with increase in degenerate space. The present study highlights the major framework change in conventional DNA writing paradigm as a sequencer itself can become a potential source of making genetic materials.

Genomic prediction unifies animal and plant breeding programs to form platforms for biological discovery.

PubMed

Hickey, John M; Chiurugwi, Tinashe; Mackay, Ian; Powell, Wayne

2017-08-30

The rate of annual yield increases for major staple crops must more than double relative to current levels in order to feed a predicted global population of 9 billion by 2050. Controlled hybridization and selective breeding have been used for centuries to adapt plant and animal species for human use. However, achieving higher, sustainable rates of improvement in yields in various species will require renewed genetic interventions and dramatic improvement of agricultural practices. Genomic prediction of breeding values has the potential to improve selection, reduce costs and provide a platform that unifies breeding approaches, biological discovery, and tools and methods. Here we compare and contrast some animal and plant breeding approaches to make a case for bringing the two together through the application of genomic selection. We propose a strategy for the use of genomic selection as a unifying approach to deliver innovative 'step changes' in the rate of genetic gain at scale.

Synthetic biology approaches: Towards sustainable exploitation of marine bioactive molecules.

PubMed

Seghal Kiran, G; Ramasamy, Pasiyappazham; Sekar, Sivasankari; Ramu, Meenatchi; Hassan, Saqib; Ninawe, A S; Selvin, Joseph

2018-06-01

The discovery of genes responsible for the production of bioactive metabolites via metabolic pathways combined with the advances in synthetic biology tools, has allowed the establishment of numerous microbial cell factories, for instance the yeast cell factories, for the manufacture of highly useful metabolites from renewable biomass. Genome mining and metagenomics are two platforms provide base-line data for reconstruction of genomes and metabolomes which is based in the development of synthetic/semi-synthetic genomes for marine natural products discovery. Engineered biofilms are being innovated on synthetic biology platform using genetic circuits and cell signalling systems as represillators controlling biofilm formation. Recombineering is a process of homologous recombination mediated genetic engineering, includes insertion, deletion or modification of any sequence specifically. Although this discipline considered new to the scientific domain, this field has now developed as promising endeavor on the accomplishment of sustainable exploitation of marine natural products. Copyright © 2018 Elsevier B.V. All rights reserved.

A multiplexable TALE-based binary expression system for in vivo cellular interaction studies.

PubMed

Toegel, Markus; Azzam, Ghows; Lee, Eunice Y; Knapp, David J H F; Tan, Ying; Fa, Ming; Fulga, Tudor A

2017-11-21

Binary expression systems have revolutionised genetic research by enabling delivery of loss-of-function and gain-of-function transgenes with precise spatial-temporal resolution in vivo. However, at present, each existing platform relies on a defined exogenous transcription activator capable of binding a unique recognition sequence. Consequently, none of these technologies alone can be used to simultaneously target different tissues or cell types in the same organism. Here, we report a modular system based on programmable transcription activator-like effector (TALE) proteins, which enables parallel expression of multiple transgenes in spatially distinct tissues in vivo. Using endogenous enhancers coupled to TALE drivers, we demonstrate multiplexed orthogonal activation of several transgenes carrying cognate variable activating sequences (VAS) in distinct neighbouring cell types of the Drosophila central nervous system. Since the number of combinatorial TALE-VAS pairs is virtually unlimited, this platform provides an experimental framework for highly complex genetic manipulation studies in vivo.

The yellow fever 17D virus as a platform for new live attenuated vaccines

PubMed Central

Bonaldo, Myrna C; Sequeira, Patrícia C; Galler, Ricardo

2014-01-01

The live-attenuated yellow fever 17D virus is one of the most outstanding human vaccines ever developed. It induces efficacious immune responses at a low production cost with a well-established manufacture process. These advantages make the YF17D virus attractive as a vector for the development of new vaccines. At the beginning of vector development studies, YF17D was genetically manipulated to express other flavivirus prM and E proteins, components of the viral envelope. While these 17D recombinants are based on the substitution of equivalent YF17D genes, other antigens from unrelated pathogens have also been successfully expressed and delivered by recombinant YF17D viruses employing alternative strategies for genetic manipulation of the YF17D genome. Herein, we discuss these strategies in terms of possibilities of single epitope or larger sequence expression and the main properties of these replication-competent viral platforms. PMID:24553128

Integration of genetically modified virus-like-particles with an optical resonator for selective bio-detection

NASA Astrophysics Data System (ADS)

Fan, X. Z.; Naves, L.; Siwak, N. P.; Brown, A.; Culver, J.; Ghodssi, R.

2015-05-01

A novel virus-like particle (TMV-VLP) receptor layer has been integrated with an optical microdisk resonator transducer for biosensing applications. This bioreceptor layer is functionalized with selective peptides that encode unique recognition affinities. Integration of bioreceptors with sensor platforms is very challenging due their very different compatibility regimes. The TMV-VLP nanoreceptor exhibits integration robustness, including the ability for self-assembly along with traditional top-down microfabrication processes. An optical microdisk resonator has been functionalized for antibody binding with this receptor, demonstrating resonant wavelength shifts of (Δλo) of 0.79 nm and 5.95 nm after primary antibody binding and enzyme-linked immunosorbent assay (ELISA), respectively, illustrating label-free sensing of this bonding event. This demonstration of label-free sensing with genetically engineered TMV-VLP shows the flexibility and utility of this receptor coating when considering integration with other existing transducer platforms.

Towards Clinical Molecular Diagnosis of Inherited Cardiac Conditions: A Comparison of Bench-Top Genome DNA Sequencers

PubMed Central

Wilkinson, Samuel L.; John, Shibu; Walsh, Roddy; Novotny, Tomas; Valaskova, Iveta; Gupta, Manu; Game, Laurence; Barton, Paul J R.; Cook, Stuart A.; Ware, James S.

2013-01-01

Background Molecular genetic testing is recommended for diagnosis of inherited cardiac disease, to guide prognosis and treatment, but access is often limited by cost and availability. Recently introduced high-throughput bench-top DNA sequencing platforms have the potential to overcome these limitations. Methodology/Principal Findings We evaluated two next-generation sequencing (NGS) platforms for molecular diagnostics. The protein-coding regions of six genes associated with inherited arrhythmia syndromes were amplified from 15 human samples using parallelised multiplex PCR (Access Array, Fluidigm), and sequenced on the MiSeq (Illumina) and Ion Torrent PGM (Life Technologies). Overall, 97.9% of the target was sequenced adequately for variant calling on the MiSeq, and 96.8% on the Ion Torrent PGM. Regions missed tended to be of high GC-content, and most were problematic for both platforms. Variant calling was assessed using 107 variants detected using Sanger sequencing: within adequately sequenced regions, variant calling on both platforms was highly accurate (Sensitivity: MiSeq 100%, PGM 99.1%. Positive predictive value: MiSeq 95.9%, PGM 95.5%). At the time of the study the Ion Torrent PGM had a lower capital cost and individual runs were cheaper and faster. The MiSeq had a higher capacity (requiring fewer runs), with reduced hands-on time and simpler laboratory workflows. Both provide significant cost and time savings over conventional methods, even allowing for adjunct Sanger sequencing to validate findings and sequence exons missed by NGS. Conclusions/Significance MiSeq and Ion Torrent PGM both provide accurate variant detection as part of a PCR-based molecular diagnostic workflow, and provide alternative platforms for molecular diagnosis of inherited cardiac conditions. Though there were performance differences at this throughput, platforms differed primarily in terms of cost, scalability, protocol stability and ease of use. Compared with current molecular genetic diagnostic tests for inherited cardiac arrhythmias, these NGS approaches are faster, less expensive, and yet more comprehensive. PMID:23861798

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 the architecture of eukaryotic genomes and are the evolutionary origin of retroviruses, including human immunodeficiency virus (HIV).

Mechanism-based strategies for protein thermostabilization.

PubMed

Mozhaev, V V

1993-03-01

Strategies for stabilizing enzymes can be derived from a two-step model of irreversible inactivation that involves preliminary reversible unfolding, followed by an irreversible step. Reversible unfolding is best prevented by covalent immobilization, whereas methods such as covalent modification of amino acid residues or 'medium engineering' (by the addition of low-molecular-weight compounds) are effective against irreversible 'incorrect' refolding. Genetic modification of the protein sequence is the most effective approach for preventing chemical deterioration.

Genetic and Genomic Toolbox of Zea mays

PubMed Central

Nannas, Natalie J.; Dawe, R. Kelly

2015-01-01

Maize has a long history of genetic and genomic tool development and is considered one of the most accessible higher plant systems. With a fully sequenced genome, a suite of cytogenetic tools, methods for both forward and reverse genetics, and characterized phenotype markers, maize is amenable to studying questions beyond plant biology. Major discoveries in the areas of transposons, imprinting, and chromosome biology came from work in maize. Moving forward in the post-genomic era, this classic model system will continue to be at the forefront of basic biological study. In this review, we outline the basics of working with maize and describe its rich genetic toolbox. PMID:25740912

Spatial and reversal learning in the Morris water maze are largely resistant to six hours of REM sleep deprivation following training

PubMed Central

Walsh, Christine M.; Booth, Victoria; Poe, Gina R.

2011-01-01

This first test of the role of REM (rapid eye movement) sleep in reversal spatial learning is also the first attempt to replicate a much cited pair of papers reporting that REM sleep deprivation impairs the consolidation of initial spatial learning in the Morris water maze. We hypothesized that REM sleep deprivation following training would impair both hippocampus-dependent spatial learning and learning a new target location within a familiar environment: reversal learning. A 6-d protocol was divided into the initial spatial learning phase (3.5 d) immediately followed by the reversal phase (2.5 d). During the 6 h following four or 12 training trials/day of initial or reversal learning phases, REM sleep was eliminated and non-REM sleep left intact using the multiple inverted flowerpot method. Contrary to our hypotheses, REM sleep deprivation during four or 12 trials/day of initial spatial or reversal learning did not affect training performance. However, some probe trial measures indicated REM sleep-deprivation–associated impairment in initial spatial learning with four trials/day and enhancement of subsequent reversal learning. In naive animals, REM sleep deprivation during normal initial spatial learning was followed by a lack of preference for the subsequent reversal platform location during the probe. Our findings contradict reports that REM sleep is essential for spatial learning in the Morris water maze and newly reveal that short periods of REM sleep deprivation do not impair concurrent reversal learning. Effects on subsequent reversal learning are consistent with the idea that REM sleep serves the consolidation of incompletely learned items. PMID:21677190

Engineered CRISPR Systems for Next Generation Gene Therapies.

PubMed

Pineda, Michael; Moghadam, Farzaneh; Ebrahimkhani, Mo R; Kiani, Samira

2017-09-15

An ideal in vivo gene therapy platform provides safe, reprogrammable, and precise strategies which modulate cell and tissue gene regulatory networks with a high temporal and spatial resolution. Clustered regularly interspaced short palindromic repeats (CRISPR), a bacterial adoptive immune system, and its CRISPR-associated protein 9 (Cas9), have gained attention for the ability to target and modify DNA sequences on demand with unprecedented flexibility and precision. The precision and programmability of Cas9 is derived from its complexation with a guide-RNA (gRNA) that is complementary to a desired genomic sequence. CRISPR systems open-up widespread applications including genetic disease modeling, functional screens, and synthetic gene regulation. The plausibility of in vivo genetic engineering using CRISPR has garnered significant traction as a next generation in vivo therapeutic. However, there are hurdles that need to be addressed before CRISPR-based strategies are fully implemented. Some key issues center on the controllability of the CRISPR platform, including minimizing genomic-off target effects and maximizing in vivo gene editing efficiency, in vivo cellular delivery, and spatial-temporal regulation. The modifiable components of CRISPR systems: Cas9 protein, gRNA, delivery platform, and the form of CRISPR system delivered (DNA, RNA, or ribonucleoprotein) have recently been engineered independently to design a better genome engineering toolbox. This review focuses on evaluating CRISPR potential as a next generation in vivo gene therapy platform and discusses bioengineering advancements that can address challenges associated with clinical translation of this emerging technology.

Alpharetroviral self-inactivating vectors produced by a superinfection-resistant stable packaging cell line allow genetic modification of primary human T lymphocytes.

PubMed

Labenski, Verena; Suerth, Julia D; Barczak, Elke; Heckl, Dirk; Levy, Camille; Bernadin, Ornellie; Charpentier, Emmanuelle; Williams, David A; Fehse, Boris; Verhoeyen, Els; Schambach, Axel

2016-08-01

Primary human T lymphocytes represent an important cell population for adoptive immunotherapies, including chimeric-antigen and T-cell receptor applications, as they have the capability to eliminate non-self, virus-infected and tumor cells. Given the increasing numbers of clinical immunotherapy applications, the development of an optimal vector platform for genetic T lymphocyte engineering, which allows cost-effective high-quality vector productions, remains a critical goal. Alpharetroviral self-inactivating vectors (ARV) have several advantages compared to other vector platforms, including a more random genomic integration pattern and reduced likelihood for inducing aberrant splicing of integrated proviruses. We developed an ARV platform for the transduction of primary human T lymphocytes. We demonstrated functional transgene transfer using the clinically relevant herpes-simplex-virus thymidine kinase variant TK.007. Proof-of-concept of alpharetroviral-mediated T-lymphocyte engineering was shown in vitro and in a humanized transplantation model in vivo. Furthermore, we established a stable, human alpharetroviral packaging cell line in which we deleted the entry receptor (SLC1A5) for RD114/TR-pseudotyped ARVs to prevent superinfection and enhance genomic integrity of the packaging cell line and viral particles. We showed that superinfection can be entirely prevented, while maintaining high recombinant virus titers. Taken together, this resulted in an improved production platform representing an economic strategy for translating the promising features of ARVs for therapeutic T-lymphocyte engineering. Copyright © 2016 Elsevier Ltd. All rights reserved.

Production of infectious dromedary camel hepatitis E virus by a reverse genetic system: Potential for zoonotic infection.

PubMed

Li, Tian-Cheng; Zhou, Xianfeng; Yoshizaki, Sayaka; Ami, Yasushi; Suzaki, Yuriko; Nakamura, Tomofumi; Takeda, Naokazu; Wakita, Takaji

2016-12-01

The pathogenicity, epidemiology and replication mechanism of dromedary camel hepatitis E virus (DcHEV), a novel hepatitis E virus (HEV), has been unclear. Here we used a reverse genetic system to produce DcHEV and examined the possibility of zoonotic infection. Capped genomic RNA derived from a synthetic DcHEV cDNA was transfected into human hepatocarcinoma cells PLC/PRF/5. The DcHEV capsid protein and RNA were detected by an enzyme-linked immunosorbent assay (ELISA) or RT-qPCR. A neutralization test for DcHEV was carried out by using antisera against HEV-like particles. DcHEV was used to inoculate two cynomolgus monkeys to examine the potential for cross-species infection. The transfection of PLC/PRF/5 cells with capped DcHEV RNA resulted in the production of infectious DcHEV. The genome sequence analysis demonstrated that both nucleotide and amino acid changes accumulated during the passages in PLC/PRF/5 cells. The cynomolgus monkeys showed serological signs of infection when DcHEV was intravenously inoculated. DcHEV was neutralized by not only anti-DcHEV-LPs antibody, but also anti-genotype 1 (G1), G3 and G4 HEV-LPs antibodies. Moreover, the monkeys immunized with DcHEV escaped the G3 HEV challenge, indicating that the serotype of DcHEV is similar to those of other human HEVs. Infectious DcHEV was produced using a reverse genetic system and propagated in PLC/PRF/5 cells. The antigenicity and immunogenicity of DcHEV are similar to those of G1, G3 and G4 HEV. DcHEV was experimentally transmitted to primates, demonstrating the possibility of a zoonotic infection by DcHEV. Dromedary camel hepatitis E virus (DcHEV) was produced by a reverse genetic system and grows well in PLC/PRF/5 cells. Cynomolgus monkeys experimentally infected with DcHEV indicated serological signs of infection, suggesting that DcHEV has the potential to cause zoonotic HEV infection. Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Reverse cascade screening of newborns for hereditary haemochromatosis: a model for other late onset diseases?

PubMed

Cadet, E; Capron, D; Gallet, M; Omanga-Léké, M-L; Boutignon, H; Julier, C; Robson, K J H; Rochette, J

2005-05-01

Genetic testing can determine those at risk for hereditary haemochromatosis (HH) caused by HFE mutations before the onset of symptoms. However, there is no optimum screening strategy, mainly owing to the variable penetrance in those who are homozygous for the HFE Cys282Tyr (C282Y) mutation. The objective of this study was to identify the majority of individuals at serious risk of developing HFE haemochromatosis before they developed life threatening complications. We first estimated the therapeutic penetrance of the C282Y mutation in people living in la Somme, France, using genetic, demographic, biochemical, and follow up data. We examined the benefits of neonatal screening on the basis of increased risk to relatives of newborns carrying one or two copies of the C282Y mutation. Between 1999 and 2002, we screened 7038 newborns from two maternity hospitals in the north of France for the C282Y and His63Asp (H63D) mutations in the HFE gene, using bloodspots collected on Guthrie cards. Family studies and genetic counselling were undertaken, based on the results of the baby's genotype. In la Somme, we found that 24% of the adults homozygous for the C282Y mutation required at least 5 g iron to be removed to restore normal iron parameters (that is, the therapeutic penetrance). In the reverse cascade screening study, we identified 19 C282Y homozygotes (1/370), 491 heterozygotes (1/14) and 166 compound heterozygotes (1/42) in 7038 newborns tested. The reverse cascade screening strategy resulted in 80 adults being screened for both mutations. We identified 10 previously unknown C282Y homozygotes of whom six (four men and two women) required venesection. Acceptance of neonatal screening was high; parents understood the risks of having HH and the benefits of early detection, but a number of parents were reluctant to take the test themselves. Neonatal screening for HH is straightforward. Reverse cascade screening increased the efficiency of detecting affected adults with undiagnosed haemochromatosis. This strategy allows almost complete coverage for HH and could be a model for efficient screening for other late onset genetic diseases.

The Corn Smut ('Huitlacoche') as a New Platform for Oral Vaccines.

PubMed

Juárez-Montiel, Margarita; Romero-Maldonado, Andrea; Monreal-Escalante, Elizabeth; Becerra-Flora, Alicia; Korban, Schuyler S; Rosales-Mendoza, Sergio; Jiménez-Bremont, Juan Francisco

2015-01-01

The development of new alternative platforms for subunit vaccine production is a priority in the biomedical field. In this study, Ustilago maydis, the causal agent of common corn smut or 'huitlacoche'has been genetically engineered to assess expression and immunogenicity of the B subunit of the cholera toxin (CTB), a relevant immunomodulatory agent in vaccinology. An oligomeric CTB recombinant protein was expressed in corn smut galls at levels of up to 1.3 mg g-1 dry weight (0.8% of the total soluble protein). Mice orally immunized with 'huitlacoche'-derived CTB showed significant humoral responses that were well-correlated with protection against challenge with the cholera toxin (CT). These findings demonstrate the feasibility of using edible corn smut as a safe, effective, and low-cost platform for production and delivery of a subunit oral vaccine. The implications of this platform in the area of molecular pharming are discussed.

The Corn Smut (‘Huitlacoche’) as a New Platform for Oral Vaccines

PubMed Central

Juárez-Montiel, Margarita; Romero-Maldonado, Andrea; Monreal-Escalante, Elizabeth; Becerra-Flora, Alicia; Korban, Schuyler S.; Rosales-Mendoza, Sergio; Jiménez-Bremont, Juan Francisco

2015-01-01

The development of new alternative platforms for subunit vaccine production is a priority in the biomedical field. In this study, Ustilago maydis, the causal agent of common corn smut or ‘huitlacoche’has been genetically engineered to assess expression and immunogenicity of the B subunit of the cholera toxin (CTB), a relevant immunomodulatory agent in vaccinology. An oligomeric CTB recombinant protein was expressed in corn smut galls at levels of up to 1.3 mg g-1 dry weight (0.8% of the total soluble protein). Mice orally immunized with ‘huitlacoche’-derived CTB showed significant humoral responses that were well-correlated with protection against challenge with the cholera toxin (CT). These findings demonstrate the feasibility of using edible corn smut as a safe, effective, and low-cost platform for production and delivery of a subunit oral vaccine. The implications of this platform in the area of molecular pharming are discussed. PMID:26207365

Xenopatients 2.0

PubMed Central

Menendez, Javier A; Alarcón, Tomás; Corominas-Faja, Bruna; Cuyàs, Elisabet; López-Bonet, Eugeni; Martin, Ángel G; Vellon, Luciano

2014-01-01

In the science-fiction thriller film Minority Report, a specialized police department called “PreCrime” apprehends criminals identified in advance based on foreknowledge provided by 3 genetically altered humans called “PreCogs”. We propose that Yamanaka stem cell technology can be similarly used to (epi)genetically reprogram tumor cells obtained directly from cancer patients and create self-evolving personalized translational platforms to foresee the evolutionary trajectory of individual tumors. This strategy yields a large stem cell population and captures the cancer genome of an affected individual, i.e., the PreCog-induced pluripotent stem (iPS) cancer cells, which are immediately available for experimental manipulation, including pharmacological screening for personalized “stemotoxic” cancer drugs. The PreCog-iPS cancer cells will re-differentiate upon orthotopic injection into the corresponding target tissues of immunodeficient mice (i.e., the PreCrime-iPS mouse avatars), and this in vivo model will run through specific cancer stages to directly explore their biological properties for drug screening, diagnosis, and personalized treatment in individual patients. The PreCog/PreCrime-iPS approach can perform sets of comparisons to directly observe changes in the cancer-iPS cell line vs. a normal iPS cell line derived from the same human genetic background. Genome editing of PreCog-iPS cells could create translational platforms to directly investigate the link between genomic expression changes and cellular malignization that is largely free from genetic and epigenetic noise and provide proof-of-principle evidence for cutting-edge “chromosome therapies” aimed against cancer aneuploidy. We might infer the epigenetic marks that correct the tumorigenic nature of the reprogrammed cancer cell population and normalize the malignant phenotype in vivo. Genetically engineered models of conditionally reprogrammable mice to transiently express the Yamanaka stemness factors following the activation of phenotypic copies of specific cancer diseases might crucially evaluate a “reprogramming cure” for cancer. A new era of xenopatients 2.0 generated via nuclear reprogramming of the epigenetic landscapes of patient-derived cancer genomes might revolutionize the current personalized translational platforms in cancer research. PMID:24406535

Xenopatients 2.0: reprogramming the epigenetic landscapes of patient-derived cancer genomes.

PubMed

Menendez, Javier A; Alarcón, Tomás; Corominas-Faja, Bruna; Cuyàs, Elisabet; López-Bonet, Eugeni; Martin, Angel G; Vellon, Luciano

2014-01-01

In the science-fiction thriller film Minority Report, a specialized police department called "PreCrime" apprehends criminals identified in advance based on foreknowledge provided by 3 genetically altered humans called "PreCogs". We propose that Yamanaka stem cell technology can be similarly used to (epi)genetically reprogram tumor cells obtained directly from cancer patients and create self-evolving personalized translational platforms to foresee the evolutionary trajectory of individual tumors. This strategy yields a large stem cell population and captures the cancer genome of an affected individual, i.e., the PreCog-induced pluripotent stem (iPS) cancer cells, which are immediately available for experimental manipulation, including pharmacological screening for personalized "stemotoxic" cancer drugs. The PreCog-iPS cancer cells will re-differentiate upon orthotopic injection into the corresponding target tissues of immunodeficient mice (i.e., the PreCrime-iPS mouse avatars), and this in vivo model will run through specific cancer stages to directly explore their biological properties for drug screening, diagnosis, and personalized treatment in individual patients. The PreCog/PreCrime-iPS approach can perform sets of comparisons to directly observe changes in the cancer-iPS cell line vs. a normal iPS cell line derived from the same human genetic background. Genome editing of PreCog-iPS cells could create translational platforms to directly investigate the link between genomic expression changes and cellular malignization that is largely free from genetic and epigenetic noise and provide proof-of-principle evidence for cutting-edge "chromosome therapies" aimed against cancer aneuploidy. We might infer the epigenetic marks that correct the tumorigenic nature of the reprogrammed cancer cell population and normalize the malignant phenotype in vivo. Genetically engineered models of conditionally reprogrammable mice to transiently express the Yamanaka stemness factors following the activation of phenotypic copies of specific cancer diseases might crucially evaluate a "reprogramming cure" for cancer. A new era of xenopatients 2.0 generated via nuclear reprogramming of the epigenetic landscapes of patient-derived cancer genomes might revolutionize the current personalized translational platforms in cancer research.

PhytoCRISP-Ex: a web-based and stand-alone application to find specific target sequences for CRISPR/CAS editing.

PubMed

Rastogi, Achal; Murik, Omer; Bowler, Chris; Tirichine, Leila

2016-07-01

With the emerging interest in phytoplankton research, the need to establish genetic tools for the functional characterization of genes is indispensable. The CRISPR/Cas9 system is now well recognized as an efficient and accurate reverse genetic tool for genome editing. Several computational tools have been published allowing researchers to find candidate target sequences for the engineering of the CRISPR vectors, while searching possible off-targets for the predicted candidates. These tools provide built-in genome databases of common model organisms that are used for CRISPR target prediction. Although their predictions are highly sensitive, the applicability to non-model genomes, most notably protists, makes their design inadequate. This motivated us to design a new CRISPR target finding tool, PhytoCRISP-Ex. Our software offers CRIPSR target predictions using an extended list of phytoplankton genomes and also delivers a user-friendly standalone application that can be used for any genome. The software attempts to integrate, for the first time, most available phytoplankton genomes information and provide a web-based platform for Cas9 target prediction within them with high sensitivity. By offering a standalone version, PhytoCRISP-Ex maintains an independence to be used with any organism and widens its applicability in high throughput pipelines. PhytoCRISP-Ex out pars all the existing tools by computing the availability of restriction sites over the most probable Cas9 cleavage sites, which can be ideal for mutant screens. PhytoCRISP-Ex is a simple, fast and accurate web interface with 13 pre-indexed and presently updating phytoplankton genomes. The software was also designed as a UNIX-based standalone application that allows the user to search for target sequences in the genomes of a variety of other species.

Sequencing and de novo assembly of visceral mass transcriptome of the critically endangered land snail Satsuma myomphala: Annotation and SSR discovery.

PubMed

Kang, Se Won; Patnaik, Bharat Bhusan; Hwang, Hee-Ju; Park, So Young; Chung, Jong Min; Song, Dae Kwon; Patnaik, Hongray Howrelia; Lee, Jae Bong; Kim, Changmu; Kim, Soonok; Park, Hong Seog; Park, Seung-Hwan; Park, Young-Su; Han, Yeon Soo; Lee, Jun Sang; Lee, Yong Seok

2017-03-01

Satsuma myomphala is critically endangered through loss of natural habitats, predation by natural enemies, and indiscriminate collection. It is a protected species in Korea but lacks genomic resources for an understanding of varied functional processes attributable to evolutionary success under natural habitats. For assessing the genetic information of S. myomphala, we performed for the first time, de novo transcriptome sequencing and functional annotation of expressed sequences using Illumina Next-Generation Sequencing (NGS) platform and bioinformatics analysis. We identified 103,774 unigenes of which 37,959, 12,890, and 17,699 were annotated in the PANM (Protostome DB), Unigene, and COG (Clusters of Orthologous Groups) databases, respectively. In addition, 14,451 unigenes were predicted under Gene Ontology functional categories, with 4581 assigned to a single category. Furthermore, 3369 sequences with 646 having Enzyme Commission (EC) numbers were mapped to 122 pathways in the Kyoto Encyclopedia of Genes and Genomes Pathway database. The prominent protein domains included the Zinc finger (C2H2-like), Reverse Transcriptase, Thioredoxin-like fold, and RNA recognition motif domain. Many unigenes with homology to immunity, defense, and reproduction-related genes were screened in the transcriptome. We also detected 3120 putative simple sequence repeats (SSRs) encompassing dinucleotide to hexanucleotide repeat motifs from >1kb unigene sequences. A list of PCR primers of SSR loci have been identified to study the genetic polymorphisms. The transcriptome data represents a valuable resource for further investigations on the species genome structure and biology. The unigenes information and microsatellites would provide an indispensable tool for conservation of the species in natural and adaptive environments. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Genomic Insight into Mechanisms of Reversion of Antibiotic Resistance in Multidrug Resistant Mycobacterium tuberculosis Induced by a Nanomolecular Iodine-Containing Complex FS-1.

PubMed

Ilin, Aleksandr I; Kulmanov, Murat E; Korotetskiy, Ilya S; Islamov, Rinat A; Akhmetova, Gulshara K; Lankina, Marina V; Reva, Oleg N

2017-01-01

Drug induced reversion of antibiotic resistance is a promising way to combat multidrug resistant infections. However, lacking knowledge of mechanisms of drug resistance reversion impedes employing this approach in medicinal therapies. Induction of antibiotic resistance reversion by a new anti-tuberculosis drug FS-1 has been reported. FS-1 was used in this work in combination with standard anti-tuberculosis antibiotics in an experiment on laboratory guinea pigs infected with an extensively drug resistant (XDR) strain Mycobacterium tuberculosis SCAID 187.0. During the experimental trial, genetic changes in the population were analyzed by sequencing of M. tuberculosis isolates followed by variant calling. In total 11 isolates obtained from different groups of infected animals at different stages of disease development and treatment were sequenced. It was found that despite the selective pressure of antibiotics, FS-1 caused a counter-selection of drug resistant variants that speeded up the recovery of the infected animals from XDR tuberculosis. Drug resistance mutations reported in the genome of the initial strain remained intact in more sensitive isolates obtained in this experiment. Variant calling in the sequenced genomes revealed that the drug resistance reversion could be associated with a general increase in genetic heterogeneity of the population of M. tuberculosis . Accumulation of mutations in PpsA and PpsE subunits of phenolpthiocerol polyketide synthase was observed in the isolates treated with FS-1 that may indicate an increase of persisting variants in the population. It was hypothesized that FS-1 caused an active counter-selection of drug resistant variants from the population by aggravating the cumulated fitness cost of the drug resistance mutations. Action of FS-1 on drug resistant bacteria exemplified the theoretically predicted induced synergy mechanism of drug resistance reversion. An experimental model to study the drug resistance reversion phenomenon is hereby introduced.

Insights from human genetic studies of lung and organ fibrosis.

PubMed

Garcia, Christine Kim

2018-01-02

Genetic investigations of fibrotic diseases, including those of late onset, often yield unanticipated insights into disease pathogenesis. This Review focuses on pathways underlying lung fibrosis that are generalizable to other organs. Herein, we discuss genetic variants subdivided into those that shorten telomeres, activate the DNA damage response, change resident protein expression or function, or affect organelle activity. Genetic studies provide a window into the downstream cascade of maladaptive responses and pathways that lead to tissue fibrosis. In addition, these studies reveal interactions between genetic variants, environmental factors, and age that influence the phenotypic spectrum of disease. The discovery of forces counterbalancing inherited risk alleles identifies potential therapeutic targets, thus providing hope for future prevention or reversal of fibrosis.

Toward understanding of the role of reversibility of phenotypic switching in the evolution of resistance to therapy

NASA Astrophysics Data System (ADS)

Horvath, D.; Brutovsky, B.

2018-06-01

Reversibility of state transitions is intensively studied topic in many scientific disciplines over many years. In cell biology, it plays an important role in epigenetic variation of phenotypes, known as phenotypic plasticity. More interestingly, the cell state reversibility is probably crucial in the adaptation of population phenotypic heterogeneity to environmental fluctuations by evolving bet-hedging strategy, which might confer to cancer cells resistance to therapy. In this article, we propose a formalization of the evolution of highly reversible states in the environments of periodic variability. Two interrelated models of heterogeneous cell populations are proposed and their behavior is studied. The first model captures selection dynamics of the cell clones for the respective levels of phenotypic reversibility. The second model focuses on the interplay between reversibility and drug resistance in the particular case of cancer. Overall, our results show that the threshold dependencies are emergent features of the investigated model with eventual therapeutic relevance. Presented examples demonstrate importance of taking into account cell to cell heterogeneity within a system of clones with different reversibility quantified by appropriately chosen genetic and epigenetic entropy measures.

Identification of Respiratory Syncytial Virus Nonstructural Protein 2 Residues Essential for Exploitation of the Host Ubiquitin System and Inhibition of Innate Immune Responses.

PubMed

Whelan, Jillian N; Tran, Kim C; van Rossum, Damian B; Teng, Michael N

2016-07-15

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in young children worldwide. The RSV nonstructural protein 2 (NS2) is a multifunctional protein that primarily acts to antagonize the innate immune system by targeting STAT2 for proteasomal degradation. We investigated the structural determinants of NS2 important for interaction with the host ubiquitin system to degrade STAT2 during infection. We found that NS2 expression enhances ubiquitination of host proteins. Bioinformatics analysis provided a platform for identification of specific residues that limit NS2-induced ubiquitination. Combinations of multiple mutations displayed an additive effect on reducing NS2-induced ubiquitination. Using a reverse genetics system, we generated recombinant RSV (rRSV) containing NS2 ubiquitin mutations, which maintained their effect on ubiquitin expression during infection. Interestingly, STAT2 degradation activity was ablated in the NS2 ubiquitin mutant rRSV. In addition, NS2 ubiquitin mutations decreased rRSV replication, indicating a correlation between NS2's ubiquitin function and antagonism of innate immune signaling to enhance viral replication. Our approach of targeting NS2 residues required for NS2 inhibition of immune responses provides a mechanism for attenuating RSV for vaccine development. RSV has been circulating globally for more than 60 years, causing severe respiratory disease in pediatric, elderly, and immunocompromised populations. Production of a safe, effective vaccine against RSV is a public health priority. The NS2 protein is an effective target for prevention and treatment of RSV due to its antagonistic activity against the innate immune system. However, NS2-deleted RSV vaccine candidates rendered RSV overattenuated or poorly immunogenic. Alternatively, we can modify essential NS2 structural features to marginally limit viral growth while maintaining immune responses, providing the necessary balance between antigenicity and safety required for an effective vaccine. We coupled bioinformatics analysis with reverse genetics to introduce mutations into RSV's negative-sense genome. In this way we constructed rRSV NS2 ubiquitin mutants that limited NS2's ability to antagonize the innate immune system, thereby attenuating rRSV growth and increasing innate immune responses. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Genetic Variation of Morphological Traits and Transpiration in an Apple Core Collection under Well-Watered Conditions: Towards the Identification of Morphotypes with High Water Use Efficiency.

PubMed

Lopez, Gerardo; Pallas, Benoît; Martinez, Sébastien; Lauri, Pierre-Éric; Regnard, Jean-Luc; Durel, Charles-Éric; Costes, Evelyne

2015-01-01

Water use efficiency (WUE) is a quantitative measurement which improvement is a major issue in the context of global warming and restrictions in water availability for agriculture. In this study, we aimed at studying the variation and genetic control of WUE and the respective role of its components (plant biomass and transpiration) in a perennial fruit crop. We explored an INRA apple core collection grown in a phenotyping platform to screen one-year-old scions for their accumulated biomass, transpiration and WUE under optimal growing conditions. Plant biomass was decompose into morphological components related to either growth or organ expansion. For each trait, nine mixed models were evaluated to account for the genetic effect and spatial heterogeneity inside the platform. The Best Linear Unbiased Predictors of genetic values were estimated after model selection. Mean broad-sense heritabilities were calculated from variance estimates. Heritability values indicated that biomass (0.76) and WUE (0.73) were under genetic control. This genetic control was lower in plant transpiration with an heritability of 0.54. Across the collection, biomass accounted for 70% of the WUE variability. A Hierarchical Ascendant Classification of the core collection indicated the existence of six groups of genotypes with contrasting morphology and WUE. Differences between morphotypes were interpreted as resulting from differences in the main processes responsible for plant growth: cell division leading to the generation of new organs and cell elongation leading to organ dimension. Although further studies will be necessary on mature trees with more complex architecture and multiple sinks such as fruits, this study is a first step for improving apple plant material for the use of water.

Genetic Variation of Morphological Traits and Transpiration in an Apple Core Collection under Well-Watered Conditions: Towards the Identification of Morphotypes with High Water Use Efficiency

PubMed Central

Lopez, Gerardo; Pallas, Benoît; Martinez, Sébastien; Lauri, Pierre-Éric; Regnard, Jean-Luc; Durel, Charles-Éric; Costes, Evelyne

2015-01-01

Water use efficiency (WUE) is a quantitative measurement which improvement is a major issue in the context of global warming and restrictions in water availability for agriculture. In this study, we aimed at studying the variation and genetic control of WUE and the respective role of its components (plant biomass and transpiration) in a perennial fruit crop. We explored an INRA apple core collection grown in a phenotyping platform to screen one-year-old scions for their accumulated biomass, transpiration and WUE under optimal growing conditions. Plant biomass was decompose into morphological components related to either growth or organ expansion. For each trait, nine mixed models were evaluated to account for the genetic effect and spatial heterogeneity inside the platform. The Best Linear Unbiased Predictors of genetic values were estimated after model selection. Mean broad-sense heritabilities were calculated from variance estimates. Heritability values indicated that biomass (0.76) and WUE (0.73) were under genetic control. This genetic control was lower in plant transpiration with an heritability of 0.54. Across the collection, biomass accounted for 70% of the WUE variability. A Hierarchical Ascendant Classification of the core collection indicated the existence of six groups of genotypes with contrasting morphology and WUE. Differences between morphotypes were interpreted as resulting from differences in the main processes responsible for plant growth: cell division leading to the generation of new organs and cell elongation leading to organ dimension. Although further studies will be necessary on mature trees with more complex architecture and multiple sinks such as fruits, this study is a first step for improving apple plant material for the use of water. PMID:26717192

A computationally identified compound antagonizes excess FGF-23 signaling in renal tubules and a mouse model of hypophosphatemia

DOE PAGES

Xiao, Zhousheng; Riccardi, Demian; Velazquez, Hector A.; ...

2016-11-22

Fibroblast growth factor–23 (FGF-23) interacts with a binary receptor complex composed of α-Klotho (α-KL) and FGF receptors (FGFRs) to regulate phosphate and vitamin D metabolism in the kidney. Excess FGF-23 production, which causes hypophosphatemia, is genetically inherited or occurs with chronic kidney disease. Among other symptoms, hypophosphatemia causes vitamin D deficiency and the bone-softening disorder rickets. Current therapeutics that target the receptor complex have limited utility clinically. In this paper, using a computationally driven, structure-based, ensemble docking and virtual high-throughput screening approach, we identified four novel compounds predicted to selectively inhibit FGF-23–induced activation of the FGFR/α-KL complex. Additional modeling andmore » functional analysis found that Zinc13407541 bound to FGF-23 and disrupted its interaction with the FGFR1/α-KL complex; experiments in a heterologous cell expression system showed that Zinc13407541 selectivity inhibited α-KL–dependent FGF-23 signaling. Zinc13407541 also inhibited FGF-23 signaling in isolated renal tubules ex vivo and partially reversed the hypophosphatemic effects of excess FGF-23 in a mouse model. Finally, these chemical probes provide a platform to develop lead compounds to treat disorders caused by excess FGF-23.« less

Rapid one-step construction of a Middle East Respiratory Syndrome (MERS-CoV) infectious clone system by homologous recombination.

PubMed

Nikiforuk, Aidan M; Leung, Anders; Cook, Bradley W M; Court, Deborah A; Kobasa, Darwyn; Theriault, Steven S

2016-10-01

Viral Infectious clone systems serve as robust platforms to study viral gene or replicative function by reverse genetics, formulate vaccines and adapt a wild type-virus to an animal host. Since the development of the first viral infectious clone system for the poliovirus, novel strategies of viral genome construction have allowed for the assembly of viral genomes across the identified viral families. However, the molecular profiles of some viruses make their genome more difficult to construct than others. Two factors that affect the difficulty of infectious clone construction are genome length and genome complexity. This work examines the available strategies for overcoming the obstacles of assembling the long and complex RNA genomes of coronaviruses and reports one-step construction of an infectious clone system for the Middle East Respiratory Syndrome coronavirus (MERS-CoV) by homologous recombination in S. cerevisiae. Future use of this methodology will shorten the time between emergence of a novel viral pathogen and construction of an infectious clone system. Completion of a viral infectious clone system facilitates further study of a virus's biology, improvement of diagnostic tests, vaccine production and the screening of antiviral compounds. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

Gene expression and the biological phenotype of papillary thyroid carcinomas.

PubMed

Delys, L; Detours, V; Franc, B; Thomas, G; Bogdanova, T; Tronko, M; Libert, F; Dumont, J E; Maenhaut, C

2007-12-13

The purpose of this paper is to correlate the molecular phenotype of papillary thyroid carcinoma (PTC) to their biological pathology. We hybridized 26 PTC on microarrays and showed that nearly 44% of the transcriptome was regulated in these tumors. We then combined our data set with two published PTC microarray studies to produce a platform- and study-independent list of PTC-associated genes. We further confirmed the mRNA regulation of 15 genes from this list by quantitative reverse transcription-PCR. Analysis of this list with statistical tools led to several conclusions: (1) there is a change in cell population with an increased expression of genes involved in the immune response, reflecting lymphocyte infiltration in the tumor compared to the normal tissue. (2) The c-jun N-terminal kinase pathway is activated by overexpression of its components. (3) The activation of ERKK1/2 by genetic alterations is supplemented by activation of the epidermal growth factor but not of the insulin-like growth factor signaling pathway. (4) There is a downregulation of immediate early genes. (5) We observed an overexpression of many proteases in accordance with tumor remodeling, and suggested a probable role of S100 proteins and annexin A2 in this process. (6) Numerous overexpressed genes favor the hypothesis of a collective migration mode of tumor cells.

Genotyping of single nucleotide polymorphism by probe-gated silica nanoparticles.

PubMed

Ercan, Meltem; Ozalp, Veli C; Tuna, Bilge G

2017-11-15

The development of simple, reliable, and rapid approaches for molecular detection of common mutations is important for prevention and early diagnosis of genetic diseases, including Thalessemia. Oligonucleotide-gated mesoporous nanoparticles-based analysis is a new platform for mutation detection that has the advantages of sensitivity, rapidity, accuracy, and convenience. A specific mutation in β-thalassemia, one of the most prevalent inherited diseases in several countries, was used as model disease in this study. An assay for detection of IVS110 point mutation (A > G reversion) was developed by designing probe-gated mesoporous silica nanoparticles (MCM-41) loaded with reporter fluorescein molecules. The silica nanoparticles were characterized by AFM, TEM and BET analysis for having 180 nm diameter and 2.83 nm pore size regular hexagonal shape. Amine group functionalized nanoparticles were analysed with FTIR technique. Mutated and normal sequence probe oligonucleotides)about 12.7 nmol per mg nanoparticles) were used to entrap reporter fluorescein molecules inside the pores and hybridization with single stranded DNA targets amplified by PCR gave different fluorescent signals for mutated targets. Samples from IVS110 mutated and normal patients resulted in statistically significant differences when the assay procedure were applied. Copyright © 2017 Elsevier Inc. All rights reserved.

A computationally identified compound antagonizes excess FGF-23 signaling in renal tubules and a mouse model of hypophosphatemia.

PubMed

Xiao, Zhousheng; Riccardi, Demian; Velazquez, Hector A; Chin, Ai L; Yates, Charles R; Carrick, Jesse D; Smith, Jeremy C; Baudry, Jerome; Quarles, L Darryl

2016-11-22

Fibroblast growth factor-23 (FGF-23) interacts with a binary receptor complex composed of α-Klotho (α-KL) and FGF receptors (FGFRs) to regulate phosphate and vitamin D metabolism in the kidney. Excess FGF-23 production, which causes hypophosphatemia, is genetically inherited or occurs with chronic kidney disease. Among other symptoms, hypophosphatemia causes vitamin D deficiency and the bone-softening disorder rickets. Current therapeutics that target the receptor complex have limited utility clinically. Using a computationally driven, structure-based, ensemble docking and virtual high-throughput screening approach, we identified four novel compounds predicted to selectively inhibit FGF-23-induced activation of the FGFR/α-KL complex. Additional modeling and functional analysis found that Zinc13407541 bound to FGF-23 and disrupted its interaction with the FGFR1/α-KL complex; experiments in a heterologous cell expression system showed that Zinc13407541 selectivity inhibited α-KL-dependent FGF-23 signaling. Zinc13407541 also inhibited FGF-23 signaling in isolated renal tubules ex vivo and partially reversed the hypophosphatemic effects of excess FGF-23 in a mouse model. These chemical probes provide a platform to develop lead compounds to treat disorders caused by excess FGF-23. Copyright © 2016, American Association for the Advancement of Science.

A computationally identified compound antagonizes excess FGF-23 signaling in renal tubules and a mouse model of hypophosphatemia

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

Xiao, Zhousheng; Riccardi, Demian; Velazquez, Hector A.

Fibroblast growth factor–23 (FGF-23) interacts with a binary receptor complex composed of α-Klotho (α-KL) and FGF receptors (FGFRs) to regulate phosphate and vitamin D metabolism in the kidney. Excess FGF-23 production, which causes hypophosphatemia, is genetically inherited or occurs with chronic kidney disease. Among other symptoms, hypophosphatemia causes vitamin D deficiency and the bone-softening disorder rickets. Current therapeutics that target the receptor complex have limited utility clinically. In this paper, using a computationally driven, structure-based, ensemble docking and virtual high-throughput screening approach, we identified four novel compounds predicted to selectively inhibit FGF-23–induced activation of the FGFR/α-KL complex. Additional modeling andmore » functional analysis found that Zinc13407541 bound to FGF-23 and disrupted its interaction with the FGFR1/α-KL complex; experiments in a heterologous cell expression system showed that Zinc13407541 selectivity inhibited α-KL–dependent FGF-23 signaling. Zinc13407541 also inhibited FGF-23 signaling in isolated renal tubules ex vivo and partially reversed the hypophosphatemic effects of excess FGF-23 in a mouse model. Finally, these chemical probes provide a platform to develop lead compounds to treat disorders caused by excess FGF-23.« less

Stage-Specific Changes in Plasmodium Metabolism Required for Differentiation and Adaptation to Different Host and Vector Environments.

PubMed

Srivastava, Anubhav; Philip, Nisha; Hughes, Katie R; Georgiou, Konstantina; MacRae, James I; Barrett, Michael P; Creek, Darren J; McConville, Malcolm J; Waters, Andrew P

2016-12-01

Malaria parasites (Plasmodium spp.) encounter markedly different (nutritional) environments during their complex life cycles in the mosquito and human hosts. Adaptation to these different host niches is associated with a dramatic rewiring of metabolism, from a highly glycolytic metabolism in the asexual blood stages to increased dependence on tricarboxylic acid (TCA) metabolism in mosquito stages. Here we have used stable isotope labelling, targeted metabolomics and reverse genetics to map stage-specific changes in Plasmodium berghei carbon metabolism and determine the functional significance of these changes on parasite survival in the blood and mosquito stages. We show that glutamine serves as the predominant input into TCA metabolism in both asexual and sexual blood stages and is important for complete male gametogenesis. Glutamine catabolism, as well as key reactions in intermediary metabolism and CoA synthesis are also essential for ookinete to oocyst transition in the mosquito. These data extend our knowledge of Plasmodium metabolism and point towards possible targets for transmission-blocking intervention strategies. Furthermore, they highlight significant metabolic differences between Plasmodium species which are not easily anticipated based on genomics or transcriptomics studies and underline the importance of integration of metabolomics data with other platforms in order to better inform drug discovery and design.

Stage-Specific Changes in Plasmodium Metabolism Required for Differentiation and Adaptation to Different Host and Vector Environments

PubMed Central

Srivastava, Anubhav; Philip, Nisha; Hughes, Katie R.; Georgiou, Konstantina; MacRae, James I.; Barrett, Michael P.; McConville, Malcolm J.

2016-01-01

Malaria parasites (Plasmodium spp.) encounter markedly different (nutritional) environments during their complex life cycles in the mosquito and human hosts. Adaptation to these different host niches is associated with a dramatic rewiring of metabolism, from a highly glycolytic metabolism in the asexual blood stages to increased dependence on tricarboxylic acid (TCA) metabolism in mosquito stages. Here we have used stable isotope labelling, targeted metabolomics and reverse genetics to map stage-specific changes in Plasmodium berghei carbon metabolism and determine the functional significance of these changes on parasite survival in the blood and mosquito stages. We show that glutamine serves as the predominant input into TCA metabolism in both asexual and sexual blood stages and is important for complete male gametogenesis. Glutamine catabolism, as well as key reactions in intermediary metabolism and CoA synthesis are also essential for ookinete to oocyst transition in the mosquito. These data extend our knowledge of Plasmodium metabolism and point towards possible targets for transmission-blocking intervention strategies. Furthermore, they highlight significant metabolic differences between Plasmodium species which are not easily anticipated based on genomics or transcriptomics studies and underline the importance of integration of metabolomics data with other platforms in order to better inform drug discovery and design. PMID:28027318

Alginic acid cell entrapment: a novel method for measuring in vivo macrophage cholesterol homeostasis

PubMed Central

Sontag, Timothy J.; Chellan, Bijoy; Bhanvadia, Clarissa V.; Getz, Godfrey S.; Reardon, Catherine A.

2015-01-01

Macrophage conversion to atherosclerotic foam cells is partly due to the balance of uptake and efflux of cholesterol. Cholesterol efflux from cells by HDL and its apoproteins for subsequent hepatic elimination is known as reverse cholesterol transport. Numerous methods have been developed to measure in vivo macrophage cholesterol efflux. Most methods do not allow for macrophage recovery for analysis of changes in cellular cholesterol status. We describe a novel method for measuring cellular cholesterol balance using the in vivo entrapment of macrophages in alginate, which retains incorporated cells while being permeable to lipoproteins. Recipient mice were injected subcutaneously with CaCl2 forming a bubble into which a macrophage/alginate suspension was injected, entrapping the macrophages. Cells were recovered after 24 h. Cellular free and esterified cholesterol mass were determined enzymatically and normalized to cellular protein. Both normal and cholesterol loaded macrophages undergo measureable changes in cell cholesterol when injected into WT and apoA-I-, LDL-receptor-, or apoE-deficient mice. Cellular cholesterol balance is dependent on initial cellular cholesterol status, macrophage cholesterol transporter expression, and apolipoprotein deficiency. Alginate entrapment allows for the in vivo measurement of macrophage cholesterol homeostasis and is a novel platform for investigating the role of genetics and therapeutic interventions in atherogenesis. PMID:25465389

Biological Targets and Mechanisms of Action of Natural Products from Marine Cyanobacteria

PubMed Central

Salvador-Reyes, Lilibeth A.

2015-01-01

Marine cyanobacteria are an ancient group of organisms and prolific producers of bioactive secondary metabolites. These compounds are presumably optimized by evolution over billions of years to exert high affinity for their intended biological target in the ecologically relevant organism but likely also possess activity in different biological contexts such as human cells. Screening of marine cyanobacterial extracts for bioactive natural products has largely focused on cancer cell viability; however, diversification of the screening platform led to the characterization of many new bioactive compounds. Targets of compounds have oftentimes been elusive if the compounds were discovered through phenotypic assays. Over the past few years, technology has advanced to determine mechanism of action (MOA) and targets through reverse chemical genetic and proteomic approaches, which has been applied to certain cyanobacterial compounds and will be discussed in this review. Some cyanobacterial molecules are the most-potent-in-class inhibitors and therefore may become valuable tools for chemical biology to probe protein function but also be templates for novel drugs, assuming in vitro potency translates into cellular and in vivo activity. Our review will focus on compounds for which the direct targets have been deciphered or which were found to target a novel pathway, and link them to disease states where target modulation may be beneficial. PMID:25571978

Fragman: an R package for fragment analysis.

PubMed

Covarrubias-Pazaran, Giovanny; Diaz-Garcia, Luis; Schlautman, Brandon; Salazar, Walter; Zalapa, Juan

2016-04-21

Determination of microsatellite lengths or other DNA fragment types is an important initial component of many genetic studies such as mutation detection, linkage and quantitative trait loci (QTL) mapping, genetic diversity, pedigree analysis, and detection of heterozygosity. A handful of commercial and freely available software programs exist for fragment analysis; however, most of them are platform dependent and lack high-throughput applicability. We present the R package Fragman to serve as a freely available and platform independent resource for automatic scoring of DNA fragment lengths diversity panels and biparental populations. The program analyzes DNA fragment lengths generated in Applied Biosystems® (ABI) either manually or automatically by providing panels or bins. The package contains additional tools for converting the allele calls to GenAlEx, JoinMap® and OneMap software formats mainly used for genetic diversity and generating linkage maps in plant and animal populations. Easy plotting functions and multiplexing friendly capabilities are some of the strengths of this R package. Fragment analysis using a unique set of cranberry (Vaccinium macrocarpon) genotypes based on microsatellite markers is used to highlight the capabilities of Fragman. Fragman is a valuable new tool for genetic analysis. The package produces equivalent results to other popular software for fragment analysis while possessing unique advantages and the possibility of automation for high-throughput experiments by exploiting the power of R.

Regulation of Meiotic Recombination

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

Gregory p. Copenhaver

Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diversemore » as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system for assaying recombination using tetrad analysis in a higher eukaryotic system (6). This system enabled the measurement of the frequency and distribution of recombination events at a genome wide level in wild type Arabidopsis (7), construction of genetic linkage maps which include positions for each centromere (8), and modeling of the strength and pattern of interference (9). This proposal extends the use of tetrad analysis in Arabidopsis by using it as the basis for assessing the phenotypes of mutants in genes important for recombination and the regulation of crossover interference and performing a novel genetic screen. In addition to broadening our knowledge of a classic genetic problem - the regulation of recombination by crossover interference - this proposal also provides broader impact by: generating pedagogical tools for use in hands-on classroom experience with genetics, building interdisciplinary collegial partnerships, and creating a platform for participation by junior scientists from underrepresented groups. There are three specific aims: (1) Isolate mutants in Arabidopsis MUS81 homologs using T-DNA and TILLING (2) Characterize recombination levels and interference in mus81 mutants (3) Execute a novel genetic screen, based on tetrad analysis, for genes that regulate meiotic recombination« less

EPI-743 reverses the progression of the pediatric mitochondrial disease--genetically defined Leigh Syndrome.

PubMed

Martinelli, Diego; Catteruccia, Michela; Piemonte, Fiorella; Pastore, Anna; Tozzi, Giulia; Dionisi-Vici, Carlo; Pontrelli, Giuseppe; Corsetti, Tiziana; Livadiotti, Susanna; Kheifets, Viktoria; Hinman, Andrew; Shrader, William D; Thoolen, Martin; Klein, Matthew B; Bertini, Enrico; Miller, Guy

2012-11-01

Genetically defined Leigh syndrome is a rare, fatal inherited neurodegenerative disorder that predominantly affects children. No treatment is available. EPI-743 is a novel small molecule developed for the treatment of Leigh syndrome and other inherited mitochondrial diseases. In compassionate use cases and in an FDA Expanded Access protocol, children with Leigh syndrome treated with EPI-743 demonstrated objective signs of neurologic and neuromuscular improvement. To confirm these initial findings, a phase 2A open label trial of EPI-743 for children with genetically-confirmed Leigh syndrome was conducted and herein we report the results. A single arm clinical trial was performed in children with genetically defined Leigh syndrome. Subjects were treated for 6 months with EPI-743 three times daily and all were eligible for a treatment extension phase. The primary objective of the trial was to arrest disease progression as assessed by neuromuscular and quality of life metrics. Results were compared to the reported natural history of the disease. Ten consecutive children, ages 1-13 years, were enrolled; they possessed seven different genetic defects. All children exhibited reversal of disease progression regardless of genetic determinant or disease severity. The primary endpoints--Newcastle Pediatric Mitochondrial Disease Scale, the Gross Motor Function Measure, and PedsQL Neuromuscular Module--demonstrated statistically significant improvement (p<0.05). In addition, all children had an improvement of one class on the Movement Disorder-Childhood Rating Scale. No significant drug-related adverse events were recorded. In comparison to the natural history of Leigh syndrome, EPI-743 improves clinical outcomes in children with genetically confirmed Leigh syndrome. Copyright © 2012 Elsevier Inc. All rights reserved.

A WebGIS platform for the monitoring of Farm Animal Genetic Resources (GENMON)

PubMed Central

Flury, Christine; Matasci, Giona; Joerin, Florent; Widmer, Ivo; Joost, Stéphane

2017-01-01

Background In 2007, the Food and Agriculture Organization of the United Nations (FAO) initiated the Global plan of action for Farm Animal Genetic Resources (FAnGR). The main goal of this plan is to reduce further loss of genetic diversity in farm animals, so as to protect and promote the diversity of farm animal resources. An important step to reach this goal is to monitor and prioritize endangered breeds in the context of conservation programs. Methodology/Web portal implementation The GENMON WebGIS platform is able to monitor FAnGR and to evaluate the degree of endangerment of livestock breeds. The system takes into account pedigree and introgression information, the geographical concentration of animals, the cryo-conservation plan and the sustainability of breeding activities based on socio-economic data as well as present and future land use conditions. A multi-criteria decision tool supports the aggregation of the multi-thematic indices mentioned above using the MACBETH method, which is based on a weighted average using satisfaction thresholds. GENMON is a monitoring tool to reach subjective decisions made by a government agency. It relies on open source software and is available at http://lasigsrv2.epfl.ch/genmon-ch. Results/Significance GENMON allows users to upload pedigree-information (animal ID, parents, birthdate, sex, location and introgression) from a specific livestock breed and to define species and/or region-specific weighting parameters and thresholds. The program then completes a pedigree analysis and derives several indices that are used to calculate an integrated score of conservation prioritization for the breeds under investigation. The score can be visualized on a geographic map and allows a fast, intuitive and regional identification of breeds in danger. Appropriate conservation actions and breeding programs can thus be undertaken in order to promote the recovery of the genetic diversity in livestock breeds in need. The use of the platform is illustrated by means of an example based on three local livestock breeds from different species in Switzerland. PMID:28453561

A WebGIS platform for the monitoring of Farm Animal Genetic Resources (GENMON).

PubMed

Duruz, Solange; Flury, Christine; Matasci, Giona; Joerin, Florent; Widmer, Ivo; Joost, Stéphane

2017-01-01

In 2007, the Food and Agriculture Organization of the United Nations (FAO) initiated the Global plan of action for Farm Animal Genetic Resources (FAnGR). The main goal of this plan is to reduce further loss of genetic diversity in farm animals, so as to protect and promote the diversity of farm animal resources. An important step to reach this goal is to monitor and prioritize endangered breeds in the context of conservation programs. The GENMON WebGIS platform is able to monitor FAnGR and to evaluate the degree of endangerment of livestock breeds. The system takes into account pedigree and introgression information, the geographical concentration of animals, the cryo-conservation plan and the sustainability of breeding activities based on socio-economic data as well as present and future land use conditions. A multi-criteria decision tool supports the aggregation of the multi-thematic indices mentioned above using the MACBETH method, which is based on a weighted average using satisfaction thresholds. GENMON is a monitoring tool to reach subjective decisions made by a government agency. It relies on open source software and is available at http://lasigsrv2.epfl.ch/genmon-ch. GENMON allows users to upload pedigree-information (animal ID, parents, birthdate, sex, location and introgression) from a specific livestock breed and to define species and/or region-specific weighting parameters and thresholds. The program then completes a pedigree analysis and derives several indices that are used to calculate an integrated score of conservation prioritization for the breeds under investigation. The score can be visualized on a geographic map and allows a fast, intuitive and regional identification of breeds in danger. Appropriate conservation actions and breeding programs can thus be undertaken in order to promote the recovery of the genetic diversity in livestock breeds in need. The use of the platform is illustrated by means of an example based on three local livestock breeds from different species in Switzerland.

Efficient assembly of full-length infectious clone of Brazilian IBDV isolate by homologous recombination in yeast

PubMed Central

Silva, J.V.J.; Arenhart, S.; Santos, H.F.; Almeida-Queiroz, S.R.; Silva, A.N.M.R.; Trevisol, I.M.; Bertani, G.R.; Gil, L.H.V.G.

2014-01-01

The Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens. Advances in molecular virology and vaccines for IBDV have been achieved by viral reverse genetics (VRG). VRG for IBDV has undergone changes over time, however all strategies used to generate particles of IBDV involves multiple rounds of amplification and need of in vitro ligation and restriction sites. The aim of this research was to build the world’s first VRG for IBDV by yeast-based homologous recombination; a more efficient, robust and simple process than cloning by in vitro ligation. The wild type IBDV (Wt-IBDV-Br) was isolated in Brazil and had its genome cloned in pJG-CMV-HDR vector by yeast-based homologous recombination. The clones were transfected into chicken embryo fibroblasts and the recovered virus (IC-IBDV-Br) showed genetic stability and similar phenotype to Wt-IBDV-Br, which were observed by nucleotide sequence, focus size/morphology and replication kinetics, respectively. Thus, IBDV reverse genetics by yeast-based homologous recombination provides tools to IBDV understanding and vaccines/viral vectors development. PMID:25763067

Epigenetic understanding of gene-environment interactions in psychiatric disorders: a new concept of clinical genetics

PubMed Central

2012-01-01

Epigenetics is a mechanism that regulates gene expression independently of the underlying DNA sequence, relying instead on the chemical modification of DNA and histone proteins. Although environmental and genetic factors were thought to be independently associated with disorders, several recent lines of evidence suggest that epigenetics bridges these two factors. Epigenetic gene regulation is essential for normal development, thus defects in epigenetics cause various rare congenital diseases. Because epigenetics is a reversible system that can be affected by various environmental factors, such as drugs, nutrition, and mental stress, the epigenetic disorders also include common diseases induced by environmental factors. In this review, we discuss the nature of epigenetic disorders, particularly psychiatric disorders, on the basis of recent findings: 1) susceptibility of the conditions to environmental factors, 2) treatment by taking advantage of their reversible nature, and 3) transgenerational inheritance of epigenetic changes, that is, acquired adaptive epigenetic changes that are passed on to offspring. These recently discovered aspects of epigenetics provide a new concept of clinical genetics. PMID:22414323

Assessment of pleiotropic transcriptome perturbations in Arabidopsis engineered for indirect insect defence.

PubMed

Houshyani, Benyamin; van der Krol, Alexander R; Bino, Raoul J; Bouwmeester, Harro J

2014-06-19

Molecular characterization is an essential step of risk/safety assessment of genetically modified (GM) crops. Holistic approaches for molecular characterization using omics platforms can be used to confirm the intended impact of the genetic engineering, but can also reveal the unintended changes at the omics level as a first assessment of potential risks. The potential of omics platforms for risk assessment of GM crops has rarely been used for this purpose because of the lack of a consensus reference and statistical methods to judge the significance or importance of the pleiotropic changes in GM plants. Here we propose a meta data analysis approach to the analysis of GM plants, by measuring the transcriptome distance to untransformed wild-types. In the statistical analysis of the transcriptome distance between GM and wild-type plants, values are compared with naturally occurring transcriptome distances in non-GM counterparts obtained from a database. Using this approach we show that the pleiotropic effect of genes involved in indirect insect defence traits is substantially equivalent to the variation in gene expression occurring naturally in Arabidopsis. Transcriptome distance is a useful screening method to obtain insight in the pleiotropic effects of genetic modification.

Flexible nanopillar-based electrochemical sensors for genetic detection of foodborne pathogens

NASA Astrophysics Data System (ADS)

Park, Yoo Min; Lim, Sun Young; Jeong, Soon Woo; Song, Younseong; Bae, Nam Ho; Hong, Seok Bok; Choi, Bong Gill; Lee, Seok Jae; Lee, Kyoung G.

2018-06-01

Flexible and highly ordered nanopillar arrayed electrodes have brought great interest for many electrochemical applications, especially to the biosensors, because of its unique mechanical and topological properties. Herein, we report an advanced method to fabricate highly ordered nanopillar electrodes produced by soft-/photo-lithography and metal evaporation. The highly ordered nanopillar array exhibited the superior electrochemical and mechanical properties in regard with the wide space to response with electrolytes, enabling the sensitive analysis. As-prepared gold and silver electrodes on nanopillar arrays exhibit great and stable electrochemical performance to detect the amplified gene from foodborne pathogen of Escherichia coli O157:H7. Additionally, lightweight, flexible, and USB-connectable nanopillar-based electrochemical sensor platform improves the connectivity, portability, and sensitivity. Moreover, we successfully confirm the performance of genetic analysis using real food, specially designed intercalator, and amplified gene from foodborne pathogens with high reproducibility (6% standard deviation) and sensitivity (10 × 1.01 CFU) within 25 s based on the square wave voltammetry principle. This study confirmed excellent mechanical and chemical characteristics of nanopillar electrodes have a great and considerable electrochemical activity to apply as genetic biosensor platform in the fields of point-of-care testing (POCT).

Reproducibility of Illumina platform deep sequencing errors allows accurate determination of DNA barcodes in cells.

PubMed

Beltman, Joost B; Urbanus, Jos; Velds, Arno; van Rooij, Nienke; Rohr, Jan C; Naik, Shalin H; Schumacher, Ton N

2016-04-02

Next generation sequencing (NGS) of amplified DNA is a powerful tool to describe genetic heterogeneity within cell populations that can both be used to investigate the clonal structure of cell populations and to perform genetic lineage tracing. For applications in which both abundant and rare sequences are biologically relevant, the relatively high error rate of NGS techniques complicates data analysis, as it is difficult to distinguish rare true sequences from spurious sequences that are generated by PCR or sequencing errors. This issue, for instance, applies to cellular barcoding strategies that aim to follow the amount and type of offspring of single cells, by supplying these with unique heritable DNA tags. Here, we use genetic barcoding data from the Illumina HiSeq platform to show that straightforward read threshold-based filtering of data is typically insufficient to filter out spurious barcodes. Importantly, we demonstrate that specific sequencing errors occur at an approximately constant rate across different samples that are sequenced in parallel. We exploit this observation by developing a novel approach to filter out spurious sequences. Application of our new method demonstrates its value in the identification of true sequences amongst spurious sequences in biological data sets.

[Biosafety issues and public concerns on recombinant influenza viruses generated in the laboratories].

PubMed

Jia, Xiaojuan; Huang, Liqin; Liu, Wenjun

2013-12-01

Understanding inter-species transmission of influenza viruses is an important research topic. Scientists try to identify and evaluate the functional factors determining the host range of influenza viruses by generating the recombinant viruses through reverse genetics in laboratories, which reveals the viruses' molecular mechanisms of infection and transmission in different species. Therefore, the reverse genetic method is a very important tool for further understanding the biology of influenza viruses and will provide the insight for the prevention and treatment of infections and transmission. However, these recombinant influenza viruses generated in laboratories will become the potential threat to the public health and the environment. In this paper, we discussed the biological safety issues of recombinant influenza viruses and suggested we should set up protocols for risk management on research activities related to recombinant highly pathogenic influenza viruses.

Generation of Recombinant Ebola Viruses Using Reverse Genetics.

PubMed

Groseth, Allison

2017-01-01

Reverse genetics systems encompass a wide array of tools aimed at recapitulating some or all of the virus life cycle. In their most complete form, full-length clone systems allow us to use plasmid-encoded versions of the ribonucleoprotein (RNP) components to initiate the transcription and replication of a plasmid-encoded version of the complete viral genome, thereby initiating the complete virus life cycle and resulting in infectious virus. As such this approach is ideal for the generation of tailor-made recombinant filoviruses, which can be used to study virus biology. In addition, the generation of tagged and particularly fluorescent or luminescent viruses can be applied as tools for both diagnostic applications and for screening to identify novel countermeasures. Here we describe the generation and basic characterization of recombinant Ebola viruses rescued from cloned cDNA using a T7-driven system.

RPPAML/RIMS: A metadata format and an information management system for reverse phase protein arrays

PubMed Central

Stanislaus, Romesh; Carey, Mark; Deus, Helena F; Coombes, Kevin; Hennessy, Bryan T; Mills, Gordon B; Almeida, Jonas S

2008-01-01

Background Reverse Phase Protein Arrays (RPPA) are convenient assay platforms to investigate the presence of biomarkers in tissue lysates. As with other high-throughput technologies, substantial amounts of analytical data are generated. Over 1000 samples may be printed on a single nitrocellulose slide. Up to 100 different proteins may be assessed using immunoperoxidase or immunoflorescence techniques in order to determine relative amounts of protein expression in the samples of interest. Results In this report an RPPA Information Management System (RIMS) is described and made available with open source software. In order to implement the proposed system, we propose a metadata format known as reverse phase protein array markup language (RPPAML). RPPAML would enable researchers to describe, document and disseminate RPPA data. The complexity of the data structure needed to describe the results and the graphic tools necessary to visualize them require a software deployment distributed between a client and a server application. This was achieved without sacrificing interoperability between individual deployments through the use of an open source semantic database, S3DB. This data service backbone is available to multiple client side applications that can also access other server side deployments. The RIMS platform was designed to interoperate with other data analysis and data visualization tools such as Cytoscape. Conclusion The proposed RPPAML data format hopes to standardize RPPA data. Standardization of data would result in diverse client applications being able to operate on the same set of data. Additionally, having data in a standard format would enable data dissemination and data analysis. PMID:19102773

Short infrared laser pulses block action potentials in neurons

NASA Astrophysics Data System (ADS)

Walsh, Alex J.; Tolstykh, Gleb P.; Martens, Stacey L.; Ibey, Bennett L.; Beier, Hope T.

2017-02-01

Short infrared laser pulses have many physiological effects on cells including the ability to stimulate action potentials in neurons. Here we show that short infrared laser pulses can also reversibly block action potentials. Primary rat hippocampal neurons were transfected with the Optopatch2 plasmid, which contains both a blue-light activated channel rhodopsin (CheRiff) and a red-light fluorescent membrane voltage reporter (QuasAr2). This optogenetic platform allows robust stimulation and recording of action potential activity in neurons in a non-contact, low noise manner. For all experiments, QuasAr2 was imaged continuously on a wide-field fluorescent microscope using a Krypton laser (647 nm) as the excitation source and an EMCCD camera operating at 1000 Hz to collect emitted fluorescence. A co-aligned Argon laser (488 nm, 5 ms at 10Hz) provided activation light for CheRiff. A 200 mm fiber delivered infrared light locally to the target neuron. Reversible action potential block in neurons was observed following a short infrared laser pulse (0.26-0.96 J/cm2; 1.37-5.01 ms; 1869 nm), with the block persisting for more than 1 s with exposures greater than 0.69 J/cm2. Action potential block was sustained for 30 s with the short infrared laser pulsed at 1-7 Hz. Full recovery of neuronal activity was observed 5-30s post-infrared exposure. These results indicate that optogenetics provides a robust platform for the study of action potential block and that short infrared laser pulses can be used for non-contact, reversible action potential block.

Research on vehicles and cargos matching model based on virtual logistics platform

NASA Astrophysics Data System (ADS)

Zhuang, Yufeng; Lu, Jiang; Su, Zhiyuan

2018-04-01

Highway less than truckload (LTL) transportation vehicles and cargos matching problem is a joint optimization problem of typical vehicle routing and loading, which is also a hot issue of operational research. This article based on the demand of virtual logistics platform, for the problem of the highway LTL transportation, the matching model of the idle vehicle and the transportation order is set up and the corresponding genetic algorithm is designed. Then the algorithm is implemented by Java. The simulation results show that the solution is satisfactory.

The impact of sex-role reversal on the diversity of the major histocompatibility complex: Insights from the seahorse (Hippocampus abdominalis)

PubMed Central

2011-01-01

Background Both natural and sexual selection are thought to influence genetic diversity, but the study of the relative importance of these two factors on ecologically-relevant traits has traditionally focused on species with conventional sex-roles, with male-male competition and female-based mate choice. With its high variability and significance in both immune function and olfactory-mediated mate choice, the major histocompatibility complex (MHC/MH) is an ideal system in which to evaluate the relative contributions of these two selective forces to genetic diversity. Intrasexual competition and mate choice are both reversed in sex-role reversed species, and sex-related differences in the detection and use of MH-odor cues are expected to influence the intensity of sexual selection in such species. The seahorse, Hippocampus abdominalis, has an exceptionally highly developed form of male parental care, with female-female competition and male mate choice. Results Here, we demonstrate that the sex-role reversed seahorse has a single MH class II beta-chain gene and that the diversity of the seahorse MHIIβ locus and its pattern of variation are comparable to those detected in species with conventional sex roles. Despite the presence of only a single gene copy, intralocus MHIIβ allelic diversity in this species exceeds that observed in species with multiple copies of this locus. The MHIIβ locus of the seahorse exhibits a novel expression domain in the male brood pouch. Conclusions The high variation found at the seahorse MHIIβ gene indicates that sex-role reversed species are capable of maintaining the high MHC diversity typical in most vertebrates. Whether such species have evolved the capacity to use MH-odor cues during mate choice is presently being investigated using mate choice experiments. If this possibility can be rejected, such systems would offer an exceptional opportunity to study the effects of natural selection in isolation, providing powerful comparative models for understanding the relative importance of selective factors in shaping patterns of genetic variation. PMID:21569286

Investigation of Magnetic Field Measurements.

DTIC Science & Technology

1983-02-28

Report) Ill. SUPPLEMENTARY NOTES IS. KEY WORDS (CoEntnue on revere side I necoseer mnd Identify by block mamber) AFGL Magnetometer Network Fluxgate ... Magnetometer Induction Coil Magnetometer Temperature Dependency of Fluxgate Automation of Testing 20. ABSTRACT (Coniniue an reverse aide If neeeeey and...data collection platforms. Support was also provided to AFGL to process the fluxgate magnetometer archive tapes in order to make the data available to

Modulating Retro-Reflectors for Space, Tracking, Acquisition and Ranging using Multiple Quantum Well Technology (Preprint)

DTIC Science & Technology

2002-01-01

feedback signals were derived from the motion of the platform rather than directly measured, though an actual spacecraft would likely utilize... large position error spikes due to target motion reversal. Of course, these tracking errors are highly dependent on the feedback gains chosen for the...Key Words: MQW Retromodulators, Modulating Retroreflector(s),Inter- spacecraft communications and navigation, space control

Repeatable Reverse Engineering with PANDA

DTIC Science & Technology

2015-12-08

that, even without 0e+00 2e+06 4e+06 6e+06 8e+06 1e+07 0 50 00 15 00 0 instruction count ta in te d co m pu ta tio n Fig. 5: Measure of tainted...AAI0807735. [8] Vitaly Chipounov, Volodymyr Kuznetsov, and George Candea. S2E: A platform for in-vivo multi-path analysis of software systems. In

Transfer of a Mycobacterium tuberculosis genotyping method, Spoligotyping, from a reverse line-blot hybridization, membrane-based assay to the Luminex multianalyte profiling system.

PubMed

Cowan, Lauren S; Diem, Lois; Brake, Mary Catherine; Crawford, Jack T

2004-01-01

Spoligotyping using Luminex technology was shown to be a highly reproducible method suitable for high-throughput analysis. Spoligotyping of 48 isolates using the traditional membrane-based assay and the Luminex assay yielded concordant results for all isolates. The Luminex platform provides greater flexibility and cost effectiveness than the membrane-based assay.

Generation of genetically modified mice using CRISPR/Cas9 and haploid embryonic stem cell systems

PubMed Central

JIN, Li-Fang; LI, Jin-Song

2016-01-01

With the development of high-throughput sequencing technology in the post-genomic era, researchers have concentrated their efforts on elucidating the relationships between genes and their corresponding functions. Recently, important progress has been achieved in the generation of genetically modified mice based on CRISPR/Cas9 and haploid embryonic stem cell (haESC) approaches, which provide new platforms for gene function analysis, human disease modeling, and gene therapy. Here, we review the CRISPR/Cas9 and haESC technology for the generation of genetically modified mice and discuss the key challenges in the application of these approaches. PMID:27469251

Dual genetic selection of synthetic riboswitches in Escherichia coli.

PubMed

Nomura, Yoko; Yokobayashi, Yohei

2014-01-01

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

gQTL: A Web Application for QTL Analysis Using the Collaborative Cross Mouse Genetic Reference Population.

PubMed

Konganti, Kranti; Ehrlich, Andre; Rusyn, Ivan; Threadgill, David W

2018-06-07

Multi-parental recombinant inbred populations, such as the Collaborative Cross (CC) mouse genetic reference population, are increasingly being used for analysis of quantitative trait loci (QTL). However specialized analytic software for these complex populations is typically built in R that works only on command-line, which limits the utility of these powerful resources for many users. To overcome analytic limitations, we developed gQTL, a web accessible, simple graphical user interface application based on the DOQTL platform in R to perform QTL mapping using data from CC mice. Copyright © 2018, G3: Genes, Genomes, Genetics.

Acute effects of cocaine and cannabis on reversal learning as a function of COMT and DRD2 genotype.

PubMed

Spronk, Desirée B; Van der Schaaf, Marieke E; Cools, Roshan; De Bruijn, Ellen R A; Franke, Barbara; van Wel, Janelle H P; Ramaekers, Johannes G; Verkes, Robbert J

2016-01-01

Long-term cannabis and cocaine use has been associated with impairments in reversal learning. However, how acute cannabis and cocaine administration affect reversal learning in humans is not known. In this study, we aimed to establish the acute effects of administration of cannabis and cocaine on valence-dependent reversal learning as a function of DRD2 Taq1A (rs1800497) and COMT Val108/158Met (rs4680) genotype. A double-blind placebo-controlled randomized 3-way crossover design was used. Sixty-one regular poly-drug users completed a deterministic reversal learning task under the influence of cocaine, cannabis, and placebo that enabled assessment of both reward- and punishment-based reversal learning. Proportion correct on the reversal learning task was increased by cocaine, but decreased by cannabis. Effects of cocaine depended on the DRD2 genotype, as increases in proportion correct were seen only in the A1 carriers, and not in the A2/A2 homozygotes. COMT genotype did not modulate drug-induced effects on reversal learning. These data indicate that acute administration of cannabis and cocaine has opposite effects on reversal learning. The effects of cocaine, but not cannabis, depend on interindividual genetic differences in the dopamine D2 receptor gene.

Stakeholder perspectives on decision-analytic modeling frameworks to assess genetic services policy.

PubMed

Guzauskas, Gregory F; Garrison, Louis P; Stock, Jacquie; Au, Sylvia; Doyle, Debra Lochner; Veenstra, David L

2013-01-01

Genetic services policymakers and insurers often make coverage decisions in the absence of complete evidence of clinical utility and under budget constraints. We evaluated genetic services stakeholder opinions on the potential usefulness of decision-analytic modeling to inform coverage decisions, and asked them to identify genetic tests for decision-analytic modeling studies. We presented an overview of decision-analytic modeling to members of the Western States Genetic Services Collaborative Reimbursement Work Group and state Medicaid representatives and conducted directed content analysis and an anonymous survey to gauge their attitudes toward decision-analytic modeling. Participants also identified and prioritized genetic services for prospective decision-analytic evaluation. Participants expressed dissatisfaction with current processes for evaluating insurance coverage of genetic services. Some participants expressed uncertainty about their comprehension of decision-analytic modeling techniques. All stakeholders reported openness to using decision-analytic modeling for genetic services assessments. Participants were most interested in application of decision-analytic concepts to multiple-disorder testing platforms, such as next-generation sequencing and chromosomal microarray. Decision-analytic modeling approaches may provide a useful decision tool to genetic services stakeholders and Medicaid decision-makers.

Demographic and genetic consequences of disturbed sex determination.

PubMed

Wedekind, Claus

2017-09-19

During sex determination, genetic and/or environmental factors determine the cascade of processes of gonad development. Many organisms, therefore, have a developmental window in which their sex determination can be sensitive to, for example, unusual temperatures or chemical pollutants. Disturbed environments can distort population sex ratios and may even cause sex reversal in species with genetic sex determination. The resulting genotype-phenotype mismatches can have long-lasting effects on population demography and genetics. I review the theoretical and empirical work in this context and explore in a simple population model the role of the fitness v yy of chromosomally aberrant YY genotypes that are a consequence of environmentally induced feminization. Low v yy is mostly beneficial for population growth. During feminization, low v yy reduces the proportion of genetic males and hence accelerates population growth, especially at low rates of feminization and at high fitness costs of the feminization itself (i.e. when feminization would otherwise not affect population dynamics much). When sex reversal ceases, low v yy mitigates the negative effects of feminization and can even prevent population extinction. Little is known about v yy in natural populations. The available models now need to be parametrized in order to better predict the long-term consequences of disturbed sex determination.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).

Genetics Home Reference: core binding factor acute myeloid leukemia

MedlinePlus

... the CBFB gene. One such rearrangement, called an inversion , involves breakage of a chromosome in two places; ... is reversed and reinserted into the chromosome. The inversion involved in CBF-AML (written as inv(16)) ...

Spatial learning and memory deficits induced by exposure to iron-56-particle radiation

NASA Technical Reports Server (NTRS)

Shukitt-Hale, B.; Casadesus, G.; McEwen, J. J.; Rabin, B. M.; Joseph, J. A.

2000-01-01

It has previously been shown that exposing rats to particles of high energy and charge (HZE) disrupts the functioning of the dopaminergic system and behaviors mediated by this system, such as motor performance and an amphetamine-induced conditioned taste aversion; these adverse behavioral and neuronal effects are similar to those seen in aged animals. Because cognition declines with age, spatial learning and memory were assessed in the Morris water maze 1 month after whole-body irradiation with 1.5 Gy of 1 GeV/nucleon high-energy (56)Fe particles, to test the cognitive behavioral consequences of radiation exposure. Irradiated rats demonstrated cognitive impairment compared to the control group as seen in their increased latencies to find the hidden platform, particularly on the reversal day when the platform was moved to the opposite quadrant. Also, the irradiated group used nonspatial strategies during the probe trials (swim with no platform), i.e. less time spent in the platform quadrant, fewer crossings of and less time spent in the previous platform location, and longer latencies to the previous platform location. These findings are similar to those seen in aged rats, suggesting that an increased release of reactive oxygen species may be responsible for the induction of radiation- and age-related cognitive deficits. If these decrements in behavior also occur in humans, they may impair the ability of astronauts to perform critical tasks during long-term space travel beyond the magnetosphere.

Leveraging annotation-based modeling with Jump.

PubMed

Bergmayr, Alexander; Grossniklaus, Michael; Wimmer, Manuel; Kappel, Gerti

2018-01-01

The capability of UML profiles to serve as annotation mechanism has been recognized in both research and industry. Today's modeling tools offer profiles specific to platforms, such as Java, as they facilitate model-based engineering approaches. However, considering the large number of possible annotations in Java, manually developing the corresponding profiles would only be achievable by huge development and maintenance efforts. Thus, leveraging annotation-based modeling requires an automated approach capable of generating platform-specific profiles from Java libraries. To address this challenge, we present the fully automated transformation chain realized by Jump, thereby continuing existing mapping efforts between Java and UML by emphasizing on annotations and profiles. The evaluation of Jump shows that it scales for large Java libraries and generates profiles of equal or even improved quality compared to profiles currently used in practice. Furthermore, we demonstrate the practical value of Jump by contributing profiles that facilitate reverse engineering and forward engineering processes for the Java platform by applying it to a modernization scenario.

Cargo-Delivery Platforms for Targeted Delivery of Inhibitor Cargos Against Botulism

PubMed Central

Wilson, Brenda A.; Ho, Mengfei

2015-01-01

Delivering therapeutic cargos to specific cell types in vivo poses many technical challenges. There is currently a plethora of drug leads and therapies against numerous diseases, ranging from small molecule compounds to nucleic acids to peptides to proteins with varying binding or enzymatic functions. Many of these candidate therapies have documented potential for mitigating or reversing disease symptoms, if only a means for gaining access to the intracellular target were available. Recent advances in our understanding of the biology of cellular uptake and transport processes and the mode of action of bacterial protein toxins have accelerated the development of toxin-based cargo-delivery vehicle platforms. This review provides an updated survey of the status of available platforms for targeted delivery of therapeutic cargos, outlining various strategies that have been used to deliver different types of cargo into cells. Particular emphasis is placed on the application of toxin-based approaches, examining critical issues that have hampered realization of post-intoxication antitoxins against botulism. PMID:25335885

Cargo-delivery platforms for targeted delivery of inhibitor cargos against botulism.

PubMed

Wilson, Brenda A; Ho, Mengfei

2014-01-01

Delivering therapeutic cargos to specific cell types in vivo poses many technical challenges. There is currently a plethora of drug leads and therapies against numerous diseases, ranging from small molecule compounds to nucleic acids to peptides to proteins with varying binding or enzymatic functions. Many of these candidate therapies have documented potential for mitigating or reversing disease symptoms, if only a means for gaining access to the intracellular target were available. Recent advances in our understanding of the biology of cellular uptake and transport processes and the mode of action of bacterial protein toxins have accelerated the development of toxin-based cargo-delivery vehicle platforms. This review provides an updated survey of the status of available platforms for targeted delivery of therapeutic cargos, outlining various strategies that have been used to deliver different types of cargo into cells. Particular emphasis is placed on the application of toxin-based approaches, examining critical issues that have hampered realization of post-intoxication antitoxins against botulism.

Genetic Modification of Human Pancreatic Progenitor Cells Through Modified mRNA.

PubMed

Lu, Song; Chow, Christie C; Zhou, Junwei; Leung, Po Sing; Tsui, Stephen K; Lui, Kathy O

2016-01-01

In this chapter, we describe a highly efficient genetic modification strategy for human pancreatic progenitor cells using modified mRNA-encoding GFP and Neurogenin-3. The properties of modified mRNA offer an invaluable platform to drive protein expression, which has broad applicability in pathway regulation, directed differentiation, and lineage specification. This approach can also be used to regulate expression of other pivotal transcription factors during pancreas development and might have potential therapeutic values in regenerative medicine.

How demanding is the brain on a reversal task under day and night conditions?

PubMed

Arias, N; Fidalgo, C; Méndez, M; Arias, J L

2015-07-23

Reversal learning has been studied as the process of learning to inhibit previously rewarded actions. These behavioral studies are usually performed during the day, when animals are in their daily period rest. However, how day or night affects spatial reversal learning and the brain regions involved in the learning process are still unknown. We conducted two experiments using the Morris Water Maze under different light-conditions: naïve group (CN, n=8), day group (DY, n=8), control DY group (CDY, n=8) night group (NG, n=8), and control NG group (CNG, n=7). Distance covered, velocity and latencies to reach the platform were examined. After completing these tasks, cytochrome c-oxidase activity (CO) in several brain limbic system structures was compared between groups. There were no behavioral differences in the time of day when the animals were trained. However, the metabolic brain consumption was higher in rats trained in the day condition. This CO increase was supported by the prefrontal cortex, thalamus, dorsal and ventral striatum, hippocampus and entorhinal cortex, revealing their role in the performance of the spatial reversal learning task. Finally, the orbitofrontal cortex has been revealed as a key structure in reversal learning execution. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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.

The near demise and subsequent revival of classical genetics for investigating Caenorhabditis elegans embryogenesis: RNAi meets next-generation DNA sequencing.

PubMed

Bowerman, Bruce

2011-10-01

Molecular genetic investigation of the early Caenorhabditis elegans embryo has contributed substantially to the discovery and general understanding of the genes, pathways, and mechanisms that regulate and execute developmental and cell biological processes. Initially, worm geneticists relied exclusively on a classical genetics approach, isolating mutants with interesting phenotypes after mutagenesis and then determining the identity of the affected genes. Subsequently, the discovery of RNA interference (RNAi) led to a much greater reliance on a reverse genetics approach: reducing the function of known genes with RNAi and then observing the phenotypic consequences. Now the advent of next-generation DNA sequencing technologies and the ensuing ease and affordability of whole-genome sequencing are reviving the use of classical genetics to investigate early C. elegans embryogenesis.

Tissue-Engineered Skeletal Muscle Organoids for Reversible Gene Therapy

NASA Technical Reports Server (NTRS)

Vandenburgh, Herman; DelTatto, Michael; Shansky, Janet; Lemaire, Julie; Chang, Albert; Payumo, Francis; Lee, Peter; Goodyear, Amy; Raven, Latasha

1996-01-01

Genetically modified murine skeletal myoblasts were tissue engineered in vitro into organ-like structures (organoids) containing only postmitotic myoribers secreting pharmacological levels of recombinant human growth hormone (rhGH). Subcutaneous organoid implantation under tension led to the rapid and stable appearance of physiological sera levels of rhGH for up to 12 weeks, whereas surgical removal led to its rapid disappearance. Reversible delivery of bioactive compounds from postmitotic cells in tissue engineered organs has several advantages over other forms of muscle gene therapy.

Tissue-Engineered Skeletal Muscle Organoids for Reversible Gene Therapy

NASA Technical Reports Server (NTRS)

Vandenburgh, Herman; DelTatto, Michael; Shansky, Janet; Lemaire, Julie; Chang, Albert; Payumo, Francis; Lee, Peter; Goodyear, Amy; Raven, Latasha

1996-01-01

Genetically modified murine skeletal myoblasts were tissue engineered in vitro into organ-like structures (organoids) containing only postmitotic myofibers secreting pharmacological levels of recombinant human growth hormone (rhGH). Subcutaneous organoid Implantation under tension led to the rapid and stable appearance of physiological sera levels of rhGH for up to 12 weeks, whereas surgical removal led to its rapid disappearance. Reversible delivery of bioactive compounds from postimtotic cells in tissue engineered organs has several advantages over other forms of muscle gene therapy.

Automated electrotransformation of Escherichia coli on a digital microfluidic platform using bioactivated magnetic beads.

PubMed

Moore, J A; Nemat-Gorgani, M; Madison, A C; Sandahl, M A; Punnamaraju, S; Eckhardt, A E; Pollack, M G; Vigneault, F; Church, G M; Fair, R B; Horowitz, M A; Griffin, P B

2017-01-01

This paper reports on the use of a digital microfluidic platform to perform multiplex automated genetic engineering (MAGE) cycles on droplets containing Escherichia coli cells. Bioactivated magnetic beads were employed for cell binding, washing, and media exchange in the preparation of electrocompetent cells in the electrowetting-on-dieletric (EWoD) platform. On-cartridge electroporation was used to deliver oligonucleotides into the cells. In addition to the optimization of a magnetic bead-based benchtop protocol for generating and transforming electrocompetent E. coli cells, we report on the implementation of this protocol in a fully automated digital microfluidic platform. Bead-based media exchange and electroporation pulse conditions were optimized on benchtop for transformation frequency to provide initial parameters for microfluidic device trials. Benchtop experiments comparing electrotransformation of free and bead-bound cells are presented. Our results suggest that dielectric shielding intrinsic to bead-bound cells significantly reduces electroporation field exposure efficiency. However, high transformation frequency can be maintained in the presence of magnetic beads through the application of more intense electroporation pulses. As a proof of concept, MAGE cycles were successfully performed on a commercial EWoD cartridge using variations of the optimal magnetic bead-based preparation procedure and pulse conditions determined by the benchtop results. Transformation frequencies up to 22% were achieved on benchtop; this frequency was matched within 1% (21%) by MAGE cycles on the microfluidic device. However, typical frequencies on the device remain lower, averaging 9% with a standard deviation of 9%. The presented results demonstrate the potential of digital microfluidics to perform complex and automated genetic engineering protocols.

Automated electrotransformation of Escherichia coli on a digital microfluidic platform using bioactivated magnetic beads

PubMed Central

Moore, J. A.; Nemat-Gorgani, M.; Madison, A. C.; Punnamaraju, S.; Eckhardt, A. E.; Pollack, M. G.; Church, G. M.; Fair, R. B.; Horowitz, M. A.; Griffin, P. B.

2017-01-01

This paper reports on the use of a digital microfluidic platform to perform multiplex automated genetic engineering (MAGE) cycles on droplets containing Escherichia coli cells. Bioactivated magnetic beads were employed for cell binding, washing, and media exchange in the preparation of electrocompetent cells in the electrowetting-on-dieletric (EWoD) platform. On-cartridge electroporation was used to deliver oligonucleotides into the cells. In addition to the optimization of a magnetic bead-based benchtop protocol for generating and transforming electrocompetent E. coli cells, we report on the implementation of this protocol in a fully automated digital microfluidic platform. Bead-based media exchange and electroporation pulse conditions were optimized on benchtop for transformation frequency to provide initial parameters for microfluidic device trials. Benchtop experiments comparing electrotransformation of free and bead-bound cells are presented. Our results suggest that dielectric shielding intrinsic to bead-bound cells significantly reduces electroporation field exposure efficiency. However, high transformation frequency can be maintained in the presence of magnetic beads through the application of more intense electroporation pulses. As a proof of concept, MAGE cycles were successfully performed on a commercial EWoD cartridge using variations of the optimal magnetic bead-based preparation procedure and pulse conditions determined by the benchtop results. Transformation frequencies up to 22% were achieved on benchtop; this frequency was matched within 1% (21%) by MAGE cycles on the microfluidic device. However, typical frequencies on the device remain lower, averaging 9% with a standard deviation of 9%. The presented results demonstrate the potential of digital microfluidics to perform complex and automated genetic engineering protocols. PMID:28191268

Automatic Compilation from High-Level Biologically-Oriented Programming Language to Genetic Regulatory Networks

PubMed Central

Beal, Jacob; Lu, Ting; Weiss, Ron

2011-01-01

Background The field of synthetic biology promises to revolutionize our ability to engineer biological systems, providing important benefits for a variety of applications. Recent advances in DNA synthesis and automated DNA assembly technologies suggest that it is now possible to construct synthetic systems of significant complexity. However, while a variety of novel genetic devices and small engineered gene networks have been successfully demonstrated, the regulatory complexity of synthetic systems that have been reported recently has somewhat plateaued due to a variety of factors, including the complexity of biology itself and the lag in our ability to design and optimize sophisticated biological circuitry. Methodology/Principal Findings To address the gap between DNA synthesis and circuit design capabilities, we present a platform that enables synthetic biologists to express desired behavior using a convenient high-level biologically-oriented programming language, Proto. The high level specification is compiled, using a regulatory motif based mechanism, to a gene network, optimized, and then converted to a computational simulation for numerical verification. Through several example programs we illustrate the automated process of biological system design with our platform, and show that our compiler optimizations can yield significant reductions in the number of genes () and latency of the optimized engineered gene networks. Conclusions/Significance Our platform provides a convenient and accessible tool for the automated design of sophisticated synthetic biological systems, bridging an important gap between DNA synthesis and circuit design capabilities. Our platform is user-friendly and features biologically relevant compiler optimizations, providing an important foundation for the development of sophisticated biological systems. PMID:21850228

Automatic compilation from high-level biologically-oriented programming language to genetic regulatory networks.

PubMed

Beal, Jacob; Lu, Ting; Weiss, Ron

2011-01-01

The field of synthetic biology promises to revolutionize our ability to engineer biological systems, providing important benefits for a variety of applications. Recent advances in DNA synthesis and automated DNA assembly technologies suggest that it is now possible to construct synthetic systems of significant complexity. However, while a variety of novel genetic devices and small engineered gene networks have been successfully demonstrated, the regulatory complexity of synthetic systems that have been reported recently has somewhat plateaued due to a variety of factors, including the complexity of biology itself and the lag in our ability to design and optimize sophisticated biological circuitry. To address the gap between DNA synthesis and circuit design capabilities, we present a platform that enables synthetic biologists to express desired behavior using a convenient high-level biologically-oriented programming language, Proto. The high level specification is compiled, using a regulatory motif based mechanism, to a gene network, optimized, and then converted to a computational simulation for numerical verification. Through several example programs we illustrate the automated process of biological system design with our platform, and show that our compiler optimizations can yield significant reductions in the number of genes (~ 50%) and latency of the optimized engineered gene networks. Our platform provides a convenient and accessible tool for the automated design of sophisticated synthetic biological systems, bridging an important gap between DNA synthesis and circuit design capabilities. Our platform is user-friendly and features biologically relevant compiler optimizations, providing an important foundation for the development of sophisticated biological systems.

Genetics educational needs in China: physicians' experience and knowledge of genetic testing.

PubMed

Li, Jing; Xu, Tengda; Yashar, Beverly M

2015-09-01

The aims of this study were to explore the relationship between physicians' knowledge and utilization of genetic testing and to explore genetics educational needs in China. An anonymous survey about experience, attitudes, and knowledge of genetic testing was conducted among physicians affiliated with Peking Union Medical College Hospital during their annual health evaluation. A personal genetics knowledge score was developed and predictors of personal genetics knowledge score were evaluated. Sixty-four physicians (33% male) completed the survey. Fifty-eight percent of them had used genetic testing in their clinical practice. Using a 4-point scale, mean knowledge scores of six common genetic testing techniques ranged from 1.7 ± 0.9 to 2.4 ± 1.0, and the average personal genetics knowledge score was 2.1 ± 0.8. In regression analysis, significant predictors of higher personal genetics knowledge score were ordering of genetic testing, utilization of pedigrees, higher medical degree, and recent genetics training (P < 0.05). Sixty-six percent of physicians indicated a desire for specialized genetic services, and 84% reported a desire for additional genetics education. This study demonstrated a sizable gap between Chinese physicians' knowledge and utilization of genetic testing. Participants had high self-perceived genetics educational needs. Development of genetics educational platforms is both warranted and desired in China.Genet Med 17 9, 757-760.

Electrostatically self-assembled polyoxometalates on molecular-dye-functionalized diamond.

PubMed

Zhong, Yu Lin; Ng, Wibowo; Yang, Jia-Xiang; Loh, Kian Ping

2009-12-30

We have successfully immobilized phosphotungstic acid (PTA), a polyoxometalate, on the surface of boron-doped diamond (BDD) surface through electrostatic self-assembly of PTA on pyridinium dye-functionalized-BDD. The inorganic/organic bilayer structure on BDD is found to exhibit fast surface-confined reversible electron transfer. The molecular dye-grafted BDD can undergo controllable electrical stripping and regeneration of PTA which can be useful for electronics or sensing applications. Furthermore, we have demonstrated the use of PTA as a molecular switch in which the direction of photocurrent from diamond to methyl viologen is reversed by the surface bound PTA. Robust photocurrent converter based on such molecular system-diamond platform can operate in corrosive medium which is not tolerated by indium tin oxide electrodes.

Supramolecular biomaterials

NASA Astrophysics Data System (ADS)

Webber, Matthew J.; Appel, Eric A.; Meijer, E. W.; Langer, Robert

2016-01-01

Polymers, ceramics and metals have historically dominated the application of materials in medicine. Yet rationally designed materials that exploit specific, directional, tunable and reversible non-covalent interactions offer unprecedented advantages: they enable modular and generalizable platforms with tunable mechanical, chemical and biological properties. Indeed, the reversible nature of supramolecular interactions gives rise to biomaterials that can sense and respond to physiological cues, or that mimic the structural and functional aspects of biological signalling. In this Review, we discuss the properties of several supramolecular biomaterials, as well as their applications in drug delivery, tissue engineering, regenerative medicine and immunology. We envision that supramolecular biomaterials will contribute to the development of new therapies that combine highly functional materials with unmatched patient- and application-specific tailoring of both material and biological properties.

A mechanical cell disruption microfluidic platform based on an on-chip micropump.

PubMed

Cheng, Yinuo; Wang, Yue; Wang, Zhiyuan; Huang, Liang; Bi, Mingzhao; Xu, Wenxiao; Wang, Wenhui; Ye, Xiongying

2017-03-01

Cell disruption plays a vital role in detection of intracellular components which contain information about genetic and disease characteristics. In this paper, we demonstrate a novel microfluidic platform based on an on-chip micropump for mechanical cell disruption and sample transport. A 50  μ l cell sample can be effectively lysed through on-chip multi-disruption in 36 s without introducing any chemical agent and suffering from clogging by cellular debris. After 30 cycles of circulating disruption, 80.6% and 90.5% cell disruption rates were achieved for the HEK293 cell sample and human natural killer cell sample, respectively. Profiting from the feature of pump-on-chip, the highly integrated platform enables more convenient and cost-effective cell disruption for the analysis of intracellular components.

A mechanical cell disruption microfluidic platform based on an on-chip micropump

PubMed Central

Cheng, Yinuo; Wang, Yue; Wang, Zhiyuan; Bi, Mingzhao; Xu, Wenxiao; Ye, Xiongying

2017-01-01

Cell disruption plays a vital role in detection of intracellular components which contain information about genetic and disease characteristics. In this paper, we demonstrate a novel microfluidic platform based on an on-chip micropump for mechanical cell disruption and sample transport. A 50 μl cell sample can be effectively lysed through on-chip multi-disruption in 36 s without introducing any chemical agent and suffering from clogging by cellular debris. After 30 cycles of circulating disruption, 80.6% and 90.5% cell disruption rates were achieved for the HEK293 cell sample and human natural killer cell sample, respectively. Profiting from the feature of pump-on-chip, the highly integrated platform enables more convenient and cost-effective cell disruption for the analysis of intracellular components. PMID:28798848

A High-Throughput Regeneration and Transformation Platform for Production of Genetically Modified Banana.

PubMed

Tripathi, Jaindra N; Oduor, Richard O; Tripathi, Leena

2015-01-01

Banana (Musa spp.) is an important staple food as well as cash crop in tropical and subtropical countries. Various bacterial, fungal, and viral diseases and pests such as nematodes are major constraints in its production and are currently destabilizing the banana production in sub-Saharan Africa. Genetic engineering is a complementary option used for incorporating useful traits in banana to bypass the long generation time, polyploidy, and sterility of most of the cultivated varieties. A robust transformation protocol for farmer preferred varieties is crucial for banana genomics and improvement. A robust and reproducible system for genetic transformation of banana using embryogenic cell suspensions (ECS) has been developed in this study. Two different types of explants (immature male flowers and multiple buds) were tested for their ability to develop ECS in several varieties of banana locally grown in Africa. ECS of banana varieties "Cavendish Williams" and "Gros Michel" were developed using multiple buds, whereas ECS of "Sukali Ndiizi" was developed using immature male flowers. Regeneration efficiency of ECS was about 20,000-50,000 plantlets per ml of settled cell volume (SCV) depending on variety. ECS of three different varieties were transformed through Agrobacterium-mediated transformation using gusA reporter gene and 20-70 independent transgenic events per ml SCV of ECS were regenerated on selective medium. The presence and integration of gusA gene in transgenic plants was confirmed by PCR, dot blot, and Southern blot analysis and expression by histochemical GUS assays. The robust transformation platform was successfully used to generate hundreds of transgenic lines with disease resistance. Such a platform will facilitate the transfer of technologies to national agricultural research systems (NARS) in Africa.

A High-Throughput Regeneration and Transformation Platform for Production of Genetically Modified Banana

PubMed Central

Tripathi, Jaindra N.; Oduor, Richard O.; Tripathi, Leena

2015-01-01

Banana (Musa spp.) is an important staple food as well as cash crop in tropical and subtropical countries. Various bacterial, fungal, and viral diseases and pests such as nematodes are major constraints in its production and are currently destabilizing the banana production in sub-Saharan Africa. Genetic engineering is a complementary option used for incorporating useful traits in banana to bypass the long generation time, polyploidy, and sterility of most of the cultivated varieties. A robust transformation protocol for farmer preferred varieties is crucial for banana genomics and improvement. A robust and reproducible system for genetic transformation of banana using embryogenic cell suspensions (ECS) has been developed in this study. Two different types of explants (immature male flowers and multiple buds) were tested for their ability to develop ECS in several varieties of banana locally grown in Africa. ECS of banana varieties “Cavendish Williams” and “Gros Michel” were developed using multiple buds, whereas ECS of “Sukali Ndiizi” was developed using immature male flowers. Regeneration efficiency of ECS was about 20,000–50,000 plantlets per ml of settled cell volume (SCV) depending on variety. ECS of three different varieties were transformed through Agrobacterium-mediated transformation using gusA reporter gene and 20–70 independent transgenic events per ml SCV of ECS were regenerated on selective medium. The presence and integration of gusA gene in transgenic plants was confirmed by PCR, dot blot, and Southern blot analysis and expression by histochemical GUS assays. The robust transformation platform was successfully used to generate hundreds of transgenic lines with disease resistance. Such a platform will facilitate the transfer of technologies to national agricultural research systems (NARS) in Africa. PMID:26635849

Whole exome sequencing is an efficient, sensitive and specific method of mutation detection in osteogenesis imperfecta and Marfan syndrome

PubMed Central

McInerney-Leo, Aideen M; Marshall, Mhairi S; Gardiner, Brooke; Coucke, Paul J; Van Laer, Lut; Loeys, Bart L; Summers, Kim M; Symoens, Sofie; West, Jennifer A; West, Malcolm J; Paul Wordsworth, B; Zankl, Andreas; Leo, Paul J; Brown, Matthew A; Duncan, Emma L

2013-01-01

Osteogenesis imperfecta (OI) and Marfan syndrome (MFS) are common Mendelian disorders. Both conditions are usually diagnosed clinically, as genetic testing is expensive due to the size and number of potentially causative genes and mutations. However, genetic testing may benefit patients, at-risk family members and individuals with borderline phenotypes, as well as improving genetic counseling and allowing critical differential diagnoses. We assessed whether whole exome sequencing (WES) is a sensitive method for mutation detection in OI and MFS. WES was performed on genomic DNA from 13 participants with OI and 10 participants with MFS who had known mutations, with exome capture followed by massive parallel sequencing of multiplexed samples. Single nucleotide polymorphisms (SNPs) and small indels were called using Genome Analysis Toolkit (GATK) and annotated with ANNOVAR. CREST, exomeCopy and exomeDepth were used for large deletion detection. Results were compared with the previous data. Specificity was calculated by screening WES data from a control population of 487 individuals for mutations in COL1A1, COL1A2 and FBN1. The target capture of five exome capture platforms was compared. All 13 mutations in the OI cohort and 9/10 in the MFS cohort were detected (sensitivity=95.6%) including non-synonymous SNPs, small indels (<10 bp), and a large UTR5/exon 1 deletion. One mutation was not detected by GATK due to strand bias. Specificity was 99.5%. Capture platforms and analysis programs differed considerably in their ability to detect mutations. Consumable costs for WES were low. WES is an efficient, sensitive, specific and cost-effective method for mutation detection in patients with OI and MFS. Careful selection of platform and analysis programs is necessary to maximize success. PMID:24501682

MyGeneFriends: A Social Network Linking Genes, Genetic Diseases, and Researchers

PubMed Central

Allot, Alexis; Chennen, Kirsley; Nevers, Yannis; Poidevin, Laetitia; Kress, Arnaud; Ripp, Raymond; Thompson, Julie Dawn; Poch, Olivier

2017-01-01

Background The constant and massive increase of biological data offers unprecedented opportunities to decipher the function and evolution of genes and their roles in human diseases. However, the multiplicity of sources and flow of data mean that efficient access to useful information and knowledge production has become a major challenge. This challenge can be addressed by taking inspiration from Web 2.0 and particularly social networks, which are at the forefront of big data exploration and human-data interaction. Objective MyGeneFriends is a Web platform inspired by social networks, devoted to genetic disease analysis, and organized around three types of proactive agents: genes, humans, and genetic diseases. The aim of this study was to improve exploration and exploitation of biological, postgenomic era big data. Methods MyGeneFriends leverages conventions popularized by top social networks (Facebook, LinkedIn, etc), such as networks of friends, profile pages, friendship recommendations, affinity scores, news feeds, content recommendation, and data visualization. Results MyGeneFriends provides simple and intuitive interactions with data through evaluation and visualization of connections (friendships) between genes, humans, and diseases. The platform suggests new friends and publications and allows agents to follow the activity of their friends. It dynamically personalizes information depending on the user’s specific interests and provides an efficient way to share information with collaborators. Furthermore, the user’s behavior itself generates new information that constitutes an added value integrated in the network, which can be used to discover new connections between biological agents. Conclusions We have developed MyGeneFriends, a Web platform leveraging conventions from popular social networks to redefine the relationship between humans and biological big data and improve human processing of biomedical data. MyGeneFriends is available at lbgi.fr/mygenefriends. PMID:28623182

Genetic diagnosis of Duchenne and Becker muscular dystrophy using next-generation sequencing technology: comprehensive mutational search in a single platform.

PubMed

Lim, Byung Chan; Lee, Seungbok; Shin, Jong-Yeon; Kim, Jong-Il; Hwang, Hee; Kim, Ki Joong; Hwang, Yong Seung; Seo, Jeong-Sun; Chae, Jong Hee

2011-11-01

Duchenne muscular dystrophy or Becker muscular dystrophy might be a suitable candidate disease for application of next-generation sequencing in the genetic diagnosis because the complex mutational spectrum and the large size of the dystrophin gene require two or more analytical methods and have a high cost. The authors tested whether large deletions/duplications or small mutations, such as point mutations or short insertions/deletions of the dystrophin gene, could be predicted accurately in a single platform using next-generation sequencing technology. A custom solution-based target enrichment kit was designed to capture whole genomic regions of the dystrophin gene and other muscular-dystrophy-related genes. A multiplexing strategy, wherein four differently bar-coded samples were captured and sequenced together in a single lane of the Illumina Genome Analyser, was applied. The study subjects were 25 16 with deficient dystrophin expression without a large deletion/duplication and 9 with a known large deletion/duplication. Nearly 100% of the exonic region of the dystrophin gene was covered by at least eight reads with a mean read depth of 107. Pathogenic small mutations were identified in 15 of the 16 patients without a large deletion/duplication. Using these 16 patients as the standard, the authors' method accurately predicted the deleted or duplicated exons in the 9 patients with known mutations. Inclusion of non-coding regions and paired-end sequence analysis enabled accurate identification by increasing the read depth and providing information about the breakpoint junction. The current method has an advantage for the genetic diagnosis of Duchenne muscular dystrophy and Becker muscular dystrophy wherein a comprehensive mutational search may be feasible using a single platform.

JRC GMO-Matrix: a web application to support Genetically Modified Organisms detection strategies.

PubMed

Angers-Loustau, Alexandre; Petrillo, Mauro; Bonfini, Laura; Gatto, Francesco; Rosa, Sabrina; Patak, Alexandre; Kreysa, Joachim

2014-12-30

The polymerase chain reaction (PCR) is the current state of the art technique for DNA-based detection of Genetically Modified Organisms (GMOs). A typical control strategy starts by analyzing a sample for the presence of target sequences (GM-elements) known to be present in many GMOs. Positive findings from this "screening" are then confirmed with GM (event) specific test methods. A reliable knowledge of which GMOs are detected by combinations of GM-detection methods is thus crucial to minimize the verification efforts. In this article, we describe a novel platform that links the information of two unique databases built and maintained by the European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) at the Joint Research Centre (JRC) of the European Commission, one containing the sequence information of known GM-events and the other validated PCR-based detection and identification methods. The new platform compiles in silico determinations of the detection of a wide range of GMOs by the available detection methods using existing scripts that simulate PCR amplification and, when present, probe binding. The correctness of the information has been verified by comparing the in silico conclusions to experimental results for a subset of forty-nine GM events and six methods. The JRC GMO-Matrix is unique for its reliance on DNA sequence data and its flexibility in integrating novel GMOs and new detection methods. Users can mine the database using a set of web interfaces that thus provide a valuable support to GMO control laboratories in planning and evaluating their GMO screening strategies. The platform is accessible at http://gmo-crl.jrc.ec.europa.eu/jrcgmomatrix/ .

Microfabricated Genomic Analysis System

NASA Technical Reports Server (NTRS)

Gonda, Steve; Elms, Rene

2005-01-01

Genetic sequencing and many genetic tests and assays require electrophoretic separation of DNA. In this technique, DNA fragments are separated by size as they migrate through a sieving gel under the influence of an applied electric field. In order to conduct these analyses on-orbit, it is essential to acquire the capability to efficiently perform electrophoresis in a microgravity environment. Conventional bench top electrophoresis equipment is large and cumbersome and does not lead itself to on-orbit utilization. Much of the previous research regarding on-orbit electrophoresis involved altering conventional electrophoresis equipment for bioprocessing, purification, and/or separation technology applications. A new and more efficient approach to on-orbit electrophoresis is the use of a microfabricated electrophoresis platform. These platforms are much smaller, less expensive to produce and operate, use less power, require smaller sample sizes (nanoliters), and achieve separation in a much shorter distance (a few centimeters instead of 10 s or 100 s of centimeters.) In contrast to previous applications, this platform would be utilized as an analytical tool for life science/medical research, environmental monitoring, and medical diagnoses. Identification of infectious agents as well as radiation related damage are significant to NASA s efforts to maintain, study, and monitor crew health during and in support of near-Earth and interplanetary missions. The capability to perform genetic assays on-orbit is imperative to conduct relevant and insightful biological and medical research, as well as continuing NASA s search for life elsewhere. This technology would provide an essential analytical tool for research conducted in a microgravity environment (Shuttle, ISS, long duration/interplanetary missions.) In addition, this technology could serve as a critical and invaluable component of a biosentinel system to monitor space environment genotoxic insults to include radiation.

Coverage Bias and Sensitivity of Variant Calling for Four Whole-genome Sequencing Technologies

PubMed Central

Lasitschka, Bärbel; Jones, David; Northcott, Paul; Hutter, Barbara; Jäger, Natalie; Kool, Marcel; Taylor, Michael; Lichter, Peter; Pfister, Stefan; Wolf, Stephan; Brors, Benedikt; Eils, Roland

2013-01-01

The emergence of high-throughput, next-generation sequencing technologies has dramatically altered the way we assess genomes in population genetics and in cancer genomics. Currently, there are four commonly used whole-genome sequencing platforms on the market: Illumina’s HiSeq2000, Life Technologies’ SOLiD 4 and its completely redesigned 5500xl SOLiD, and Complete Genomics’ technology. A number of earlier studies have compared a subset of those sequencing platforms or compared those platforms with Sanger sequencing, which is prohibitively expensive for whole genome studies. Here we present a detailed comparison of the performance of all currently available whole genome sequencing platforms, especially regarding their ability to call SNVs and to evenly cover the genome and specific genomic regions. Unlike earlier studies, we base our comparison on four different samples, allowing us to assess the between-sample variation of the platforms. We find a pronounced GC bias in GC-rich regions for Life Technologies’ platforms, with Complete Genomics performing best here, while we see the least bias in GC-poor regions for HiSeq2000 and 5500xl. HiSeq2000 gives the most uniform coverage and displays the least sample-to-sample variation. In contrast, Complete Genomics exhibits by far the smallest fraction of bases not covered, while the SOLiD platforms reveal remarkable shortcomings, especially in covering CpG islands. When comparing the performance of the four platforms for calling SNPs, HiSeq2000 and Complete Genomics achieve the highest sensitivity, while the SOLiD platforms show the lowest false positive rate. Finally, we find that integrating sequencing data from different platforms offers the potential to combine the strengths of different technologies. In summary, our results detail the strengths and weaknesses of all four whole-genome sequencing platforms. It indicates application areas that call for a specific sequencing platform and disallow other platforms. This helps to identify the proper sequencing platform for whole genome studies with different application scopes. PMID:23776689

Mendelian randomization in nutritional epidemiology

PubMed Central

Qi, Lu

2013-01-01

Nutritional epidemiology aims to identify dietary and lifestyle causes for human diseases. Causality inference in nutritional epidemiology is largely based on evidence from studies of observational design, and may be distorted by unmeasured or residual confounding and reverse causation. Mendelian randomization is a recently developed methodology that combines genetic and classical epidemiological analysis to infer causality for environmental exposures, based on the principle of Mendel’s law of independent assortment. Mendelian randomization uses genetic variants as proxiesforenvironmentalexposuresofinterest.AssociationsderivedfromMendelian randomization analysis are less likely to be affected by confounding and reverse causation. During the past 5 years, a body of studies examined the causal effects of diet/lifestyle factors and biomarkers on a variety of diseases. The Mendelian randomization approach also holds considerable promise in the study of intrauterine influences on offspring health outcomes. However, the application of Mendelian randomization in nutritional epidemiology has some limitations. PMID:19674341

Transposable elements in sexual and ancient asexual taxa

PubMed Central

Arkhipova, Irina; Meselson, Matthew

2000-01-01

Sexual reproduction allows deleterious transposable elements to proliferate in populations, whereas the loss of sex, by preventing their spread, has been predicted eventually to result in a population free of such elements [Hickey, D. A. (1982) Genetics 101, 519–531]. We tested this expectation by screening representatives of a majority of animal phyla for LINE-like and gypsy-like reverse transcriptases and mariner/Tc1-like transposases. All species tested positive for reverse transcriptases except rotifers of the class Bdelloidea, the largest eukaryotic taxon in which males, hermaphrodites, and meiosis are unknown and for which ancient asexuality is supported by molecular genetic evidence. Mariner-like transposases are distributed sporadically among species and are present in bdelloid rotifers. The remarkable lack of LINE-like and gypsy-like retrotransposons in bdelloids and their ubiquitous presence in other taxa support the view that eukaryotic retrotransposons are sexually transmitted nuclear parasites and that bdelloid rotifers evolved asexually. PMID:11121049

Interactions Between Genetics, Lifestyle, and Environmental Factors for Healthcare.

PubMed

Lin, Yuxin; Chen, Jiajia; Shen, Bairong

2017-01-01

The occurrence and progression of diseases are strongly associated with a combination of genetic, lifestyle, and environmental factors. Understanding the interplay between genetic and nongenetic components provides deep insights into disease pathogenesis and promotes personalized strategies for people healthcare. Recently, the paradigm of systems medicine, which integrates biomedical data and knowledge at multidimensional levels, is considered to be an optimal way for disease management and clinical decision-making in the era of precision medicine. In this chapter, epigenetic-mediated genetics-lifestyle-environment interactions within specific diseases and different ethnic groups are systematically discussed, and data sources, computational models, and translational platforms for systems medicine research are sequentially presented. Moreover, feasible suggestions on precision healthcare and healthy longevity are kindly proposed based on the comprehensive review of current studies.

Genetics and Genomics of Acute Neurologic Disorders.

PubMed

Maserati, Megan; Alexander, Sheila A

2018-01-01

Neurologic diseases and injuries are complex and multifactorial, making risk prediction, targeted treatment modalities, and outcome prognostication difficult and elusive. Genetics and genomics have affected clinical practice in many aspects in medicine, particularly cancer treatment. Advancements in knowledge of genetic and genomic variability in neurologic disease and injury are growing rapidly. Although these data are not yet ready for use in clinical practice, research continues to progress and elucidate information that eventually will provide answers to complex neurologic questions and serve as a platform to provide individualized care plans aimed at improving outcomes. This article provides a focused review of relevant literature on genetics, genomics, and common complex neurologic disease and injury likely to be seen in the acute care setting. ©2018 American Association of Critical-Care Nurses.

Genome Wide Identification of SARS-CoV Susceptibility Loci Using the Collaborative Cross

PubMed Central

Gralinski, Lisa E.; Ferris, Martin T.; Aylor, David L.; Whitmore, Alan C.; Green, Richard; Frieman, Matthew B.; Deming, Damon; Menachery, Vineet D.; Miller, Darla R.; Buus, Ryan J.; Bell, Timothy A.; Churchill, Gary A.; Threadgill, David W.; Katze, Michael G.; McMillan, Leonard; Valdar, William; Heise, Mark T.; Pardo-Manuel de Villena, Fernando; Baric, Ralph S.

2015-01-01

New systems genetics approaches are needed to rapidly identify host genes and genetic networks that regulate complex disease outcomes. Using genetically diverse animals from incipient lines of the Collaborative Cross mouse panel, we demonstrate a greatly expanded range of phenotypes relative to classical mouse models of SARS-CoV infection including lung pathology, weight loss and viral titer. Genetic mapping revealed several loci contributing to differential disease responses, including an 8.5Mb locus associated with vascular cuffing on chromosome 3 that contained 23 genes and 13 noncoding RNAs. Integrating phenotypic and genetic data narrowed this region to a single gene, Trim55, an E3 ubiquitin ligase with a role in muscle fiber maintenance. Lung pathology and transcriptomic data from mice genetically deficient in Trim55 were used to validate its role in SARS-CoV-induced vascular cuffing and inflammation. These data establish the Collaborative Cross platform as a powerful genetic resource for uncovering genetic contributions of complex traits in microbial disease severity, inflammation and virus replication in models of outbred populations. PMID:26452100

Testicular Differentiation Occurs in Absence of R-spondin1 and Sox9 in Mouse Sex Reversals

PubMed Central

Pauper, Eva; Gregoire, Elodie P.; Klopfenstein, Muriel; de Rooij, Dirk G.; Mark, Manuel; Schedl, Andreas; Ghyselinck, Norbert B.; Chaboissier, Marie-Christine

2012-01-01

In mammals, male sex determination is governed by SRY-dependent activation of Sox9, whereas female development involves R-spondin1 (RSPO1), an activator of the WNT/beta-catenin signaling pathway. Genetic analyses in mice have demonstrated Sry and Sox9 to be both required and sufficient to induce testicular development. These genes are therefore considered as master regulators of the male pathway. Indeed, female-to-male sex reversal in XX Rspo1 mutant mice correlates with Sox9 expression, suggesting that this transcription factor induces testicular differentiation in pathological conditions. Unexpectedly, here we show that testicular differentiation can occur in XX mutants lacking both Rspo1 and Sox9 (referred to as XX Rspo1KOSox9cKO ), indicating that Sry and Sox9 are dispensable to induce female-to-male sex reversal. Molecular analyses show expression of both Sox8 and Sox10, suggesting that activation of Sox genes other than Sox9 can induce male differentiation in Rspo1KOSox9cKO mice. Moreover, since testis development occurs in XY Rspo1KOSox9cKO mice, our data show that Rspo1 is the main effector for male-to-female sex reversal in XY Sox9cKO mice. Thus, Rspo1 is an essential activator of ovarian development not only in normal situations, but also in sex reversal situations. Taken together these data demonstrate that both male and female sex differentiation is induced by distinct, active, genetic pathways. The dogma that considers female differentiation as a default pathway therefore needs to be definitively revised. PMID:23300469

Regulation of Sex Determination in Mice by a Non-coding Genomic Region

PubMed Central

Arboleda, Valerie A.; Fleming, Alice; Barseghyan, Hayk; Délot, Emmanuèle; Sinsheimer, Janet S.; Vilain, Eric

2014-01-01

To identify novel genomic regions that regulate sex determination, we utilized the powerful C57BL/6J-YPOS (B6-YPOS) model of XY sex reversal where mice with autosomes from the B6 strain and a Y chromosome from a wild-derived strain, Mus domesticus poschiavinus (YPOS), show complete sex reversal. In B6-YPOS, the presence of a 55-Mb congenic region on chromosome 11 protects from sex reversal in a dose-dependent manner. Using mouse genetic backcross designs and high-density SNP arrays, we narrowed the congenic region to a 1.62-Mb genomic region on chromosome 11 that confers 80% protection from B6-YPOS sex reversal when one copy is present and complete protection when two copies are present. It was previously believed that the protective congenic region originated from the 129S1/SviMJ (129) strain. However, genomic analysis revealed that this region is not derived from 129 and most likely is derived from the semi-inbred strain POSA. We show that the small 1.62-Mb congenic region that protects against B6-YPOS sex reversal is located within the Sox9 promoter and promotes the expression of Sox9, thereby driving testis development within the B6-YPOS background. Through 30 years of backcrossing, this congenic region was maintained, as it promoted male sex determination and fertility despite the female-promoting B6-YPOS genetic background. Our findings demonstrate that long-range enhancer regions are critical to developmental processes and can be used to identify the complex interplay between genome variants, epigenetics, and developmental gene regulation. PMID:24793290

Transfer of a Mycobacterium tuberculosis Genotyping Method, Spoligotyping, from a Reverse Line-Blot Hybridization, Membrane-Based Assay to the Luminex Multianalyte Profiling System

PubMed Central

Cowan, Lauren S.; Diem, Lois; Brake, Mary Catherine; Crawford, Jack T.

2004-01-01

Spoligotyping using Luminex technology was shown to be a highly reproducible method suitable for high-throughput analysis. Spoligotyping of 48 isolates using the traditional membrane-based assay and the Luminex assay yielded concordant results for all isolates. The Luminex platform provides greater flexibility and cost effectiveness than the membrane-based assay. PMID:14715809

Salinity-gradient energy driven microbial electrosynthesis of value-added chemicals from CO2 reduction.

PubMed

Li, Xiaohu; Angelidaki, Irini; Zhang, Yifeng

2018-06-14

Biological conversion of CO 2 to value-added chemicals and biofuels has emerged as an attractive strategy to address the energy and environmental concerns caused by the over-reliance on fossil fuels. In this study, an innovative microbial reverse-electrodialysis electrolysis cell (MREC), which combines the strengths of reverse electrodialysis (RED) and microbial electrosynthesis technology platforms, was developed to achieve efficient CO 2 -to-value chemicals bioconversion by using the salinity gradient energy as driven energy sources. In the MREC, maximum acetate and ethanol concentrations of 477.5 ± 33.2 and 46.2 ± 8.2 mg L -1 were obtained at the cathode, catalyzed by Sporomusa ovata with production rates of 165.79 ± 11.52 and 25.11 ± 4.46 mmol m -2 d -1 , respectively. Electron balance analysis indicates that 94.4 ± 3.9% of the electrons derived from wastewater and salinity gradient were recovered in acetate and ethanol. This work for the first time proved the potential of innovative MREC configuration has the potential as an efficient technology platform for simultaneous CO 2 capture and electrosynthesis of valuable chemicals. Copyright © 2018 Elsevier Ltd. All rights reserved.

The societal opportunities and challenges of genome editing.

PubMed

Carroll, Dana; Charo, R Alta

2015-11-05

The genome editing platforms currently in use have revolutionized the field of genetics. At an accelerating rate, these tools are entering areas with direct impact on human well being. Here, we discuss applications in agriculture and in medicine, and examine some associated societal issues.

Genetic manipulation of STEP reverses behavioral abnormalities in a fragile X syndrome mouse model.

PubMed

Goebel-Goody, S M; Wilson-Wallis, E D; Royston, S; Tagliatela, S M; Naegele, J R; Lombroso, P J

2012-07-01

Fragile X syndrome (FXS), the most common inherited form of intellectual disability and prevailing known genetic basis of autism, is caused by an expansion in the Fmr1 gene that prevents transcription and translation of fragile X mental retardation protein (FMRP). FMRP binds to and controls translation of mRNAs downstream of metabotropic glutamate receptor (mGluR) activation. Recent work shows that FMRP interacts with the transcript encoding striatal-enriched protein tyrosine phosphatase (STEP; Ptpn5). STEP opposes synaptic strengthening and promotes synaptic weakening by dephosphorylating its substrates, including ERK1/2, p38, Fyn and Pyk2, and subunits of N-methyl-d-aspartate (NMDA) and AMPA receptors. Here, we show that basal levels of STEP are elevated and mGluR-dependent STEP synthesis is absent in Fmr1(KO) mice. We hypothesized that the weakened synaptic strength and behavioral abnormalities reported in FXS may be linked to excess levels of STEP. To test this hypothesis, we reduced or eliminated STEP genetically in Fmr1(KO) mice and assessed mice in a battery of behavioral tests. In addition to attenuating audiogenic seizures and seizure-induced c-Fos activation in the periaqueductal gray, genetically reducing STEP in Fmr1(KO) mice reversed characteristic social abnormalities, including approach, investigation and anxiety. Loss of STEP also corrected select nonsocial anxiety-related behaviors in Fmr1(KO) mice, such as light-side exploration in the light/dark box. Our findings indicate that genetically reducing STEP significantly diminishes seizures and restores select social and nonsocial anxiety-related behaviors in Fmr1(KO) mice, suggesting that strategies to inhibit STEP activity may be effective for treating patients with FXS. © 2012 The Authors. Genes, Brain and Behavior © 2012 Blackwell Publishing Ltd and International Behavioural and Neural Genetics Society.

Distinct virulence of Rift Valley fever phlebovirus strains from different genetic lineages in a mouse model.

PubMed

Ikegami, Tetsuro; Balogh, Aaron; Nishiyama, Shoko; Lokugamage, Nandadeva; Saito, Tais B; Morrill, John C; Shivanna, Vinay; Indran, Sabarish V; Zhang, Lihong; Smith, Jennifer K; Perez, David; Juelich, Terry L; Morozov, Igor; Wilson, William C; Freiberg, Alexander N; Richt, Juergen A

2017-01-01

Rift Valley fever phlebovirus (RVFV) causes high rates of abortions and fetal malformations in ruminants, and hemorrhagic fever, encephalitis, or blindness in humans. Viral transmission occurs via mosquito vectors in endemic areas, which necessitates regular vaccination of susceptible livestock animals to prevent the RVF outbreaks. Although ZH501 strain has been used as a challenge strain for past vaccine efficacy studies, further characterization of other RVFV strains is important to optimize ruminant and nonhuman primate RVFV challenge models. This study aimed to characterize the virulence of wild-type RVFV strains belonging to different genetic lineages in outbred CD1 mice. Mice were intraperitoneally infected with 1x103 PFU of wild-type ZH501, Kenya 9800523, Kenya 90058, Saudi Arabia 200010911, OS1, OS7, SA75, Entebbe, or SA51 strains. Among them, mice infected with SA51, Entebbe, or OS7 strain showed rapid dissemination of virus in livers and peracute necrotic hepatitis at 2-3 dpi. Recombinant SA51 (rSA51) and Zinga (rZinga) strains were recovered by reverse genetics, and their virulence was also tested in CD1 mice. The rSA51 strain reproduced peracute RVF disease in mice, whereas the rZinga strain showed a similar virulence with that of rZH501 strain. This study showed that RVFV strains in different genetic lineages display distinct virulence in outbred mice. Importantly, since wild-type RVFV strains contain defective-interfering RNA or various genetic subpopulations during passage from original viral isolations, recombinant RVFV strains generated by reverse genetics will be better suitable for reproducible challenge studies for vaccine development as well as pathological studies.

Distinct virulence of Rift Valley fever phlebovirus strains from different genetic lineages in a mouse model

PubMed Central

Balogh, Aaron; Nishiyama, Shoko; Lokugamage, Nandadeva; Saito, Tais B.; Morrill, John C.; Shivanna, Vinay; Indran, Sabarish V.; Zhang, Lihong; Smith, Jennifer K.; Perez, David; Juelich, Terry L.; Morozov, Igor; Wilson, William C.; Freiberg, Alexander N.; Richt, Juergen A.

2017-01-01

Rift Valley fever phlebovirus (RVFV) causes high rates of abortions and fetal malformations in ruminants, and hemorrhagic fever, encephalitis, or blindness in humans. Viral transmission occurs via mosquito vectors in endemic areas, which necessitates regular vaccination of susceptible livestock animals to prevent the RVF outbreaks. Although ZH501 strain has been used as a challenge strain for past vaccine efficacy studies, further characterization of other RVFV strains is important to optimize ruminant and nonhuman primate RVFV challenge models. This study aimed to characterize the virulence of wild-type RVFV strains belonging to different genetic lineages in outbred CD1 mice. Mice were intraperitoneally infected with 1x103 PFU of wild-type ZH501, Kenya 9800523, Kenya 90058, Saudi Arabia 200010911, OS1, OS7, SA75, Entebbe, or SA51 strains. Among them, mice infected with SA51, Entebbe, or OS7 strain showed rapid dissemination of virus in livers and peracute necrotic hepatitis at 2–3 dpi. Recombinant SA51 (rSA51) and Zinga (rZinga) strains were recovered by reverse genetics, and their virulence was also tested in CD1 mice. The rSA51 strain reproduced peracute RVF disease in mice, whereas the rZinga strain showed a similar virulence with that of rZH501 strain. This study showed that RVFV strains in different genetic lineages display distinct virulence in outbred mice. Importantly, since wild-type RVFV strains contain defective-interfering RNA or various genetic subpopulations during passage from original viral isolations, recombinant RVFV strains generated by reverse genetics will be better suitable for reproducible challenge studies for vaccine development as well as pathological studies. PMID:29267298

The remarkable frequency of human immunodeficiency virus type 1 genetic recombination.

PubMed

Onafuwa-Nuga, Adewunmi; Telesnitsky, Alice

2009-09-01

The genetic diversity of human immunodeficiency virus type 1 (HIV-1) results from a combination of point mutations and genetic recombination, and rates of both processes are unusually high. This review focuses on the mechanisms and outcomes of HIV-1 genetic recombination and on the parameters that make recombination so remarkably frequent. Experimental work has demonstrated that the process that leads to recombination--a copy choice mechanism involving the migration of reverse transcriptase between viral RNA templates--occurs several times on average during every round of HIV-1 DNA synthesis. Key biological factors that lead to high recombination rates for all retroviruses are the recombination-prone nature of their reverse transcription machinery and their pseudodiploid RNA genomes. However, HIV-1 genes recombine even more frequently than do those of many other retroviruses. This reflects the way in which HIV-1 selects genomic RNAs for coencapsidation as well as cell-to-cell transmission properties that lead to unusually frequent associations between distinct viral genotypes. HIV-1 faces strong and changeable selective conditions during replication within patients. The mode of HIV-1 persistence as integrated proviruses and strong selection for defective proviruses in vivo provide conditions for archiving alleles, which can be resuscitated years after initial provirus establishment. Recombination can facilitate drug resistance and may allow superinfecting HIV-1 strains to evade preexisting immune responses, thus adding to challenges in vaccine development. These properties converge to provide HIV-1 with the means, motive, and opportunity to recombine its genetic material at an unprecedented high rate and to allow genetic recombination to serve as one of the highest barriers to HIV-1 eradication.

A functional metagenomic approach for expanding the synthetic biology toolbox for biomass conversion

PubMed Central

Sommer, Morten OA; Church, George M; Dantas, Gautam

2010-01-01

Sustainable biofuel alternatives to fossil fuel energy are hampered by recalcitrance and toxicity of biomass substrates to microbial biocatalysts. To address this issue, we present a culture-independent functional metagenomic platform for mining Nature's vast enzymatic reservoir and show its relevance to biomass conversion. We performed functional selections on 4.7 Gb of metagenomic fosmid libraries and show that genetic elements conferring tolerance toward seven important biomass inhibitors can be identified. We select two metagenomic fosmids that improve the growth of Escherichia coli by 5.7- and 6.9-fold in the presence of inhibitory concentrations of syringaldehyde and 2-furoic acid, respectively, and identify the individual genes responsible for these tolerance phenotypes. Finally, we combine the individual genes to create a three-gene construct that confers tolerance to mixtures of these important biomass inhibitors. This platform presents a route for expanding the repertoire of genetic elements available to synthetic biology and provides a starting point for efforts to engineer robust strains for biofuel generation. PMID:20393580

Computer-Assisted Transgenesis of Caenorhabditis elegans for Deep Phenotyping

PubMed Central

Gilleland, Cody L.; Falls, Adam T.; Noraky, James; Heiman, Maxwell G.; Yanik, Mehmet F.

2015-01-01

A major goal in the study of human diseases is to assign functions to genes or genetic variants. The model organism Caenorhabditis elegans provides a powerful tool because homologs of many human genes are identifiable, and large collections of genetic vectors and mutant strains are available. However, the delivery of such vector libraries into mutant strains remains a long-standing experimental bottleneck for phenotypic analysis. Here, we present a computer-assisted microinjection platform to streamline the production of transgenic C. elegans with multiple vectors for deep phenotyping. Briefly, animals are immobilized in a temperature-sensitive hydrogel using a standard multiwell platform. Microinjections are then performed under control of an automated microscope using precision robotics driven by customized computer vision algorithms. We demonstrate utility by phenotyping the morphology of 12 neuronal classes in six mutant backgrounds using combinations of neuron-type-specific fluorescent reporters. This technology can industrialize the assignment of in vivo gene function by enabling large-scale transgenic engineering. PMID:26163188

PHYLOSCANNER: Inferring Transmission from Within- and Between-Host Pathogen Genetic Diversity

PubMed Central

Hall, Matthew; Ratmann, Oliver; Bonsall, David; Golubchik, Tanya; de Cesare, Mariateresa; Gall, Astrid; Cornelissen, Marion; Fraser, Christophe

2018-01-01

Abstract A central feature of pathogen genomics is that different infectious particles (virions and bacterial cells) within an infected individual may be genetically distinct, with patterns of relatedness among infectious particles being the result of both within-host evolution and transmission from one host to the next. Here, we present a new software tool, phyloscanner, which analyses pathogen diversity from multiple infected hosts. phyloscanner provides unprecedented resolution into the transmission process, allowing inference of the direction of transmission from sequence data alone. Multiply infected individuals are also identified, as they harbor subpopulations of infectious particles that are not connected by within-host evolution, except where recombinant types emerge. Low-level contamination is flagged and removed. We illustrate phyloscanner on both viral and bacterial pathogens, namely HIV-1 sequenced on Illumina and Roche 454 platforms, HCV sequenced with the Oxford Nanopore MinION platform, and Streptococcus pneumoniae with sequences from multiple colonies per individual. phyloscanner is available from https://github.com/BDI-pathogens/phyloscanner. PMID:29186559

Micro-algae come of age as a platform for recombinant protein production

PubMed Central

Specht, Elizabeth; Miyake-Stoner, Shigeki

2010-01-01

A complete set of genetic tools is still being developed for the micro-alga Chlamydomonas reinhardtii. Yet even with this incomplete set, this photosynthetic single-celled plant has demonstrated significant promise as a platform for recombinant protein expression. In recent years, techniques have been developed that allow for robust expression of genes from both the nuclear and plastid genome. With these advances, many research groups have examined the pliability of this and other micro-algae as biological machines capable of producing recombinant peptides and proteins. This review describes recent successes in recombinant protein production in Chlamydomonas, including production of complex mammalian therapeutic proteins and monoclonal antibodies at levels sufficient for production at economic parity with existing production platforms. These advances have also shed light on the details of algal protein production at the molecular level, and provide insight into the next steps for optimizing micro-algae as a useful platform for the production of therapeutic and industrially relevant recombinant proteins. PMID:20556634

Continuous pCO2 time series from Ocean Networks Canada cabled observatories at the northeast Pacific shelf edge and in the sub-tidal Arctic

NASA Astrophysics Data System (ADS)

Juniper, S. Kim; Sastri, Akash; Mihaly, Steven; Duke, Patrick; Else, Brent; Thomas, Helmuth; Miller, Lisa

2017-04-01

Marine pCO2 sensor technology has progressed to the point where months-long time series from remotely-deployed pCO2 sensors can be used to document seasonal and higher frequency variability in pCO2 and its relationship to oceanographic processes. Ocean Networks Canada recently deployed pCO2 sensors on two cabled platforms: a bottom-moored (400 m depth), vertical profiler at the edge of the northeast Pacific continental shelf off Vancouver Island, Canada, and a subtidal seafloor platform in the Canadian High Arctic (69˚ N) at Cambridge Bay, Nunavut. Both platforms streamed continuous data to a shore-based archive from Pro-Oceanus pCO2 sensors and other oceanographic instruments. The vertical profiler time series revealed substantial intrusions of corrosive (high CO2/low O2), saltier, colder water masses during the summertime upwelling season and during winter-time reversals of along-slope currents. Step-wise profiles during the downcast provided the most reliable pCO2 data, permitting the sensor to equilibrate to the broad range of pCO2 concentrations encountered over the 400 metre depth interval. The Arctic pCO2 sensor was deployed in August 2015. Reversing seasonal trends in pCO2 and dissolved oxygen values can be related to the changing balance of photosynthesis and respiration under sea ice, as influenced by irradiance. Correlation of pCO2 and dissolved oxygen sensor data and the collection of calibration samples have permitted evaluation of sensor performance in relation to operational conditions encountered in vertical profiling and lengthy exposure to subzero seawater.

An in vitro recombination-based reverse genetic system for rapid mutagenesis of structural genes of the Japanese encephalitis virus.

PubMed

Du, Ruikun; Wang, Manli; Hu, Zhihong; Wang, Hualin; Deng, Fei

2015-10-01

Japanese encephalitis virus (JEV) is one of the most common pathogens of severe viral encephalitis, which is a severe threat to human health. Despite instability of the JEV genome in bacteria, many strategies have been developed to establish molecular clone systems of JEV, providing convenient tools for studying the virus life cycle and virus-host interactions. In this study, we adapted an In-Fusion enzyme-based in vitro recombination method to construct a reverse genetic system of JEV, thereby providing a rapid approach to introduce mutations into the structural genes. A truncated genome without the structural genes was constructed as the backbone, and the complementary segment containing the structural genes was recombined in vitro, which was then transfected directly into virus-permissive cells. The progeny of the infectious virus was successfully detected in the supernatant of the transfected cells, and showed an identical phenotype to its parental virus. To provide a proof-of-principle, the 12 conserved cysteine residues in the envelope (E) protein of JEV were respectively mutated using this approach, and all mutations resulted in a complete failure to generate infectious virus. However, a leucine-tophenylanine mutation at amino acid 107 of the E protein did not interfere with the production of the infectious virus. These results suggested that all 12 cysteines in the E protein are essential for the JEV life cycle. In summary, a novel reverse genetic system of JEV was established for rapidly introducing mutations into structural genes, which will serve as a useful tool for functional studies.

Epigenetics meets endocrinology

PubMed Central

Zhang, Xiang; Ho, Shuk-Mei

2014-01-01

Although genetics determines endocrine phenotypes, it cannot fully explain the great variability and reversibility of the system in response to environmental changes. Evidence now suggests that epigenetics, i.e. heritable but reversible changes in gene function without changes in nucleotide sequence, links genetics and environment in shaping endocrine function. Epigenetic mechanisms, including DNA methylation, histone modification, and microRNA, partition the genome into active and inactive domains based on endogenous and exogenous environmental changes and developmental stages, creating phenotype plasticity that can explain interindividual and population endocrine variability. We will review the current understanding of epigenetics in endocrinology, specifically, the regulation by epigenetics of the three levels of hormone action (synthesis and release, circulating and target tissue levels, and target-organ responsiveness) and the epigenetic action of endocrine disruptors. We will also discuss the impacts of hormones on epigenetics. We propose a three-dimensional model (genetics, environment, and developmental stage) to explain the phenomena related to progressive changes in endocrine functions with age, the early origin of endocrine disorders, phenotype discordance between monozygotic twins, rapid shifts in disease patterns among populations experiencing major lifestyle changes such as immigration, and the many endocrine disruptions in contemporary life. We emphasize that the key for understanding epigenetics in endocrinology is the identification, through advanced high-throughput screening technologies, of plasticity genes or loci that respond directly to a specific environmental stimulus. Investigations to determine whether epigenetic changes induced by today's lifestyles or environmental `exposures' can be inherited and are reversible should open doors for applying epigenetics to the prevention and treatment of endocrine disorders. PMID:21322125

The Role of Drosophila Merlin in the Control of Mitosis Exit and Development

DTIC Science & Technology

2006-07-01

schwannomas and is associated with mutations in the tumor suppressor gene called the neurofibromatosis type 2 (NF2) gene (Chang et al., 2005; Neff...been shown to associate with endocytic compartments and because mutations in the genes , such as clathrin and ff16, that are known to be important... mutations in the Drosophila homologues of the human Neurofibromatosis 2 and yeast CDC42 genes using a simple and efficient reverse-genetic method. Genetics

Practical aspects of mutagenicity testing strategy: an industrial perspective.

PubMed

Gollapudi, B B; Krishna, G

2000-11-20

Genetic toxicology studies play a central role in the development and marketing of new chemicals for pharmaceutical, agricultural, industrial, and consumer use. During the discovery phase of product development, rapid screening tests that require minimal amounts of test materials are used to assist in the design and prioritization of new molecules. At this stage, a modified Salmonella reverse mutation assay and an in vitro micronucleus test with mammalian cell culture are frequently used for screening. Regulatory genetic toxicology studies are conducted with a short list of compounds using protocols that conform to various international guidelines. A set of four assays usually constitutes the minimum test battery that satisfies global requirements. This set includes a bacterial reverse mutation assay, an in vitro cytogenetic test with mammalian cell culture, an in vitro gene mutation assay in mammalian cell cultures, and an in vivo rodent bone marrow micronucleus test. Supplementary studies are conducted in certain instances either as a follow-up to the findings from this initial testing battery and/or to satisfy a regulatory requirement. Currently available genetic toxicology assays have helped the scientific and industrial community over the past several decades in evaluating the mutagenic potential of chemical agents. The emerging field of toxicogenomics has the potential to redefine our ability to study the response of cells to genetic damage and hence our ability to study threshold phenomenon.

Analysis and visualization of Arabidopsis thaliana GWAS using web 2.0 technologies.

PubMed

Huang, Yu S; Horton, Matthew; Vilhjálmsson, Bjarni J; Seren, Umit; Meng, Dazhe; Meyer, Christopher; Ali Amer, Muhammad; Borevitz, Justin O; Bergelson, Joy; Nordborg, Magnus

2011-01-01

With large-scale genomic data becoming the norm in biological studies, the storing, integrating, viewing and searching of such data have become a major challenge. In this article, we describe the development of an Arabidopsis thaliana database that hosts the geographic information and genetic polymorphism data for over 6000 accessions and genome-wide association study (GWAS) results for 107 phenotypes representing the largest collection of Arabidopsis polymorphism data and GWAS results to date. Taking advantage of a series of the latest web 2.0 technologies, such as Ajax (Asynchronous JavaScript and XML), GWT (Google-Web-Toolkit), MVC (Model-View-Controller) web framework and Object Relationship Mapper, we have created a web-based application (web app) for the database, that offers an integrated and dynamic view of geographic information, genetic polymorphism and GWAS results. Essential search functionalities are incorporated into the web app to aid reverse genetics research. The database and its web app have proven to be a valuable resource to the Arabidopsis community. The whole framework serves as an example of how biological data, especially GWAS, can be presented and accessed through the web. In the end, we illustrate the potential to gain new insights through the web app by two examples, showcasing how it can be used to facilitate forward and reverse genetics research. Database URL: http://arabidopsis.usc.edu/

Integration of maternal genome into the neonate genome through breast milk mRNA transcripts and reverse transcriptase.

PubMed

Irmak, M Kemal; Oztas, Yesim; Oztas, Emin

2012-06-07

Human milk samples contain microvesicles similar to the retroviruses. These microvesicles contain mRNA transcripts and possess reverse transcriptase activity. They contain about 14,000 transcripts representing the milk transcriptome. Microvesicles are also enriched with proteins related to "caveolar-mediated endocytosis signaling" pathway. It has recently been reported that microvesicles could be transferred to other cells by endocytosis and their RNA content can be translated and be functional in their new location. A significant percentage of the mammalian genome appears to be the product of reverse transcription, containing sequences whose characteristics point to RNA as a template precursor. These are mobile elements that move by way of transposition and are called retrotransposons. We thought that retrotransposons may stem from about 14,000 transcriptome of breast milk microvesicles, and reviewed the literature.The enhanced acceptance of maternal allografts in children who were breast-fed and tolerance to the maternal MHC antigens after breastfeeding may stem from RNAs of the breast milk microvesicles that can be taken up by the breastfed infant and receiving maternal genomic information. We conclude that milk microvesicles may transfer genetic signals from mother to neonate during breastfeeding. Moreover, transfer of wild type RNA from a healthy wet-nurse to the suckling neonate through the milk microvesicles and its subsequent reverse transcription and integration into the neonate genome could result in permanent correction of the clinical manifestations in genetic diseases.

Integration of maternal genome into the neonate genome through breast milk mRNA transcripts and reverse transcriptase

PubMed Central

2012-01-01

Human milk samples contain microvesicles similar to the retroviruses. These microvesicles contain mRNA transcripts and possess reverse transcriptase activity. They contain about 14,000 transcripts representing the milk transcriptome. Microvesicles are also enriched with proteins related to “caveolar-mediated endocytosis signaling” pathway. It has recently been reported that microvesicles could be transferred to other cells by endocytosis and their RNA content can be translated and be functional in their new location. A significant percentage of the mammalian genome appears to be the product of reverse transcription, containing sequences whose characteristics point to RNA as a template precursor. These are mobile elements that move by way of transposition and are called retrotransposons. We thought that retrotransposons may stem from about 14,000 transcriptome of breast milk microvesicles, and reviewed the literature. The enhanced acceptance of maternal allografts in children who were breast-fed and tolerance to the maternal MHC antigens after breastfeeding may stem from RNAs of the breast milk microvesicles that can be taken up by the breastfed infant and receiving maternal genomic information. We conclude that milk microvesicles may transfer genetic signals from mother to neonate during breastfeeding. Moreover, transfer of wild type RNA from a healthy wet-nurse to the suckling neonate through the milk microvesicles and its subsequent reverse transcription and integration into the neonate genome could result in permanent correction of the clinical manifestations in genetic diseases. PMID:22676860

Solving TSP problem with improved genetic algorithm

NASA Astrophysics Data System (ADS)

Fu, Chunhua; Zhang, Lijun; Wang, Xiaojing; Qiao, Liying

2018-05-01

The TSP is a typical NP problem. The optimization of vehicle routing problem (VRP) and city pipeline optimization can use TSP to solve; therefore it is very important to the optimization for solving TSP problem. The genetic algorithm (GA) is one of ideal methods in solving it. The standard genetic algorithm has some limitations. Improving the selection operator of genetic algorithm, and importing elite retention strategy can ensure the select operation of quality, In mutation operation, using the adaptive algorithm selection can improve the quality of search results and variation, after the chromosome evolved one-way evolution reverse operation is added which can make the offspring inherit gene of parental quality improvement opportunities, and improve the ability of searching the optimal solution algorithm.

Lactococcus lactis-based vaccines: current status and future perspectives.

PubMed

Bahey-El-Din, Mohammed; Gahan, Cormac G M

2011-01-01

Lactococcus lactis offers significant potential as a platform for the delivery of vaccines especially via mucosal routes of administration. The organism has an established history of safe use in the food industry and is highly amenable to genetic manipulation, with many systems available for efficient production of secreted and surface-expressed proteins. Here we describe the benefits of using this organism as a vaccine delivery platform and outline how L. lactis based antigen delivery may be improved. Finally we discuss the safe use of L. lactis vectors and outline the potential for use of biological containment systems and killed lactococcal preparations.

A platform for evolving intelligently interactive adversaries.

PubMed

Fogel, David B; Hays, Timothy J; Johnson, Douglas R

2006-07-01

Entertainment software developers face significant challenges in designing games with broad appeal. One of the challenges concerns creating nonplayer (computer-controlled) characters that can adapt their behavior in light of the current and prospective situation, possibly emulating human behaviors. This adaptation should be inherently novel, unrepeatable, yet within the bounds of realism. Evolutionary algorithms provide a suitable method for generating such behaviors. This paper provides background on the entertainment software industry, and details a prior and current effort to create a platform for evolving nonplayer characters with genetic and behavioral traits within a World War I combat flight simulator.

Use of a platform in an automated open-field to enhance assessment of anxiety-like behaviors in mice.

PubMed

Pogorelov, Vladimir M; Lanthorn, Thomas H; Savelieva, Katerina V

2007-05-15

The present report describes a setup for simultaneously measuring anxiety-like behaviors and locomotor activity in mice. Animals are placed in a brightly lit, standard automated open-field (OF) in which a rectangular ceramic platform 8 cm high covers one quadrant of the floor. Mice preferred to stay under the platform, avoiding the area with bright illumination. Activities under and outside the platform were measured for 5 min. Chlordiazepoxide and buspirone dose-dependently increased time spent outside the platform (L-Time) and the light distance to total OF distance ratio (L:T-TD) in both genders without changing total OF distance. By contrast, amphetamine decreased L-Time and L:T-TD in males, thus displaying an anxiogenic effect. Imipramine was without selective effect on L-Time or L:T-TD, but decreased total OF distance at the highest dose indicative of a sedative effect. Drug effects were also evaluated in the OF without platform using conventional anxiety measures. Introduction of the platform into the OF apparatus strongly enhanced the sensitivity to anxiolytics. Comparison of strains differing in activity or anxiety levels showed that L-Time and L:T-TD can be used as measures of anxiety-like behavior independent of locomotor activity. Changes in motor activity are reflected in the total distance traveled under and outside the platform. Therefore, the platform test is fully automated, sensitive to both anxiolytic and anxiogenic effects of drugs and genetic phenotypes with little evidence of gender-specific responses, and can be easily utilized by most laboratories measuring behavior.

Soul on Silicon.

ERIC Educational Resources Information Center

Kurzweil, Raymond C.

1994-01-01

Summarizes recent advances in computer simulation and "reverse engineering" technologies, highlighting the Human Genome Project to scan the human genetic code; artificial retina chips to copy the human retina's neural organization; high-speed, high-resolution Magnetic Resonance Imaging scanners; and the virtual book. Discusses…

Crime and Child-Rearing.

ERIC Educational Resources Information Center

Roth, Byron M.

1996-01-01

Examines the notion that heredity plays a powerful role in criminal behavior, including genetic evidence that can allow for antisocial behavior. Reviews suggestions for reversing rising crime rates in light of the hereditary connection, policy development, family cohesion, and child raising. (GR)

Characterization of Hepatitis C Virus (HCV) Envelope Diversification from Acute to Chronic Infection within a Sexually Transmitted HCV Cluster by Using Single-Molecule, Real-Time Sequencing

PubMed Central

Ho, Cynthia K. Y.; Raghwani, Jayna; Koekkoek, Sylvie; Liang, Richard H.; Van der Meer, Jan T. M.; Van Der Valk, Marc; De Jong, Menno; Pybus, Oliver G.

2016-01-01

ABSTRACT In contrast to other available next-generation sequencing platforms, PacBio single-molecule, real-time (SMRT) sequencing has the advantage of generating long reads albeit with a relatively higher error rate in unprocessed data. Using this platform, we longitudinally sampled and sequenced the hepatitis C virus (HCV) envelope genome region (1,680 nucleotides [nt]) from individuals belonging to a cluster of sexually transmitted cases. All five subjects were coinfected with HIV-1 and a closely related strain of HCV genotype 4d. In total, 50 samples were analyzed by using SMRT sequencing. By using 7 passes of circular consensus sequencing, the error rate was reduced to 0.37%, and the median number of sequences was 612 per sample. A further reduction of insertions was achieved by alignment against a sample-specific reference sequence. However, in vitro recombination during PCR amplification could not be excluded. Phylogenetic analysis supported close relationships among HCV sequences from the four male subjects and subsequent transmission from one subject to his female partner. Transmission was characterized by a strong genetic bottleneck. Viral genetic diversity was low during acute infection and increased upon progression to chronicity but subsequently fluctuated during chronic infection, caused by the alternate detection of distinct coexisting lineages. SMRT sequencing combines long reads with sufficient depth for many phylogenetic analyses and can therefore provide insights into within-host HCV evolutionary dynamics without the need for haplotype reconstruction using statistical algorithms. IMPORTANCE Next-generation sequencing has revolutionized the study of genetically variable RNA virus populations, but for phylogenetic and evolutionary analyses, longer sequences than those generated by most available platforms, while minimizing the intrinsic error rate, are desired. Here, we demonstrate for the first time that PacBio SMRT sequencing technology can be used to generate full-length HCV envelope sequences at the single-molecule level, providing a data set with large sequencing depth for the characterization of intrahost viral dynamics. The selection of consensus reads derived from at least 7 full circular consensus sequencing rounds significantly reduced the intrinsic high error rate of this method. We used this method to genetically characterize a unique transmission cluster of sexually transmitted HCV infections, providing insight into the distinct evolutionary pathways in each patient over time and identifying the transmission-associated genetic bottleneck as well as fluctuations in viral genetic diversity over time, accompanied by dynamic shifts in viral subpopulations. PMID:28077634

The transformative potential of an integrative approach to pregnancy.

PubMed

Eidem, Haley R; McGary, Kriston L; Capra, John A; Abbot, Patrick; Rokas, Antonis

2017-09-01

Complex traits typically involve diverse biological pathways and are shaped by numerous genetic and environmental factors. Pregnancy-associated traits and pathologies are further complicated by extensive communication across multiple tissues in two individuals, interactions between two genomes-maternal and fetal-that obscure causal variants and lead to genetic conflict, and rapid evolution of pregnancy-associated traits across mammals and in the human lineage. Given the multi-faceted complexity of human pregnancy, integrative approaches that synthesize diverse data types and analyses harbor tremendous promise to identify the genetic architecture and environmental influences underlying pregnancy-associated traits and pathologies. We review current research that addresses the extreme complexities of traits and pathologies associated with human pregnancy. We find that successful efforts to address the many complexities of pregnancy-associated traits and pathologies often harness the power of many and diverse types of data, including genome-wide association studies, evolutionary analyses, multi-tissue transcriptomic profiles, and environmental conditions. We propose that understanding of pregnancy and its pathologies will be accelerated by computational platforms that provide easy access to integrated data and analyses. By simplifying the integration of diverse data, such platforms will provide a comprehensive synthesis that transcends many of the inherent challenges present in studies of pregnancy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Transgenic barley: a prospective tool for biotechnology and agriculture.

PubMed

Mrízová, Katarína; Holasková, Edita; Öz, M Tufan; Jiskrová, Eva; Frébort, Ivo; Galuszka, Petr

2014-01-01

Barley (Hordeum vulgare L.) is one of the founder crops of agriculture, and today it is the fourth most important cereal grain worldwide. Barley is used as malt in brewing and distilling industry, as an additive for animal feed, and as a component of various food and bread for human consumption. Progress in stable genetic transformation of barley ensures a potential for improvement of its agronomic performance or use of barley in various biotechnological and industrial applications. Recently, barley grain has been successfully used in molecular farming as a promising bioreactor adapted for production of human therapeutic proteins or animal vaccines. In addition to development of reliable transformation technologies, an extensive amount of various barley genetic resources and tools such as sequence data, microarrays, genetic maps, and databases has been generated. Current status on barley transformation technologies including gene transfer techniques, targets, and progeny stabilization, recent trials for improvement of agricultural traits and performance of barley, especially in relation to increased biotic and abiotic stress tolerance, and potential use of barley grain as a protein production platform have been reviewed in this study. Overall, barley represents a promising tool for both agricultural and biotechnological transgenic approaches, and is considered an ancient but rediscovered crop as a model industrial platform for molecular farming. © 2013 Elsevier Inc. All rights reserved.

Molecular genetic improvements of cyanobacteria to enhance the industrial potential of the microbe: A review.

PubMed

Johnson, Tylor J; Gibbons, Jaimie L; Gu, Liping; Zhou, Ruanbao; Gibbons, William R

2016-11-01

The rapid increase in worldwide population coupled with the increasing demand for fossil fuels has led to an increased urgency to develop sustainable sources of energy and chemicals from renewable resources. Using microorganisms to produce high-value chemicals and next-generation biofuels is one sustainable option and is the focus of much current research. Cyanobacteria are ideal platform organisms for chemical and biofuel production because they can be genetically engineered to produce a broad range of products directly from CO 2 , H 2 O, and sunlight, and require minimal nutrient inputs. The purpose of this review is to provide an overview on advances that have been or could be made to improve strains of cyanobacteria for industrial purposes. First, the benefits of using cyanobacteria as a platform for chemical and biofuel production are discussed. Next, an overview of cyanobacterial strain improvements by genetic engineering is provided. Finally, mutagenesis techniques to improve the industrial potential of cyanobacteria are described. Along with providing an overview on various areas of research that are currently being investigated to improve the industrial potential of cyanobacteria, this review aims to elucidate potential targets for future research involving cyanobacteria as an industrial microorganism. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1357-1371, 2016. © 2016 American Institute of Chemical Engineers.

The GMOseek matrix: a decision support tool for optimizing the detection of genetically modified plants.

PubMed

Block, Annette; Debode, Frédéric; Grohmann, Lutz; Hulin, Julie; Taverniers, Isabel; Kluga, Linda; Barbau-Piednoir, Elodie; Broeders, Sylvia; Huber, Ingrid; Van den Bulcke, Marc; Heinze, Petra; Berben, Gilbert; Busch, Ulrich; Roosens, Nancy; Janssen, Eric; Žel, Jana; Gruden, Kristina; Morisset, Dany

2013-08-22

Since their first commercialization, the diversity of taxa and the genetic composition of transgene sequences in genetically modified plants (GMOs) are constantly increasing. To date, the detection of GMOs and derived products is commonly performed by PCR-based methods targeting specific DNA sequences introduced into the host genome. Information available regarding the GMOs' molecular characterization is dispersed and not appropriately organized. For this reason, GMO testing is very challenging and requires more complex screening strategies and decision making schemes, demanding in return the use of efficient bioinformatics tools relying on reliable information. The GMOseek matrix was built as a comprehensive, online open-access tabulated database which provides a reliable, comprehensive and user-friendly overview of 328 GMO events and 247 different genetic elements (status: 18/07/2013). The GMOseek matrix is aiming to facilitate GMO detection from plant origin at different phases of the analysis. It assists in selecting the targets for a screening analysis, interpreting the screening results, checking the occurrence of a screening element in a group of selected GMOs, identifying gaps in the available pool of GMO detection methods, and designing a decision tree. The GMOseek matrix is an independent database with effective functionalities in a format facilitating transferability to other platforms. Data were collected from all available sources and experimentally tested where detection methods and certified reference materials (CRMs) were available. The GMOseek matrix is currently a unique and very valuable tool with reliable information on GMOs from plant origin and their present genetic elements that enables further development of appropriate strategies for GMO detection. It is flexible enough to be further updated with new information and integrated in different applications and platforms.

Isolation and Screening of Thermophilic Bacilli from Compost for Electrotransformation and Fermentation: Characterization of Bacillus smithii ET 138 as a New Biocatalyst

PubMed Central

Bosma, Elleke F.; van de Weijer, Antonius H. P.; Daas, Martinus J. A.; van der Oost, John; de Vos, Willem M.

2015-01-01

Thermophilic bacteria are regarded as attractive production organisms for cost-efficient conversion of renewable resources to green chemicals, but their genetic accessibility is a major bottleneck in developing them into versatile platform organisms. In this study, we aimed to isolate thermophilic, facultatively anaerobic bacilli that are genetically accessible and have potential as platform organisms. From compost, we isolated 267 strains that produced acids from C5 and C6 sugars at temperatures of 55°C or 65°C. Subsequently, 44 strains that showed the highest production of acids were screened for genetic accessibility by electroporation. Two Geobacillus thermodenitrificans isolates and one Bacillus smithii isolate were found to be transformable with plasmid pNW33n. Of these, B. smithii ET 138 was the best-performing strain in laboratory-scale fermentations and was capable of producing organic acids from glucose as well as from xylose. It is an acidotolerant strain able to produce organic acids until a lower limit of approximately pH 4.5. As genetic accessibility of B. smithii had not been described previously, six other B. smithii strains from the DSMZ culture collection were tested for electroporation efficiencies, and we found the type strain DSM 4216T and strain DSM 460 to be transformable. The transformation protocol for B. smithii isolate ET 138 was optimized to obtain approximately 5 × 103 colonies per μg plasmid pNW33n. Genetic accessibility combined with robust acid production capacities on C5 and C6 sugars at a relatively broad pH range make B. smithii ET 138 an attractive biocatalyst for the production of lactic acid and potentially other green chemicals. PMID:25556192

The GMOseek matrix: a decision support tool for optimizing the detection of genetically modified plants

PubMed Central

2013-01-01

Background Since their first commercialization, the diversity of taxa and the genetic composition of transgene sequences in genetically modified plants (GMOs) are constantly increasing. To date, the detection of GMOs and derived products is commonly performed by PCR-based methods targeting specific DNA sequences introduced into the host genome. Information available regarding the GMOs’ molecular characterization is dispersed and not appropriately organized. For this reason, GMO testing is very challenging and requires more complex screening strategies and decision making schemes, demanding in return the use of efficient bioinformatics tools relying on reliable information. Description The GMOseek matrix was built as a comprehensive, online open-access tabulated database which provides a reliable, comprehensive and user-friendly overview of 328 GMO events and 247 different genetic elements (status: 18/07/2013). The GMOseek matrix is aiming to facilitate GMO detection from plant origin at different phases of the analysis. It assists in selecting the targets for a screening analysis, interpreting the screening results, checking the occurrence of a screening element in a group of selected GMOs, identifying gaps in the available pool of GMO detection methods, and designing a decision tree. The GMOseek matrix is an independent database with effective functionalities in a format facilitating transferability to other platforms. Data were collected from all available sources and experimentally tested where detection methods and certified reference materials (CRMs) were available. Conclusions The GMOseek matrix is currently a unique and very valuable tool with reliable information on GMOs from plant origin and their present genetic elements that enables further development of appropriate strategies for GMO detection. It is flexible enough to be further updated with new information and integrated in different applications and platforms. PMID:23965170

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

FAVR (Filtering and Annotation of Variants that are Rare): methods to facilitate the analysis of rare germline genetic variants from massively parallel sequencing datasets

PubMed Central

2013-01-01

Background Characterising genetic diversity through the analysis of massively parallel sequencing (MPS) data offers enormous potential to significantly improve our understanding of the genetic basis for observed phenotypes, including predisposition to and progression of complex human disease. Great challenges remain in resolving genetic variants that are genuine from the millions of artefactual signals. Results FAVR is a suite of new methods designed to work with commonly used MPS analysis pipelines to assist in the resolution of some of the issues related to the analysis of the vast amount of resulting data, with a focus on relatively rare genetic variants. To the best of our knowledge, no equivalent method has previously been described. The most important and novel aspect of FAVR is the use of signatures in comparator sequence alignment files during variant filtering, and annotation of variants potentially shared between individuals. The FAVR methods use these signatures to facilitate filtering of (i) platform and/or mapping-specific artefacts, (ii) common genetic variants, and, where relevant, (iii) artefacts derived from imbalanced paired-end sequencing, as well as annotation of genetic variants based on evidence of co-occurrence in individuals. We applied conventional variant calling applied to whole-exome sequencing datasets, produced using both SOLiD and TruSeq chemistries, with or without downstream processing by FAVR methods. We demonstrate a 3-fold smaller rare single nucleotide variant shortlist with no detected reduction in sensitivity. This analysis included Sanger sequencing of rare variant signals not evident in dbSNP131, assessment of known variant signal preservation, and comparison of observed and expected rare variant numbers across a range of first cousin pairs. The principles described herein were applied in our recent publication identifying XRCC2 as a new breast cancer risk gene and have been made publically available as a suite of software tools. Conclusions FAVR is a platform-agnostic suite of methods that significantly enhances the analysis of large volumes of sequencing data for the study of rare genetic variants and their influence on phenotypes. PMID:23441864

Molecular Genetic Analysis of Chlamydia Species.

PubMed

Sixt, Barbara S; Valdivia, Raphael H

2016-09-08

Species of Chlamydia are the etiologic agent of endemic blinding trachoma, the leading cause of bacterial sexually transmitted diseases, significant respiratory pathogens, and a zoonotic threat. Their dependence on an intracellular growth niche and their peculiar developmental cycle are major challenges to elucidating their biology and virulence traits. The last decade has seen tremendous advances in our ability to perform a molecular genetic analysis of Chlamydia species. Major achievements include the generation of large collections of mutant strains, now available for forward- and reverse-genetic applications, and the introduction of a system for plasmid-based transformation enabling complementation of mutations; expression of foreign, modified, or reporter genes; and even targeted gene disruptions. This review summarizes the current status of the molecular genetic toolbox for Chlamydia species and highlights new insights into their biology and new challenges in the nascent field of Chlamydia genetics.

DNAGear--a free software for spa type identification in Staphylococcus aureus.

PubMed

AL-Tam, Faroq; Brunel, Anne-Sophie; Bouzinbi, Nicolas; Corne, Philippe; Bañuls, Anne-Laure; Shahbazkia, Hamid Reza

2012-11-19

Staphylococcus aureus is both human commensal and an important human pathogen, responsible for community-acquired and nosocomial infections ranging from superficial wound infections to invasive infections, such as osteomyelitis, bacteremia and endocarditis, pneumonia or toxin shock syndrome with a mortality rate up to 40%. S. aureus reveals a high genetic polymorphism and detecting the genotypes is extremely useful to manage and prevent possible outbreaks and to understand the route of infection. One of current and expanded typing method is based on the X region of the spa gene composed of a succession of repeats of 21 to 27 bp. More than 10000 types are known. Extracting the repeats is impossible by hand and needs a dedicated software. Unfortunately the only software on the market is a commercial program from Ridom. This article presents DNAGear, a free and open source software with a user friendly interface written all in Java on top of NetBeans Platform to perform spa typing, detecting new repeats and new spa types and synchronizing automatically the files with the open access database. The installation is easy and the application is platform independent. In fact, the SPA identification is a formal regular expression matching problem and the results are 100% exact. As the program is using Java embedded modules written over string manipulation of well established algorithms, the exactitude of the solution is perfectly established. DNAGear is able to identify the types of the S. aureus sequences and detect both new types and repeats. Comparing to manual processing, which is time consuming and error prone, this application saves a lot of time and effort and gives very reliable results. Additionally, the users do not need to prepare the forward-reverse sequences manually, or even by using additional tools. They can simply create them in DNAGear and perform the typing task. In short, researchers who do not have commercial software will benefit a lot from this application.

Using C. elegans Forward and Reverse Genetics to Identify New Compounds with Anthelmintic Activity

PubMed Central

Mathew, Mark D.; Mathew, Neal D.; Miller, Angela; Simpson, Mike; Au, Vinci; Garland, Stephanie; Gestin, Marie; Edgley, Mark L.; Flibotte, Stephane; Balgi, Aruna; Chiang, Jennifer; Giaever, Guri; Dean, Pamela; Tung, Audrey; Roberge, Michel; Roskelley, Calvin; Forge, Tom; Nislow, Corey; Moerman, Donald

2016-01-01

Background The lack of new anthelmintic agents is of growing concern because it affects human health and our food supply, as both livestock and plants are affected. Two principal factors contribute to this problem. First, nematode resistance to anthelmintic drugs is increasing worldwide and second, many effective nematicides pose environmental hazards. In this paper we address this problem by deploying a high throughput screening platform for anthelmintic drug discovery using the nematode Caenorhabditis elegans as a surrogate for infectious nematodes. This method offers the possibility of identifying new anthelmintics in a cost-effective and timely manner. Methods/Principal findings Using our high throughput screening platform we have identified 14 new potential anthelmintics by screening more than 26,000 compounds from the Chembridge and Maybridge chemical libraries. Using phylogenetic profiling we identified a subset of the 14 compounds as potential anthelmintics based on the relative sensitivity of C. elegans when compared to yeast and mammalian cells in culture. We showed that a subset of these compounds might employ mechanisms distinct from currently used anthelmintics by testing diverse drug resistant strains of C. elegans. One of these newly identified compounds targets mitochondrial complex II, and we used structural analysis of the target to suggest how differential binding of this compound may account for its different effects in nematodes versus mammalian cells. Conclusions/Significance The challenge of anthelmintic drug discovery is exacerbated by several factors; including, 1) the biochemical similarity between host and parasite genomes, 2) the geographic location of parasitic nematodes and 3) the rapid development of resistance. Accordingly, an approach that can screen large compound collections rapidly is required. C. elegans as a surrogate parasite offers the ability to screen compounds rapidly and, equally importantly, with specificity, thus reducing the potential toxicity of these compounds to the host and the environment. We believe this approach will help to replenish the pipeline of potential nematicides. PMID:27755544

DNAGear- a free software for spa type identification in Staphylococcus aureus

PubMed Central

2012-01-01

Background Staphylococcus aureus is both human commensal and an important human pathogen, responsible for community-acquired and nosocomial infections ranging from superficial wound infections to invasive infections, such as osteomyelitis, bacteremia and endocarditis, pneumonia or toxin shock syndrome with a mortality rate up to 40%. S. aureus reveals a high genetic polymorphism and detecting the genotypes is extremely useful to manage and prevent possible outbreaks and to understand the route of infection. One of current and expanded typing method is based on the X region of the spa gene composed of a succession of repeats of 21 to 27 bp. More than 10000 types are known. Extracting the repeats is impossible by hand and needs a dedicated software. Unfortunately the only software on the market is a commercial program from Ridom. Findings This article presents DNAGear, a free and open source software with a user friendly interface written all in Java on top of NetBeans Platform to perform spa typing, detecting new repeats and new spa types and synchronizing automatically the files with the open access database. The installation is easy and the application is platform independent. In fact, the SPA identification is a formal regular expression matching problem and the results are 100% exact. As the program is using Java embedded modules written over string manipulation of well established algorithms, the exactitude of the solution is perfectly established. Conclusions DNAGear is able to identify the types of the S. aureus sequences and detect both new types and repeats. Comparing to manual processing, which is time consuming and error prone, this application saves a lot of time and effort and gives very reliable results. Additionally, the users do not need to prepare the forward-reverse sequences manually, or even by using additional tools. They can simply create them in DNAGear and perform the typing task. In short, researchers who do not have commercial software will benefit a lot from this application. PMID:23164452

Toward a 3D model of human brain development for studying gene/environment interactions

PubMed Central

2013-01-01

This project aims to establish and characterize an in vitro model of the developing human brain for the purpose of testing drugs and chemicals. To accurately assess risk, a model needs to recapitulate the complex interactions between different types of glial cells and neurons in a three-dimensional platform. Moreover, human cells are preferred over cells from rodents to eliminate cross-species differences in sensitivity to chemicals. Previously, we established conditions to culture rat primary cells as three-dimensional aggregates, which will be humanized and evaluated here with induced pluripotent stem cells (iPSCs). The use of iPSCs allows us to address gene/environment interactions as well as the potential of chemicals to interfere with epigenetic mechanisms. Additionally, iPSCs afford us the opportunity to study the effect of chemicals during very early stages of brain development. It is well recognized that assays for testing toxicity in the developing brain must consider differences in sensitivity and susceptibility that arise depending on the time of exposure. This model will reflect critical developmental processes such as proliferation, differentiation, lineage specification, migration, axonal growth, dendritic arborization and synaptogenesis, which will probably display differences in sensitivity to different types of chemicals. Functional endpoints will evaluate the complex cell-to-cell interactions that are affected in neurodevelopment through chemical perturbation, and the efficacy of drug intervention to prevent or reverse phenotypes. The model described is designed to assess developmental neurotoxicity effects on unique processes occurring during human brain development by leveraging human iPSCs from diverse genetic backgrounds, which can be differentiated into different cell types of the central nervous system. Our goal is to demonstrate the feasibility of the personalized model using iPSCs derived from individuals with neurodevelopmental disorders caused by known mutations and chromosomal aberrations. Notably, such a human brain model will be a versatile tool for more complex testing platforms and strategies as well as research into central nervous system physiology and pathology. PMID:24564953

SPE-IMS-MS: An automated platform for sub-sixty second surveillance of endogenous metabolites and xenobiotics in biofluids

DOE PAGES

Zhang, Xing; Romm, Michelle; Zheng, Xueyun; ...

2016-12-29

Characterization of endogenous metabolites and xenobiotics is essential to deconvoluting the genetic and environmental causes of disease. However, surveillance of chemical exposure and disease-related changes in large cohorts requires an analytical platform that offers rapid measurement, high sensitivity, efficient separation, broad dynamic range, and application to an expansive chemical space. Here in this article, we present a novel platform for small molecule analyses that addresses these requirements by combining solid-phase extraction with ion mobility spectrometry and mass spectrometry (SPE-IMS-MS). This platform is capable of performing both targeted and global measurements of endogenous metabolites and xenobiotics in human biofluids with highmore » reproducibility (CV ≤ 3%), sensitivity (LODs in the pM range in biofluids) and throughput (10-s sample-to-sample duty cycle). We report application of this platform to the analysis of human urine from patients with and without type 1 diabetes, where we observed statistically significant variations in the concentration of disaccharides and previously unreported chemical isomers. Lastly, this SPE-IMS-MS platform overcomes many of the current challenges of large-scale metabolomic and exposomic analyses and offers a viable option for population and patient cohort screening in an effort to gain insights into disease processes and human environmental chemical exposure.« less

SPE-IMS-MS: An automated platform for sub-sixty second surveillance of endogenous metabolites and xenobiotics in biofluids.

PubMed

Zhang, Xing; Romm, Michelle; Zheng, Xueyun; Zink, Erika M; Kim, Young-Mo; Burnum-Johnson, Kristin E; Orton, Daniel J; Apffel, Alex; Ibrahim, Yehia M; Monroe, Matthew E; Moore, Ronald J; Smith, Jordan N; Ma, Jian; Renslow, Ryan S; Thomas, Dennis G; Blackwell, Anne E; Swinford, Glenn; Sausen, John; Kurulugama, Ruwan T; Eno, Nathan; Darland, Ed; Stafford, George; Fjeldsted, John; Metz, Thomas O; Teeguarden, Justin G; Smith, Richard D; Baker, Erin S

2016-12-01

Characterization of endogenous metabolites and xenobiotics is essential to deconvoluting the genetic and environmental causes of disease. However, surveillance of chemical exposure and disease-related changes in large cohorts requires an analytical platform that offers rapid measurement, high sensitivity, efficient separation, broad dynamic range, and application to an expansive chemical space. Here, we present a novel platform for small molecule analyses that addresses these requirements by combining solid-phase extraction with ion mobility spectrometry and mass spectrometry (SPE-IMS-MS). This platform is capable of performing both targeted and global measurements of endogenous metabolites and xenobiotics in human biofluids with high reproducibility (CV 6 3%), sensitivity (LODs in the pM range in biofluids) and throughput (10-s sample-to-sample duty cycle). We report application of this platform to the analysis of human urine from patients with and without type 1 diabetes, where we observed statistically significant variations in the concentration of disaccharides and previously unreported chemical isomers. This SPE-IMS-MS platform overcomes many of the current challenges of large-scale metabolomic and exposomic analyses and offers a viable option for population and patient cohort screening in an effort to gain insights into disease processes and human environmental chemical exposure.

SPE-IMS-MS: An automated platform for sub-sixty second surveillance of endogenous metabolites and xenobiotics in biofluids

PubMed Central

Zhang, Xing; Romm, Michelle; Zheng, Xueyun; Zink, Erika M.; Kim, Young-Mo; Burnum-Johnson, Kristin E.; Orton, Daniel J.; Apffel, Alex; Ibrahim, Yehia M.; Monroe, Matthew E.; Moore, Ronald J.; Smith, Jordan N.; Ma, Jian; Renslow, Ryan S.; Thomas, Dennis G.; Blackwell, Anne E.; Swinford, Glenn; Sausen, John; Kurulugama, Ruwan T.; Eno, Nathan; Darland, Ed; Stafford, George; Fjeldsted, John; Metz, Thomas O.; Teeguarden, Justin G.; Smith, Richard D.; Baker, Erin S.

2017-01-01

Characterization of endogenous metabolites and xenobiotics is essential to deconvoluting the genetic and environmental causes of disease. However, surveillance of chemical exposure and disease-related changes in large cohorts requires an analytical platform that offers rapid measurement, high sensitivity, efficient separation, broad dynamic range, and application to an expansive chemical space. Here, we present a novel platform for small molecule analyses that addresses these requirements by combining solid-phase extraction with ion mobility spectrometry and mass spectrometry (SPE-IMS-MS). This platform is capable of performing both targeted and global measurements of endogenous metabolites and xenobiotics in human biofluids with high reproducibility (CV 6 3%), sensitivity (LODs in the pM range in biofluids) and throughput (10-s sample-to-sample duty cycle). We report application of this platform to the analysis of human urine from patients with and without type 1 diabetes, where we observed statistically significant variations in the concentration of disaccharides and previously unreported chemical isomers. This SPE-IMS-MS platform overcomes many of the current challenges of large-scale metabolomic and exposomic analyses and offers a viable option for population and patient cohort screening in an effort to gain insights into disease processes and human environmental chemical exposure. PMID:29276770

Detection of Inter-Lineage Natural Recombination in Avian Paramyxovirus Serotype 1 Using Simplified Deep Sequencing Platform

PubMed Central

Satharasinghe, Dilan A.; Murulitharan, Kavitha; Tan, Sheau W.; Yeap, Swee K.; Munir, Muhammad; Ideris, Aini; Omar, Abdul R.

2016-01-01

Newcastle disease virus (NDV) is a prototype member of avian paramyxovirus serotype 1 (APMV-1), which causes severe and contagious disease in the commercial poultry and wild birds. Despite extensive vaccination programs and other control measures, the disease remains endemic around the globe especially in Asia, Africa, and the Middle East. Being a single serotype, genotype II based vaccines remained most acceptable means of immunization. However, the evidence is emerging on failures of vaccines mainly due to evolving nature of the virus and higher genetic gaps between vaccine and field strains of APMV-1. Most of the epidemiological and genetic characterizations of APMVs are based on conventional methods, which are prone to mask the diverse population of viruses in complex samples. In this study, we report the application of a simple, robust, and less resource-demanding methodology for the whole genome sequencing of NDV, using next-generation sequencing (NGS) on the Illumina MiSeq platform. Using this platform, we sequenced full genomes of five virulent Malaysian NDV strains collected during 2004–2013. All isolates clustered within highly prevalent lineage 5 (specifically in lineage 5a); however, a significantly greater genetic divergence was observed in isolates collected from 2004 to 2011. Interestingly, genetic characterization of one isolate collected in 2013 (IBS025/13) shown natural recombination between lineage 2 and lineage 5. In the event of recombination, the isolate (IBS025/13) carried nucleocapsid protein consist of 55–1801 nucleotides (nts) and near-complete phosphoprotein (1804–3254 nts) genes of lineage 2 whereas surface glycoproteins (fusion, hemagglutinin-neuraminidase) and large polymerase of lineage 5. Additionally, the recombinant virus has a genome size of 15,186 nts which is characteristics for the old genotypes I–IV isolated from 1930 to 1960. Taken together, we report the occurrence of a natural recombination in circulating strains of NDV in commercial poultry using NGS methodology. These findings will not only highlight the potential of RNA viruses to evolve but also to consider the application of NGS in revealing the genetic diversity of these viruses in clinical materials. Factors that drive these evolutionary events and subsequent impact of these divergences on clinical outcome of the disease warrant future investigations. PMID:27965642

Detection of Inter-Lineage Natural Recombination in Avian Paramyxovirus Serotype 1 Using Simplified Deep Sequencing Platform.

PubMed

Satharasinghe, Dilan A; Murulitharan, Kavitha; Tan, Sheau W; Yeap, Swee K; Munir, Muhammad; Ideris, Aini; Omar, Abdul R

2016-01-01

Newcastle disease virus (NDV) is a prototype member of avian paramyxovirus serotype 1 (APMV-1), which causes severe and contagious disease in the commercial poultry and wild birds. Despite extensive vaccination programs and other control measures, the disease remains endemic around the globe especially in Asia, Africa, and the Middle East. Being a single serotype, genotype II based vaccines remained most acceptable means of immunization. However, the evidence is emerging on failures of vaccines mainly due to evolving nature of the virus and higher genetic gaps between vaccine and field strains of APMV-1. Most of the epidemiological and genetic characterizations of APMVs are based on conventional methods, which are prone to mask the diverse population of viruses in complex samples. In this study, we report the application of a simple, robust, and less resource-demanding methodology for the whole genome sequencing of NDV, using next-generation sequencing (NGS) on the Illumina MiSeq platform. Using this platform, we sequenced full genomes of five virulent Malaysian NDV strains collected during 2004-2013. All isolates clustered within highly prevalent lineage 5 (specifically in lineage 5a); however, a significantly greater genetic divergence was observed in isolates collected from 2004 to 2011. Interestingly, genetic characterization of one isolate collected in 2013 (IBS025/13) shown natural recombination between lineage 2 and lineage 5. In the event of recombination, the isolate (IBS025/13) carried nucleocapsid protein consist of 55-1801 nucleotides (nts) and near-complete phosphoprotein (1804-3254 nts) genes of lineage 2 whereas surface glycoproteins (fusion, hemagglutinin-neuraminidase) and large polymerase of lineage 5. Additionally, the recombinant virus has a genome size of 15,186 nts which is characteristics for the old genotypes I-IV isolated from 1930 to 1960. Taken together, we report the occurrence of a natural recombination in circulating strains of NDV in commercial poultry using NGS methodology. These findings will not only highlight the potential of RNA viruses to evolve but also to consider the application of NGS in revealing the genetic diversity of these viruses in clinical materials. Factors that drive these evolutionary events and subsequent impact of these divergences on clinical outcome of the disease warrant future investigations.

The group II intron maturase: a reverse transcriptase and splicing factor go hand in hand.

PubMed

Zhao, Chen; Pyle, Anna Marie

2017-12-01

The splicing of group II introns in vivo requires the assistance of a multifunctional intron encoded protein (IEP, or maturase). Each IEP is also a reverse-transcriptase enzyme that enables group II introns to behave as mobile genetic elements. During splicing or retro-transposition, each group II intron forms a tight, specific complex with its own encoded IEP, resulting in a highly reactive holoenzyme. This review focuses on the structural basis for IEP function, as revealed by recent crystal structures of an IEP reverse transcriptase domain and cryo-EM structures of an IEP-intron complex. These structures explain how the same IEP scaffold is utilized for intron recognition, splicing and reverse transcription, while providing a physical basis for understanding the evolutionary transformation of the IEP into the eukaryotic splicing factor Prp8. Copyright © 2017 Elsevier Ltd. All rights reserved.

Muscular dystrophy in a dish: engineered human skeletal muscle mimetics for disease modeling and drug discovery

PubMed Central

Smith, Alec S.T.; Davis, Jennifer; Lee, Gabsang; Mack, David L.

2016-01-01

Engineered in vitro models using human cells, particularly patient-derived induced pluripotent stem cells (iPSCs), offer a potential solution to issues associated with the use of animals for studying disease pathology and drug efficacy. Given the prevalence of muscle diseases in human populations, an engineered tissue model of human skeletal muscle could provide a biologically accurate platform to study basic muscle physiology, disease progression, and drug efficacy and/or toxicity. Such platforms could be used as phenotypic drug screens to identify compounds capable of alleviating or reversing congenital myopathies, such as Duchene muscular dystrophy (DMD). Here, we review current skeletal muscle modeling technologies with a specific focus on efforts to generate biomimetic systems for investigating the pathophysiology of dystrophic muscle. PMID:27109386

Genetics and genomics in bioenergy and bioproducts

Treesearch

James S. McLaren; Thomas W. Jeffries

2003-01-01

There is now widespread acknowledgment that renewable bioresources, as a platform for raw inputs and as a source of powerful biocatalysts, have considerable potential to support sustainable economic growth, increase national energy security, strengthen rural communities, and minimize anthropogenic effects on the environment. Clearly, the transition to renewable...

Proximal hyperspectral sensing and data analysis approaches for field-based plant phenomics

USDA-ARS?s Scientific Manuscript database

Field-based plant phenomics requires robust crop sensing platforms and data analysis tools to successfully identify cultivars that exhibit phenotypes with high agronomic and economic importance. Such efforts will lead to genetic improvements that maintain high crop yield with concomitant tolerance t...

The plastid genome as a platform for the expression of microbial resistance genes

USDA-ARS?s Scientific Manuscript database

In recent years, our fundamental understanding of host-microbe interaction has developed considerably. We have begun to tease out the genetic components that influence host resistance to microbial colonization. The use of advancing molecular technologies such as microarray expression profiling and...

Could Rice Endosperm Be the Answer for Inexpensive HIV Protection? | Poster

Cancer.gov

According to a study in Plant Biotechnology Journal, genetically modified rice could be an inexpensive production platform for microbicides that inhibit HIV entry into target cells. Such a method could be one sustainable option for poverty-stricken countries with high rates of AIDS.

Developing Single Nucleotide Polymorphism markers for the identification of Coffee germplasm

USDA-ARS?s Scientific Manuscript database

Coffee is one of the most widely consumed beverages that represent a multibillion dollar global industry. Accurate identification of coffee cultivars is essential for efficient management, exchange and use of coffee genetic resources. So far a universal platform that can allow data comparison across...

Independently segregating simple sequence repeats (SSR) alleles in polyploid sugarcane

USDA-ARS?s Scientific Manuscript database

The complex nuclear genomic and flower structures of sugarcane cultivars (Saccharum hybrids spp., 2n = 10x = 100 – 130) render sugarcane a difficult subject for genetics research. Using a capillary electrophoresis- and fluorescence-labeling-based SSR genotyping platform, the segregation of a multi-a...

Genetic manipulation for inherited neurodegenerative diseases: myth or reality?

PubMed Central

Yu-Wai-Man, Patrick

2016-01-01

Rare genetic diseases affect about 7% of the general population and over 7000 distinct clinical syndromes have been described with the majority being due to single gene defects. This review will provide a critical overview of genetic strategies that are being pioneered to halt or reverse disease progression in inherited neurodegenerative diseases. This field of research covers a vast area and only the most promising treatment paradigms will be discussed with a particular focus on inherited eye diseases, which have paved the way for innovative gene therapy paradigms, and mitochondrial diseases, which are currently generating a lot of debate centred on the bioethics of germline manipulation. PMID:27002113

An in vivo multiplexed small molecule screening platform

PubMed Central

Yang, Dian; Ogasawara, Daisuke; Dix, Melissa M.; Rogers, Zoë N.; Chuang, Chen-Hua; McFarland, Christopher D.; Chiou, Shin-Heng; Brown, J. Mark; Cravatt, Benjamin F.; Bogyo, Matthew; Winslow, Monte M.

2016-01-01

Phenotype-based small molecule screening is a powerful method to identify regulators of cellular function. However, such screens are generally performed in vitro using conditions that do not necessarily model complex physiological conditions or disease states. Here, we use molecular cell barcoding to enable direct in vivo phenotypic screening of libraries of small molecules. The multiplexed nature of this approach allows rapid in vivo analysis of hundreds to thousands of compounds. Using this platform, we screened >700 covalent inhibitors directed towards hydrolases for their effect on pancreatic cancer metastatic seeding. We identified multiple hits and confirmed the relevant target of one compound as the lipase ABHD6. Pharmacological and genetic studies confirmed the role of this enzyme as a regulator of metastatic fitness. Our results highlight the applicability of this multiplexed screening platform for investigating complex processes in vivo. PMID:27617390

Acylphloroglucinol biosynthesis in strawberry fruit

USDA-ARS?s Scientific Manuscript database

Phenolics have health-promoting properties and are a major group of metabolites in fruit crops. Through reverse genetic analysis of the functions of four ripening-related genes in the octoploid strawberry, Fragaria ×ananassa, we discovered four acylphloroglucinol (APG)-glucosides as native strawberr...

Reinventing potato at the diploid level

USDA-ARS?s Scientific Manuscript database

The outcrossing polyploidy nature of cultivated potato has hindered the use of genomics resources to dissect the genetic basis of agronomically important traits. Reversion to the diploid level allows us to apply powerful tools toward this effort. Parthenogenesis generates diploid cultivated potato, ...

GB3.0: a platform for plant bio-design that connects functional DNA elements with associated biological data

PubMed Central

Vazquez-Vilar, Marta; Quijano-Rubio, Alfredo; Fernandez-del-Carmen, Asun; Sarrion-Perdigones, Alejandro; Ochoa-Fernandez, Rocio; Ziarsolo, Peio

2017-01-01

Abstract Modular DNA assembly simplifies multigene engineering in Plant Synthetic Biology. Furthermore, the recent adoption of a common syntax to facilitate the exchange of plant DNA parts (phytobricks) is a promising strategy to speed up genetic engineering. Following this lead, here, we present a platform for plant biodesign that incorporates functional descriptions of phytobricks obtained under pre-defined experimental conditions, and systematically registers the resulting information as metadata for documentation. To facilitate the handling of functional descriptions, we developed a new version (v3.0) of the GoldenBraid (GB) webtool that integrates the experimental data and displays it in the form of datasheets. We report the use of the Luciferase/Renilla (Luc/Ren) transient agroinfiltration assay in Nicotiana benthamiana as a standard to estimate relative transcriptional activities conferred by regulatory phytobricks, and show the consistency and reproducibility of this method in the characterization of a synthetic phytobrick based on the CaMV35S promoter. Furthermore, we illustrate the potential for combinatorial optimization and incremental innovation of the GB3.0 platform in two separate examples, (i) the development of a collection of orthogonal transcriptional regulators based on phiC31 integrase and (ii) the design of a small genetic circuit that connects a glucocorticoid switch to a MYB/bHLH transcriptional activation module. PMID:28053117

Impact of Genetic Deletion of Platform Apolipoproteins on the Size Distribution of the Murine Lipoproteome

PubMed Central

Gordon, Scott M.; Li, Hailong; Zhu, Xiaoting; Tso, Patrick; Reardon, Catherine A.; Shah, Amy S.; Lu, L. Jason; Davidson, W. Sean

2016-01-01

Given their association with cardiovascular disease protection, there has been intense interest in understanding the biology of high density lipoproteins (HDL). HDL is actually a family of diverse particle types, each made up of discrete - but as yet undetermined – combinations of proteins drawn from up to 95 lipophilic plasma proteins. The abundant apolipoproteins (apo) of the A class (apoA-I, apoA-II and apoA-IV) have been proposed to act as organizing platforms for auxiliary proteins, but this concept has not been systematically evaluated. We assessed the impact of genetic knock down of each platform protein on the particle size distribution of auxiliary HDL proteins. Loss of apoA-I or apoA-II massively reduced HDL lipids and changed the plasma size pattern and/or abundance of several plasma proteins. Surprisingly though, many HDL proteins were not affected, suggesting they assemble on lipid particles in the absence of apoA-I or apoA-II. In contrast, apoA-IV ablation had minor effects on plasma lipids and proteins, suggesting that it forms particles that largely exclude other apolipoproteins. Overall, the data indicate that distinct HDL subpopulations exist that do not contain, nor depend on, apoA-I, apoA-II or apoA-IV and these contribute substantially to the proteomic diversity of HDL. PMID:27385375

Barley whole exome capture: a tool for genomic research in the genus Hordeum and beyond

PubMed Central

Mascher, Martin; Richmond, Todd A; Gerhardt, Daniel J; Himmelbach, Axel; Clissold, Leah; Sampath, Dharanya; Ayling, Sarah; Steuernagel, Burkhard; Pfeifer, Matthias; D'Ascenzo, Mark; Akhunov, Eduard D; Hedley, Pete E; Gonzales, Ana M; Morrell, Peter L; Kilian, Benjamin; Blattner, Frank R; Scholz, Uwe; Mayer, Klaus FX; Flavell, Andrew J; Muehlbauer, Gary J; Waugh, Robbie; Jeddeloh, Jeffrey A; Stein, Nils

2013-01-01

Advanced resources for genome-assisted research in barley (Hordeum vulgare) including a whole-genome shotgun assembly and an integrated physical map have recently become available. These have made possible studies that aim to assess genetic diversity or to isolate single genes by whole-genome resequencing and in silico variant detection. However such an approach remains expensive given the 5 Gb size of the barley genome. Targeted sequencing of the mRNA-coding exome reduces barley genomic complexity more than 50-fold, thus dramatically reducing this heavy sequencing and analysis load. We have developed and employed an in-solution hybridization-based sequence capture platform to selectively enrich for a 61.6 megabase coding sequence target that includes predicted genes from the genome assembly of the cultivar Morex as well as publicly available full-length cDNAs and de novo assembled RNA-Seq consensus sequence contigs. The platform provides a highly specific capture with substantial and reproducible enrichment of targeted exons, both for cultivated barley and related species. We show that this exome capture platform provides a clear path towards a broader and deeper understanding of the natural variation residing in the mRNA-coding part of the barley genome and will thus constitute a valuable resource for applications such as mapping-by-sequencing and genetic diversity analyzes. PMID:23889683

GlobAl Distribution of GEnetic Traits (GADGET) web server: polygenic trait scores worldwide.

PubMed

Chande, Aroon T; Wang, Lu; Rishishwar, Lavanya; Conley, Andrew B; Norris, Emily T; Valderrama-Aguirre, Augusto; Jordan, I King

2018-05-18

Human populations from around the world show striking phenotypic variation across a wide variety of traits. Genome-wide association studies (GWAS) are used to uncover genetic variants that influence the expression of heritable human traits; accordingly, population-specific distributions of GWAS-implicated variants may shed light on the genetic basis of human phenotypic diversity. With this in mind, we developed the GlobAl Distribution of GEnetic Traits web server (GADGET http://gadget.biosci.gatech.edu). The GADGET web server provides users with a dynamic visual platform for exploring the relationship between worldwide genetic diversity and the genetic architecture underlying numerous human phenotypes. GADGET integrates trait-implicated single nucleotide polymorphisms (SNPs) from GWAS, with population genetic data from the 1000 Genomes Project, to calculate genome-wide polygenic trait scores (PTS) for 818 phenotypes in 2504 individual genomes. Population-specific distributions of PTS are shown for 26 human populations across 5 continental population groups, with traits ordered based on the extent of variation observed among populations. Users of GADGET can also upload custom trait SNP sets to visualize global PTS distributions for their own traits of interest.

A differential mobility spectrometry/mass spectrometry platform for the rapid detection and quantitation of DNA adduct dG-ABP.

PubMed

Kafle, Amol; Klaene, Joshua; Hall, Adam B; Glick, James; Coy, Stephen L; Vouros, Paul

2013-07-15

There is continued interest in exploring new analytical technologies for the detection and quantitation of DNA adducts, biomarkers which provide direct evidence of exposure and genetic damage in cells. With the goal of reducing clean-up steps and improving sample throughput, a Differential Mobility Spectrometry/Mass Spectrometry (DMS/MS) platform has been introduced for adduct analysis. A DMS/MS platform has been utilized for the analysis of dG-ABP, the deoxyguanosine adduct of the bladder carcinogen 4-aminobiphenyl (4-ABP). After optimization of the DMS parameters, each sample was analyzed in just 30 s following a simple protein precipitation step of the digested DNA. A detection limit of one modification in 10^6 nucleosides has been achieved using only 2 µg of DNA. A brief comparison (quantitative and qualitative) with liquid chromatography/mass spectrometry is also presented highlighting the advantages of using the DMS/MS method as a high-throughput platform. The data presented demonstrate the successful application of a DMS/MS/MS platform for the rapid quantitation of DNA adducts using, as a model analyte, the deoxyguanosine adduct of the bladder carcinogen 4-aminobiphenyl. Copyright © 2013 John Wiley & Sons, Ltd.

A substitution in the transmembrane region of the glycoprotein leads to an unstable attenuation of Machupo virus.

PubMed

Patterson, Michael; Koma, Takaaki; Seregin, Alexey; Huang, Cheng; Miller, Milagros; Smith, Jennifer; Yun, Nadezhda; Smith, Jeanon; Paessler, Slobodan

2014-09-01

Machupo virus (MACV) is the etiologic agent of Bolivian hemorrhagic fever (BHF). Utilizing a reverse-genetics system recently developed, we report the rescue of a rationally modified recombinant MACV containing a single mutation in the transmembrane region of the glycoprotein. Following challenge of susceptible mice, we identified a significant reduction in virulence in the novel virus. We also identified an instability leading to reversion of the single mutation to a wild-type genotype. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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.

Continuous throughput and long-term observation of single-molecule FRET without immobilization.

PubMed

Tyagi, Swati; VanDelinder, Virginia; Banterle, Niccolò; Fuertes, Gustavo; Milles, Sigrid; Agez, Morgane; Lemke, Edward A

2014-03-01

We present an automated microfluidic platform that performs multisecond observation of single molecules with millisecond time resolution while bypassing the need for immobilization procedures. With this system, we confine biomolecules to a thin excitation field by reversibly collapsing microchannels to nanochannels. We demonstrate the power of our method by studying a variety of complex nucleic acid and protein systems, including DNA Holliday junctions, nucleosomes and human transglutaminase 2.

Explore, Visualize, and Analyze Functional Cancer Proteomic Data Using the Cancer Proteome Atlas. | Office of Cancer Genomics

Cancer.gov

Reverse-phase protein arrays (RPPA) represent a powerful functional proteomic approach to elucidate cancer-related molecular mechanisms and to develop novel cancer therapies. To facilitate community-based investigation of the large-scale protein expression data generated by this platform, we have developed a user-friendly, open-access bioinformatic resource, The Cancer Proteome Atlas (TCPA, http://tcpaportal.org), which contains two separate web applications.

Establishing Chlamydomonas reinhardtii as an industrial biotechnology host

PubMed Central

Scaife, Mark A; Nguyen, Ginnie TDT; Rico, Juan; Lambert, Devinn; Helliwell, Katherine E; Smith, Alison G

2015-01-01

Microalgae constitute a diverse group of eukaryotic unicellular organisms that are of interest for pure and applied research. Owing to their natural synthesis of value-added natural products microalgae are emerging as a source of sustainable chemical compounds, proteins and metabolites, including but not limited to those that could replace compounds currently made from fossil fuels. For the model microalga, Chlamydomonas reinhardtii, this has prompted a period of rapid development so that this organism is poised for exploitation as an industrial biotechnology platform. The question now is how best to achieve this? Highly advanced industrial biotechnology systems using bacteria and yeasts were established in a classical metabolic engineering manner over several decades. However, the advent of advanced molecular tools and the rise of synthetic biology provide an opportunity to expedite the development of C. reinhardtii as an industrial biotechnology platform, avoiding the process of incremental improvement. In this review we describe the current status of genetic manipulation of C. reinhardtii for metabolic engineering. We then introduce several concepts that underpin synthetic biology, and show how generic parts are identified and used in a standard manner to achieve predictable outputs. Based on this we suggest that the development of C. reinhardtii as an industrial biotechnology platform can be achieved more efficiently through adoption of a synthetic biology approach. Significance Statement Chlamydomonas reinhardtii offers potential as a host for the production of high value compounds for industrial biotechnology. Synthetic biology provides a mechanism to generate generic, well characterised tools for application in the rational genetic manipulation of organisms: if synthetic biology principles were adopted for manipulation of C. reinhardtii, development of this microalga as an industrial biotechnology platform would be expedited. PMID:25641561

A Novel Drug Delivery Vesicle Development to Reverse Neurodegeneration: Analysis of the Interactions among Protein, Graphene Oxide and Liposome

NASA Astrophysics Data System (ADS)

Miraz, Md Alamin

In this study, Liposome was decorated with graphene oxide (GO) to synthesize fully-biocompatible theranostic vesicle that can carry bovine serum albumin (BSA) as a model protein. Graphene oxide has been studied as one of the most promising platforms for promoting the growth and repair of neurons. Our graphene oxide based structure could account for the high efficiency of protein loading and deliver to the damaged neuron cell which can reverse the neurodegeneration associated with Alzheimer's disease. The resultant vesicle exhibited high stability in aqueous solution. We investigated the protein adsorption capacity and protein interaction to carbon-based nanomaterials. The Liposome, graphene oxide and bovine serum albumin (BSA) are all biocompatible and hence will not trigger an immune response in vivo.

Sex Reversal in C57BL/6J XY Mice Caused by Increased Expression of Ovarian Genes and Insufficient Activation of the Testis Determining Pathway

PubMed Central

Correa, Stephanie M.; Washburn, Linda L.; Kahlon, Ravi S.; Musson, Michelle C.; Bouma, Gerrit J.; Eicher, Eva M.; Albrecht, Kenneth H.

2012-01-01

Sex reversal can occur in XY humans with only a single functional WT1 or SF1 allele or a duplication of the chromosome region containing WNT4. In contrast, XY mice with only a single functional Wt1, Sf1, or Wnt4 allele, or mice that over-express Wnt4 from a transgene, reportedly are not sex-reversed. Because genetic background plays a critical role in testis differentiation, particularly in C57BL/6J (B6) mice, we tested the hypothesis that Wt1, Sf1, and Wnt4 are dosage sensitive in B6 XY mice. We found that reduced Wt1 or Sf1 dosage in B6 XYB6 mice impaired testis differentiation, but no ovarian tissue developed. If, however, a YAKR chromosome replaced the YB6 chromosome, these otherwise genetically identical B6 XY mice developed ovarian tissue. In contrast, reduced Wnt4 dosage increased the amount of testicular tissue present in Sf1+/− B6 XYAKR, Wt1+/− B6 XYAKR, B6 XYPOS, and B6 XYAKR fetuses. We propose that Wt1B6 and Sf1B6 are hypomorphic alleles of testis-determining pathway genes and that Wnt4B6 is a hypermorphic allele of an ovary-determining pathway gene. The latter hypothesis is supported by the finding that expression of Wnt4 and four other genes in the ovary-determining pathway are elevated in normal B6 XX E12.5 ovaries. We propose that B6 mice are sensitive to XY sex reversal, at least in part, because they carry Wt1B6 and/or Sf1B6 alleles that compromise testis differentiation and a Wnt4B6 allele that promotes ovary differentiation and thereby antagonizes testis differentiation. Addition of a “weak” Sry allele, such as the one on the YPOS chromosome, to the sensitized B6 background results in inappropriate development of ovarian tissue. We conclude that Wt1, Sf1, and Wnt4 are dosage-sensitive in mice, this dosage-sensitivity is genetic background-dependant, and the mouse strains described here are good models for the investigation of human dosage-sensitive XY sex reversal. PMID:22496664

Maize rayado fino virus-like particles expressed in tobacco plants: a new platform for cysteine selective bioconjugation peptide display

USDA-ARS?s Scientific Manuscript database

The ability of plant virus coat proteins to self-assemble into virus-like particles (VLPs), coupled with unique properties including three-dimensional structures, orthogonal reactivities, suitability for genetic manipulation and chemical bio-conjugation, provide potential utility in nanotechnology a...

Triacetic acid lactone production in industrial Saccharomyces yeast strains

USDA-ARS?s Scientific Manuscript database

Triacetic acid lactone (TAL) is a potential platform chemical that can be produced in yeast. To evaluate the potential for industrial yeast strains to produce TAL, the g2ps1 gene encoding 2-pyrone synthase was transformed into thirteen industrial yeast strains of varied genetic background. TAL produ...

An improved consensus linkage map of barley based on flow-sorted chromosomes and SNP markers

USDA-ARS?s Scientific Manuscript database

Recent advances in high-throughput genotyping have made it easier to combine information from different mapping populations into consensus genetic maps, which provide increased marker density and genome coverage compared to individual maps. Previously, a SNP-based genotyping platform was developed a...

New genetic tools to improve citrus fruit quality and drive consumer demand

USDA-ARS?s Scientific Manuscript database

Chemical and genomic dissection of important components underlying fruit quality has led toward the development of new tools to make the creation and selection of citrus cultivars improved in quality attributes more targeted and efficient. The use of SNP platforms and other technologies have resulte...

Genome editing: progress and challenges for medical applications.

PubMed

Carroll, Dana

2016-11-15

The development of the CRISPR-Cas platform for genome editing has greatly simplified the process of making targeted genetic modifications. Applications of genome editing are expected to have a substantial impact on human therapies through the development of better animal models, new target discovery, and direct therapeutic intervention.

Multi-locus mixed model analysis of stem rust resistance in a worldwide collection of winter wheat

USDA-ARS?s Scientific Manuscript database

Genome-wide association mapping is a powerful tool for dissecting the relationship between phenotypes and genetic variants in diverse populations. With improved cost efficiency of high-throughput genotyping platforms, association mapping is a desirable method to mine populations for favorable allele...

In Situ Gene Therapy via AAV-CRISPR-Cas9-Mediated Targeted Gene Regulation.

PubMed

Moreno, Ana M; Fu, Xin; Zhu, Jie; Katrekar, Dhruva; Shih, Yu-Ru V; Marlett, John; Cabotaje, Jessica; Tat, Jasmine; Naughton, John; Lisowski, Leszek; Varghese, Shyni; Zhang, Kang; Mali, Prashant

2018-04-25

Development of efficacious in vivo delivery platforms for CRISPR-Cas9-based epigenome engineering will be critical to enable the ability to target human diseases without permanent modification of the genome. Toward this, we utilized split-Cas9 systems to develop a modular adeno-associated viral (AAV) vector platform for CRISPR-Cas9 delivery to enable the full spectrum of targeted in situ gene regulation functionalities, demonstrating robust transcriptional repression (up to 80%) and activation (up to 6-fold) of target genes in cell culture and mice. We also applied our platform for targeted in vivo gene-repression-mediated gene therapy for retinitis pigmentosa. Specifically, we engineered targeted repression of Nrl, a master regulator of rod photoreceptor determination, and demonstrated Nrl knockdown mediates in situ reprogramming of rod cells into cone-like cells that are resistant to retinitis pigmentosa-specific mutations, with concomitant prevention of secondary cone loss. Furthermore, we benchmarked our results from Nrl knockdown with those from in vivo Nrl knockout via gene editing. Taken together, our AAV-CRISPR-Cas9 platform for in vivo epigenome engineering enables a robust approach to target disease in a genomically scarless and potentially reversible manner. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Systematic Optimization of Long Gradient Chromatography Mass Spectrometry for Deep Analysis of Brain Proteome

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

Wang, Hong; Yang, Yanling; Li, Yuxin

2015-02-06

Development of high resolution liquid chromatography (LC) is essential for improving the sensitivity and throughput of mass spectrometry (MS)-based proteomics. Here we present systematic optimization of a long gradient LC-MS/MS platform to enhance protein identification from a complex mixture. The platform employed an in-house fabricated, reverse phase column (100 μm x 150 cm) coupled with Q Exactive MS. The column was capable of achieving a peak capacity of approximately 700 in a 720 min gradient of 10-45% acetonitrile. The optimal loading level was about 6 micrograms of peptides, although the column allowed loading as many as 20 micrograms. Gas phasemore » fractionation of peptide ions further increased the number of peptide identification by ~10%. Moreover, the combination of basic pH LC pre-fractionation with the long gradient LC-MS/MS platform enabled the identification of 96,127 peptides and 10,544 proteins at 1% protein false discovery rate in a postmortem brain sample of Alzheimer’s disease. As deep RNA sequencing of the same specimen suggested that ~16,000 genes were expressed, current analysis covered more than 60% of the expressed proteome. Further improvement strategies of the LC/LC-MS/MS platform were also discussed.« less

A wearable fingernail chemical sensing platform: pH sensing at your fingertips.

PubMed

Kim, Jayoung; Cho, Thomas N; Valdés-Ramírez, Gabriela; Wang, Joseph

2016-04-01

This article demonstrates an example of a wearable chemical sensor based on a fingernail platform. Fingernails represent an attractive wearable platform, merging beauty products with chemical sensing, to enable monitoring of our surrounding environment. The new colorimetric pH fingernail sensor relies on coating artificial nails with a recognition layer consisted of pH indicators entrapped in a polyvinyl chloride (PVC) matrix. Such color changing fingernails offer fast and reversible response to pH changes, repeated use, and intense color change detected easily with naked eye. The PVC matrix prevents leaching out of the indicator molecules from the fingernail sensor toward such repeated use. The limited narrow working pH range of a single pH indicator has been addressed by multiplexing three different pH indicators: bromothymol blue (pH 6.0-7.6), bromocresol green (pH 3.8-5.4), and cresol red (pH 7.2-8.8), as demonstrated for analyses of real-life samples of acidic, neutral, and basic character. The new concept of an optical wearable chemical sensor on fingernail platforms can be expanded towards diverse analytes for various applications in connection to the judicious design of the recognition layer. Copyright © 2016 Elsevier B.V. All rights reserved.

FPscope: a field-portable high-resolution microscope using a cellphone lens.

PubMed

Dong, Siyuan; Guo, Kaikai; Nanda, Pariksheet; Shiradkar, Radhika; Zheng, Guoan

2014-10-01

The large consumer market has made cellphone lens modules available at low-cost and in high-quality. In a conventional cellphone camera, the lens module is used to demagnify the scene onto the image plane of the camera, where image sensor is located. In this work, we report a 3D-printed high-resolution Fourier ptychographic microscope, termed FPscope, which uses a cellphone lens in a reverse manner. In our platform, we replace the image sensor with sample specimens, and use the cellphone lens to project the magnified image to the detector. To supersede the diffraction limit of the lens module, we use an LED array to illuminate the sample from different incident angles and synthesize the acquired images using the Fourier ptychographic algorithm. As a demonstration, we use the reported platform to acquire high-resolution images of resolution target and biological specimens, with a maximum synthetic numerical aperture (NA) of 0.5. We also show that, the depth-of-focus of the reported platform is about 0.1 mm, orders of magnitude longer than that of a conventional microscope objective with a similar NA. The reported platform may enable healthcare accesses in low-resource settings. It can also be used to demonstrate the concept of computational optics for educational purposes.

Biomarker discovery for colon cancer using a 761 gene RT-PCR assay.

PubMed

Clark-Langone, Kim M; Wu, Jenny Y; Sangli, Chithra; Chen, Angela; Snable, James L; Nguyen, Anhthu; Hackett, James R; Baker, Joffre; Yothers, Greg; Kim, Chungyeul; Cronin, Maureen T

2007-08-15

Reverse transcription PCR (RT-PCR) is widely recognized to be the gold standard method for quantifying gene expression. Studies using RT-PCR technology as a discovery tool have historically been limited to relatively small gene sets compared to other gene expression platforms such as microarrays. We have recently shown that TaqMan RT-PCR can be scaled up to profile expression for 192 genes in fixed paraffin-embedded (FPE) clinical study tumor specimens. This technology has also been used to develop and commercialize a widely used clinical test for breast cancer prognosis and prediction, the Onco typeDX assay. A similar need exists in colon cancer for a test that provides information on the likelihood of disease recurrence in colon cancer (prognosis) and the likelihood of tumor response to standard chemotherapy regimens (prediction). We have now scaled our RT-PCR assay to efficiently screen 761 biomarkers across hundreds of patient samples and applied this process to biomarker discovery in colon cancer. This screening strategy remains attractive due to the inherent advantages of maintaining platform consistency from discovery through clinical application. RNA was extracted from formalin fixed paraffin embedded (FPE) tissue, as old as 28 years, from 354 patients enrolled in NSABP C-01 and C-02 colon cancer studies. Multiplexed reverse transcription reactions were performed using a gene specific primer pool containing 761 unique primers. PCR was performed as independent TaqMan reactions for each candidate gene. Hierarchal clustering demonstrates that genes expected to co-express form obvious, distinct and in certain cases very tightly correlated clusters, validating the reliability of this technical approach to biomarker discovery. We have developed a high throughput, quantitatively precise multi-analyte gene expression platform for biomarker discovery that approaches low density DNA arrays in numbers of genes analyzed while maintaining the high specificity, sensitivity and reproducibility that are characteristics of RT-PCR. Biomarkers discovered using this approach can be transferred to a clinical reference laboratory setting without having to re-validate the assay on a second technology platform.

46,XX males: a case series based on clinical and genetics evaluation.

PubMed

Mohammadpour Lashkari, F; Totonchi, M; Zamanian, M R; Mansouri, Z; Sadighi Gilani, M A; Sabbaghian, M; Mohseni Meybodi, A

2017-09-01

46,XX male sex reversal syndrome is one of the rarest sex chromosomal aberrations. The presence of SRY gene on one of the X chromosomes is the most frequent cause of this syndrome. Based on Y chromosome profile, there are SRY-positive and SRY-negative forms. The purpose of our study was to report first case series of Iranian patients and describe the different clinical appearances based on their genetic component. From the 8,114 azoospermic and severe oligozoospermic patients referred to Royan institute, we diagnosed 57 cases as sex reversal patients. Based on the endocrinological history, we performed karyotyping, SRY and AZF microdeletion screening. Patients had a female karyotype. According to available hormonal reports of 37 patients, 16 cases had low levels of testosterone (43.2%). On the other hand, 15 males were SRY positive (90.2%), while they lacked the spermatogenic factors encoding genes on Yq. Commencing the testicular differentiation in males, the SRY gene is considered to be very important in this process. Due to homogeneous results of karyotyping and AZF deletion, there are both positive and negative SRY cases that show similar sex reversal phenotypes. Evidences show that there could be diverse phenotypic differences that could be raised from various reasons. © 2016 Blackwell Verlag GmbH.

Chronic skin inflammation accelerates macrophage cholesterol crystal formation and atherosclerosis

PubMed Central

Ng, Qimin; Sanda, Gregory E.; Dey, Amit K.; Teague, Heather L.; Sorokin, Alexander V.; Dagur, Pradeep K.; Silverman, Joanna I.; Harrington, Charlotte L.; Rodante, Justin A.; Rose, Shawn M.; Varghese, Nevin J.; Belur, Agastya D.; Goyal, Aditya; Gelfand, Joel M.; Springer, Danielle A.; Bleck, Christopher K.E.; Thomas, Crystal L.; Yu, Zu-Xi; Winge, Mårten C.G.; Kruth, Howard S.; Marinkovich, M. Peter; Joshi, Aditya A.; Playford, Martin P.; Mehta, Nehal N.

2018-01-01

Inflammation is critical to atherogenesis. Psoriasis is a chronic inflammatory skin disease that accelerates atherosclerosis in humans and provides a compelling model to understand potential pathways linking these diseases. A murine model capturing the vascular and metabolic diseases in psoriasis would accelerate our understanding and provide a platform to test emerging therapies. We aimed to characterize a new murine model of skin inflammation (Rac1V12) from a cardiovascular standpoint to identify novel atherosclerotic signaling pathways modulated in chronic skin inflammation. The RacV12 psoriasis mouse resembled the human disease state, including presence of systemic inflammation, dyslipidemia, and cardiometabolic dysfunction. Psoriasis macrophages had a proatherosclerotic phenotype with increased lipid uptake and foam cell formation, and also showed a 6-fold increase in cholesterol crystal formation. We generated a triple-genetic K14-RacV12–/+/Srb1–/–/ApoER61H/H mouse and confirmed psoriasis accelerates atherogenesis (~7-fold increase). Finally, we noted a 60% reduction in superoxide dismutase 2 (SOD2) expression in human psoriasis macrophages. When SOD2 activity was restored in macrophages, their proatherogenic phenotype reversed. We demonstrate that the K14-RacV12 murine model captures the cardiometabolic dysfunction and accelerates vascular disease observed in chronic inflammation and that skin inflammation induces a proatherosclerotic macrophage phenotype with impaired SOD2 function, which associated with accelerated atherogenesis. PMID:29321372

Multiplexed MRM-based assays for the quantitation of proteins in mouse plasma and heart tissue.

PubMed

Percy, Andrew J; Michaud, Sarah A; Jardim, Armando; Sinclair, Nicholas J; Zhang, Suping; Mohammed, Yassene; Palmer, Andrea L; Hardie, Darryl B; Yang, Juncong; LeBlanc, Andre M; Borchers, Christoph H

2017-04-01

The mouse is the most commonly used laboratory animal, with more than 14 million mice being used for research each year in North America alone. The number and diversity of mouse models is increasing rapidly through genetic engineering strategies, but detailed characterization of these models is still challenging because most phenotypic information is derived from time-consuming histological and biochemical analyses. To expand the biochemists' toolkit, we generated a set of targeted proteomic assays for mouse plasma and heart tissue, utilizing bottom-up LC/MRM-MS with isotope-labeled peptides as internal standards. Protein quantitation was performed using reverse standard curves, with LC-MS platform and curve performance evaluated by quality control standards. The assays comprising the final panel (101 peptides for 81 proteins in plasma; 227 peptides for 159 proteins in heart tissue) have been rigorously developed under a fit-for-purpose approach and utilize stable-isotope labeled peptides for every analyte to provide high-quality, precise relative quantitation. In addition, the peptides have been tested to be interference-free and the assay is highly multiplexed, with reproducibly determined protein concentrations spanning >4 orders of magnitude. The developed assays have been used in a small pilot study to demonstrate their application to molecular phenotyping or biomarker discovery/verification studies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of the maize gravitropism gene lazy plant1 by a transposon-tagging genome resequencing strategy.

PubMed

Howard, Thomas P; Hayward, Andrew P; Tordillos, Anthony; Fragoso, Christopher; Moreno, Maria A; Tohme, Joe; Kausch, Albert P; Mottinger, John P; Dellaporta, Stephen L

2014-01-01

Since their initial discovery, transposons have been widely used as mutagens for forward and reverse genetic screens in a range of organisms. The problems of high copy number and sequence divergence among related transposons have often limited the efficiency at which tagged genes can be identified. A method was developed to identity the locations of Mutator (Mu) transposons in the Zea mays genome using a simple enrichment method combined with genome resequencing to identify transposon junction fragments. The sequencing library was prepared from genomic DNA by digesting with a restriction enzyme that cuts within a perfectly conserved motif of the Mu terminal inverted repeats (TIR). Paired-end reads containing Mu TIR sequences were computationally identified and chromosomal sequences flanking the transposon were mapped to the maize reference genome. This method has been used to identify Mu insertions in a number of alleles and to isolate the previously unidentified lazy plant1 (la1) gene. The la1 gene is required for the negatively gravitropic response of shoots and mutant plants lack the ability to sense gravity. Using bioinformatic and fluorescence microscopy approaches, we show that the la1 gene encodes a cell membrane and nuclear localized protein. Our Mu-Taq method is readily adaptable to identify the genomic locations of any insertion of a known sequence in any organism using any sequencing platform.

Selection of antigenic markers on a GFP-C{kappa} fusion scaffold with high sensitivity by eukaryotic ribosome display

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

Yang Yongmin; IgE Therapeutics, Inc., San Diego, CA 92121-2233; Barankiewicz, Teresa J.

2007-07-27

Ribosome display is a cell-free system permitting gene selection through the physical association of genetic material (mRNA) and its phenotypic (protein) product. While often used to select single-chain antibodies from large libraries by panning against immobilized antigens, we have adapted ribosome display for use in the 'reverse' format in order to select high affinity antigenic determinants against solid-phase antibody. To create an antigenic scaffold, DNA encoding green fluorescent protein (GFP) was fused to a light chain constant domain (C{kappa}) with stop codon deleted, and with 5' signals (T7 promoter, Kozak) enabling coupled transcription/translation in a eukaryotic cell-free system. Epitopes onmore » either GFP (5') or C{kappa} (3') were selected by anti-GFP or anti-C{kappa} antibodies, respectively, coupled to magnetic beads. After selection, mRNA was amplified directly from protein-ribosome-mRNA (PRM) complexes by in situ PCR followed by internal amplification and reassembly PCR. As little as 10 fg of the 1 kb DNA construct, i.e. approximately 7500 molecules, could be recovered following a single round of interaction with solid-phase anti-GFP antibody. This platform is highly specific and sensitive for the antigen-antibody interaction and may permit selection and reshaping of high affinity antigenic variants of scaffold proteins.« less

Identification of the Maize Gravitropism Gene lazy plant1 by a Transposon-Tagging Genome Resequencing Strategy

PubMed Central

Howard, Thomas P.; Hayward, Andrew P.; Tordillos, Anthony; Fragoso, Christopher; Moreno, Maria A.; Tohme, Joe; Kausch, Albert P.; Mottinger, John P.; Dellaporta, Stephen L.

2014-01-01

Since their initial discovery, transposons have been widely used as mutagens for forward and reverse genetic screens in a range of organisms. The problems of high copy number and sequence divergence among related transposons have often limited the efficiency at which tagged genes can be identified. A method was developed to identity the locations of Mutator (Mu) transposons in the Zea mays genome using a simple enrichment method combined with genome resequencing to identify transposon junction fragments. The sequencing library was prepared from genomic DNA by digesting with a restriction enzyme that cuts within a perfectly conserved motif of the Mu terminal inverted repeats (TIR). Paired-end reads containing Mu TIR sequences were computationally identified and chromosomal sequences flanking the transposon were mapped to the maize reference genome. This method has been used to identify Mu insertions in a number of alleles and to isolate the previously unidentified lazy plant1 (la1) gene. The la1 gene is required for the negatively gravitropic response of shoots and mutant plants lack the ability to sense gravity. Using bioinformatic and fluorescence microscopy approaches, we show that the la1 gene encodes a cell membrane and nuclear localized protein. Our Mu-Taq method is readily adaptable to identify the genomic locations of any insertion of a known sequence in any organism using any sequencing platform. PMID:24498020

Contribution of genetics to ecological restoration.

PubMed

Mijangos, Jose Luis; Pacioni, Carlo; Spencer, Peter B S; Craig, Michael D

2015-01-01

Ecological restoration of degraded ecosystems has emerged as a critical tool in the fight to reverse and ameliorate the current loss of biodiversity and ecosystem services. Approaches derived from different genetic disciplines are extending the theoretical and applied frameworks on which ecological restoration is based. We performed a search of scientific articles and identified 160 articles that employed a genetic approach within a restoration context to shed light on the links between genetics and restoration. These articles were then classified on whether they examined association between genetics and fitness or the application of genetics in demographic studies, and on the way the studies informed restoration practice. Although genetic research in restoration is rapidly growing, we found that studies could make better use of the extensive toolbox developed by applied fields in genetics. Overall, 41% of reviewed studies used genetic information to evaluate or monitor restoration, and 59% provided genetic information to guide prerestoration decision-making processes. Reviewed studies suggest that restoration practitioners often overlook the importance of including genetic aspects within their restoration goals. Even though there is a genetic basis influencing the provision of ecosystem services, few studies explored this relationship. We provide a view of research gaps, future directions and challenges in the genetics of restoration. © 2014 John Wiley & Sons Ltd.

The magnetic low of central Europe: analysis and interpretation by a multi scale approach.

NASA Astrophysics Data System (ADS)

Milano, Maurizio; Fedi, Maurizio

2016-04-01

The objective of this work is an interpretation of the European magnetic low (EML) which is the main magnetic anomaly characterizing the magnetic field of central Europe at high-altitude, extending from the eastern France to Poland and placed above the main geological boundary of Europe, the Trans European Suture Zone (TESZ), that separates the western and thinner Paleozoic platform from the eastern and thicker Precambrian platform. In particular, the EML has a relative magnetic high north-east of it, showing a reverse dipolar behavior that many authors tried to interpret in past also by high-altitude satellite exploration. We used an aeromagnetic dataset and employed a level-to-level upward continuation from 1 km up to 200 km, following a multiscale approach thanks to which the anomalies generated by sources placed at different depths can be discriminated. Low-altitude magnetic maps show a complex pattern of high-frequency anomalies up to an altitude of 50 km; then, increasing the altitude up to 200 km, the field simplifies gradually. In order to interpret the anomalies we generated the maps of the total gradient (|T|) of the field at each upward continued altitude, thanks to its property in localizing in a very simple way the edges of the sources and their horizontal position without specifying a priori information about source parameters. From the total gradient maps at low altitude we obtained information about the position of shallow and localized sources producing patterns of small anomalies. In central Europe, most of them have a reverse dipolar behavior, being related probably to metasedimentary rocks in the upper crust containing pyrrhotite and a strong remament component. At higher altitude the total gradient maps has been useful to give a more complex explanation of the EML taking in consideration the results obtained in previous studies. The maps at 150-200 km show that the maximum amplitude of |T| is exactly localized along the TESZ in the NW-SE direction. So, a simple contact model was performed in order to demonstrate that the main source that generates the EML is the complex fault system of the TESZ. However, the |T| maxima are positioned not only along the suture zone, but also in Central Europe, showing that the contributions to the EML derive also from sources placed in the Paleozoic platform with a reverse dipolar aspect. From these results it appears that the contributions responsible for the nature of this anomaly are to be reconnected first to the presence of the TESZ, which puts in contact two different platforms with different thicknesses, and also to the presence of bodies with a strong remanent component, which characterize part of the Central European crust.