Sample records for expression system based
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Kim, Seong Keun; Lee, Dae-Hee; Kim, Oh Cheol; Kim, Jihyun F; Yoon, Sung Ho
2017-09-15
Most inducible expression systems suffer from growth defects, leaky basal induction, and inhomogeneous expression levels within a host cell population. These difficulties are most prominent with the overproduction of membrane proteins that are toxic to host cells. Here, we developed an Escherichia coli inducible expression system for membrane protein production based on titrated expression of a mutant lac repressor (mLacI). Performance of the mLacI inducible system was evaluated in conjunction with commonly used lac operator-based expression vectors using a T7 or tac promoter. Remarkably, expression of a target gene can be titrated by the dose-dependent addition of l-rhamnose, and the expression levels were homogeneous in the cell population. The developed system was successfully applied to overexpress three membrane proteins that were otherwise difficult to produce in E. coli. This gene expression control system can be easily applied to a broad range of existing protein expression systems and should be useful in constructing genetic circuits that require precise output signals.
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Martin, G A; Kawaguchi, R; Lam, Y; DeGiovanni, A; Fukushima, M; Mutter, W
2001-10-01
The Rapid Translation System (RTS 500) (Roche Molecular Biochemicals) is a high-yield protein expression system that utilizes an enhanced E. coli lysate for an in vitro transcription/translation reaction. In contrast to conventional transcription/translation, this system allows protein expression to continue for more than 24 h. We demonstrated the utility of the RTS 500 by expressing different soluble and active proteins that generally pose problems in cell-based expression systems. We first expressed GFP-lunasin, a fusion protein that, because of its toxicity, has been impossible to produce in whole cells. The second protein we expressed, human interleukin-2 (IL-2), is generally difficult to produce, either as the native molecule or as a GSTfusion protein, in a soluble form in bacteria. Finally, we demonstrated the capacity of the RTS 500 to co-express proteins, by the simultaneous production of GFP and CAT in a single reaction. This new technology appears to be particularly usefulfor the convenient production of preparative amounts (100-900 microg) of proteins that are toxic or insoluble in cell-based systems.
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2017-01-01
Experiments in synthetic biology and microbiology can benefit from protein expression systems with low cell-to-cell variability (noise) and expression levels precisely tunable across a useful dynamic range. Despite advances in understanding the molecular biology of microbial gene regulation, many experiments employ protein-expression systems exhibiting high noise and nearly all-or-none responses to induction. I present an expression system that incorporates elements known to reduce gene expression noise: negative autoregulation and bicistronic transcription. I show by stochastic simulation that while negative autoregulation can produce a more gradual response to induction, bicistronic expression of a repressor and gene of interest can be necessary to reduce noise below the extrinsic limit. I synthesized a plasmid-based system incorporating these principles and studied its properties in Escherichia coli cells, using flow cytometry and fluorescence microscopy to characterize induction dose-response, induction/repression kinetics and gene expression noise. By varying ribosome binding site strengths, expression levels from 55–10,740 molecules/cell were achieved with noise below the extrinsic limit. Individual strains are inducible across a dynamic range greater than 20-fold. Experimental comparison of different regulatory networks confirmed that bicistronic autoregulation reduces noise, and revealed unexpectedly high noise for a conventional expression system with a constitutively expressed transcriptional repressor. I suggest a hybrid, low-noise expression system to increase the dynamic range. PMID:29084263
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Druzinec, Damir; Salzig, Denise; Brix, Alexander; Kraume, Matthias; Vilcinskas, Andreas; Kollewe, Christian; Czermak, Peter
2013-01-01
Due to the increasing use of insect cell based expression systems in research and industrial recombinant protein production, the development of efficient and reproducible production processes remains a challenging task. In this context, the application of online monitoring techniques is intended to ensure high and reproducible product qualities already during the early phases of process development. In the following chapter, the most common transient and stable insect cell based expression systems are briefly introduced. Novel applications of insect cell based expression systems for the production of insect derived antimicrobial peptides/proteins (AMPs) are discussed using the example of G. mellonella derived gloverin. Suitable in situ sensor techniques for insect cell culture monitoring in disposable and common bioreactor systems are outlined with respect to optical and capacitive sensor concepts. Since scale up of production processes is one of the most critical steps in process development, a conclusive overview is given about scale up aspects for industrial insect cell culture processes.
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Definition of Contravariant Velocity Components
NASA Technical Reports Server (NTRS)
Hung, Ching-moa; Kwak, Dochan (Technical Monitor)
2002-01-01
In this paper we have reviewed the basics of tensor analysis in an attempt to clarify some misconceptions regarding contravariant and covariant vector components as used in fluid dynamics. We have indicated that contravariant components are components of a given vector expressed as a unique combination of the covariant base vector system and, vice versa, that the covariant components are components of a vector expressed with the contravariant base vector system. Mathematically, expressing a vector with a combination of base vector is a decomposition process for a specific base vector system. Hence, the contravariant velocity components are decomposed components of velocity vector along the directions of coordinate lines, with respect to the covariant base vector system. However, the contravariant (and covariant) components are not physical quantities. Their magnitudes and dimensions are controlled by their corresponding covariant (and contravariant) base vectors.
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BEAT: A Web-Based Boolean Expression Fault-Based Test Case Generation Tool
ERIC Educational Resources Information Center
Chen, T. Y.; Grant, D. D.; Lau, M. F.; Ng, S. P.; Vasa, V. R.
2006-01-01
BEAT is a Web-based system that generates fault-based test cases from Boolean expressions. It is based on the integration of our several fault-based test case selection strategies. The generated test cases are considered to be fault-based, because they are aiming at the detection of particular faults. For example, when the Boolean expression is in…
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Heiss, Silvia; Hörmann, Angelika; Tauer, Christopher; Sonnleitner, Margot; Egger, Esther; Grabherr, Reingard; Heinl, Stefan
2016-03-10
Engineering lactic acid bacteria (LAB) is of growing importance for food and feed industry as well as for in vivo vaccination or the production of recombinant proteins in food grade organisms. Often, expression of a transgene is only desired at a certain time point or period, e.g. to minimize the metabolic burden for the host cell or to control the expression time span. For this purpose, inducible expression systems are preferred, though cost and availability of the inducing agent must be feasible. We selected the plasmid free strain Lactobacillus plantarum 3NSH for testing and characterization of novel inducible promoters/repressor systems. Their feasibility in recombinant protein production was evaluated. Expression of the reporter protein mCherry was monitored with the BioLector(®) micro-fermentation system. Reporter gene mCherry expression was compared under the control of different promoter/repressor systems: PlacA (an endogenous promoter/repressor system derived from L. plantarum 3NSH), PxylA (a promoter/repressor system derived from Bacillus megaterium DSMZ 319) and PlacSynth (synthetic promoter and codon-optimized repressor gene based on the Escherichia coli lac operon). We observed that PlacA was inducible solely by lactose, but not by non-metabolizable allolactose analoga. PxylA was inducible by xylose, yet showed basal expression under non-induced conditions. Growth on galactose (as compared to exponential growth phase on glucose) reduced basal mCherry expression at non-induced conditions. PlacSynth was inducible with TMG (methyl β-D-thiogalactopyranoside) and IPTG (isopropyl β-D-1-thiogalactopyranoside), but also showed basal expression without inducer. The promoter PlacSynth was used for establishment of a dual plasmid expression system, based on T7 RNA polymerase driven expression in L. plantarum. Comparative Western blot supported BioLector(®) micro-fermentation measurements. Conclusively, overall expression levels were moderate (compared to a constitutive promoter). We evaluated different inducible promoters, as well as an orthologous expression system, for controlled gene expression in L. plantarum. Furthermore, here we provide proof of concept for a T7 RNA polymerase based expression system for L. plantarum. Thereby we expanded the molecular toolbox for an industrial relevant and generally regarded as safe (GRAS) strain.
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Peck, Grantley R; Bowden, Timothy R; Shiell, Brian J; Michalski, Wojtek P
2014-01-01
EnBase (BioSilta, Finland) is a microbial cultivation system that replicates fed-batch systems through sustained release of glucose by enzymatic degradation of a polymeric substrate. Achievable bacterial cell densities and recombinant capripoxvirus protein expression levels, solubility, and antigenicity using the EnBase system were assessed. BL21-AI Escherichia coli expressing capripoxvirus proteins achieved up to eightfold higher cell densities when grown in EnBase media compared with standard media. Greater yields of capripoxvirus proteins were attained using EnBase media, either through increases in the amount of expressed protein per cell in conjunction with higher cell density or through the increase in cell density alone. Addition of EnBase booster enhanced protein yield for one of the proteins tested but reduced yield for the other. However, the amount of soluble forms of the capripoxvirus proteins tested was not different from that observed from cultures grown under standard conditions. Purified capripoxvirus proteins expressed using EnBase or standard media were assessed for their performance by enzyme-linked immunosorbent assay (ELISA) and were shown to be equally capable of specifically binding capripoxvirus antibodies.
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Gao, Wen; Yin, Jun; Bao, Lichen; Wang, Qun; Hou, Shan; Yue, Yali; Yao, Wenbing; Gao, Xiangdong
2018-05-18
Escherichia coli extracellular expression systems have a number of advantages over other systems, such as lower pyrogen levels and a simple purification process. Various approaches, such as the generation of leaky mutants via chromosomal engineering, have been explored for this expression system. However, extracellular protein yields in leaky mutants are relatively low compared to that in intracellular expression systems and therefore need to be improved. In this work, we describe the construction, characterization, and mechanism of enhanced extracellular expression in Escherichia coli. On the basis of the localizations, functions, and transcription levels of cell envelope proteins, we systematically elucidated the effects of multiple gene deletions on cell growth and extracellular expression using modified CRISPR/Cas9-based genome editing and a FlAsH labeling assay. High extracellular yields of heterologous proteins of different sizes were obtained by screening multiple gene mutations. The enhancement of extracellular secretion was associated with the derepression of translation and translocation. This work utilized universal methods in the design of extracellular expression systems for genes not directly associated with protein synthesis that were used to generate strains with higher protein expression capability. We anticipate that extracellular expression systems may help to shed light on the poorly understood aspects of these secretion processes as well as to further assist in the construction of engineered prokaryotic cells for efficient extracellular production of heterologous proteins.
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Designing a Syntax-Based Retrieval System for Supporting Language Learning
ERIC Educational Resources Information Center
Tsao, Nai-Lung; Kuo, Chin-Hwa; Wible, David; Hung, Tsung-Fu
2009-01-01
In this paper, we propose a syntax-based text retrieval system for on-line language learning and use a fast regular expression search engine as its main component. Regular expression searches provide more scalable querying and search results than keyword-based searches. However, without a well-designed index scheme, the execution time of regular…
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Royo, Jose Luis; Moreno-Ruiz, Emilia; Cebolla, Angel; Santero, Eduardo
2005-03-16
In our laboratory we have analyzed different factors to maximize the yield in heterologous protein expression for long-term cultivation, by combination of an efficient cascade expression system and stable integration in the bacterial chromosome. In this work, we have explored this system for the production of indigo dye as a model for biotechnological production, by expressing in Escherichia coli the thnA1A2A3A4 genes from Sphingomonas macrogolitabida strain TFA, which encode the components of a tetralin dioxygenase activity. We compared Ptac, and the Pm-based cascade expression circuit in a multicopy plasmid and stably integrated into the bacterial chromosome. Plasmid-based expression systems resulted in instability of indigo production when serially diluted batch experiments were performed without a selective pressure. This problem was solved by integrating the expression module in the chromosome. Despite the gene dosage reduction, the synergic effect of the cascade expression system produced comparable expression to the dioxygenase activity in the plasmid configuration but could be stably maintained for at least 5 days. Here, we show that the cascade amplification circuit integrated in the chromosome could be an excellent system for tight control and stable production of recombinant products.
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Yang, Laurence; Tan, Justin; O'Brien, Edward J; Monk, Jonathan M; Kim, Donghyuk; Li, Howard J; Charusanti, Pep; Ebrahim, Ali; Lloyd, Colton J; Yurkovich, James T; Du, Bin; Dräger, Andreas; Thomas, Alex; Sun, Yuekai; Saunders, Michael A; Palsson, Bernhard O
2015-08-25
Finding the minimal set of gene functions needed to sustain life is of both fundamental and practical importance. Minimal gene lists have been proposed by using comparative genomics-based core proteome definitions. A definition of a core proteome that is supported by empirical data, is understood at the systems-level, and provides a basis for computing essential cell functions is lacking. Here, we use a systems biology-based genome-scale model of metabolism and expression to define a functional core proteome consisting of 356 gene products, accounting for 44% of the Escherichia coli proteome by mass based on proteomics data. This systems biology core proteome includes 212 genes not found in previous comparative genomics-based core proteome definitions, accounts for 65% of known essential genes in E. coli, and has 78% gene function overlap with minimal genomes (Buchnera aphidicola and Mycoplasma genitalium). Based on transcriptomics data across environmental and genetic backgrounds, the systems biology core proteome is significantly enriched in nondifferentially expressed genes and depleted in differentially expressed genes. Compared with the noncore, core gene expression levels are also similar across genetic backgrounds (two times higher Spearman rank correlation) and exhibit significantly more complex transcriptional and posttranscriptional regulatory features (40% more transcription start sites per gene, 22% longer 5'UTR). Thus, genome-scale systems biology approaches rigorously identify a functional core proteome needed to support growth. This framework, validated by using high-throughput datasets, facilitates a mechanistic understanding of systems-level core proteome function through in silico models; it de facto defines a paleome.
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Irla, Marta; Heggeset, Tonje M B; Nærdal, Ingemar; Paul, Lidia; Haugen, Tone; Le, Simone B; Brautaset, Trygve; Wendisch, Volker F
2016-01-01
Bacillus methanolicus is a thermophilic methylotroph able to overproduce amino acids from methanol, a substrate not used for human or animal nutrition. Based on our previous RNA-seq analysis a mannitol inducible promoter and a putative mannitol activator gene mtlR were identified. The mannitol inducible promoter was applied for controlled gene expression using fluorescent reporter proteins and a flow cytometry analysis, and improved by changing the -35 promoter region and by co-expression of the mtlR regulator gene. For independent complementary gene expression control, the heterologous xylose-inducible system from B. megaterium was employed and a two-plasmid gene expression system was developed. Four different replicons for expression vectors were compared with respect to their copy number and stability. As an application example, methanol-based production of cadaverine was shown to be improved from 11.3 to 17.5 g/L when a heterologous lysine decarboxylase gene cadA was expressed from a theta-replicating rather than a rolling-circle replicating vector. The current work on inducible promoter systems and compatible theta- or rolling circle-replicating vectors is an important extension of the poorly developed B. methanolicus genetic toolbox, valuable for genetic engineering and further exploration of this bacterium.
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Irla, Marta; Heggeset, Tonje M. B.; Nærdal, Ingemar; Paul, Lidia; Haugen, Tone; Le, Simone B.; Brautaset, Trygve; Wendisch, Volker F.
2016-01-01
Bacillus methanolicus is a thermophilic methylotroph able to overproduce amino acids from methanol, a substrate not used for human or animal nutrition. Based on our previous RNA-seq analysis a mannitol inducible promoter and a putative mannitol activator gene mtlR were identified. The mannitol inducible promoter was applied for controlled gene expression using fluorescent reporter proteins and a flow cytometry analysis, and improved by changing the -35 promoter region and by co-expression of the mtlR regulator gene. For independent complementary gene expression control, the heterologous xylose-inducible system from B. megaterium was employed and a two-plasmid gene expression system was developed. Four different replicons for expression vectors were compared with respect to their copy number and stability. As an application example, methanol-based production of cadaverine was shown to be improved from 11.3 to 17.5 g/L when a heterologous lysine decarboxylase gene cadA was expressed from a theta-replicating rather than a rolling-circle replicating vector. The current work on inducible promoter systems and compatible theta- or rolling circle-replicating vectors is an important extension of the poorly developed B. methanolicus genetic toolbox, valuable for genetic engineering and further exploration of this bacterium. PMID:27713731
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Development of an Improved Mammalian Overexpression Method for Human CD62L
Brown, Haley A.; Roth, Gwynne; Holzapfel, Genevieve; Shen, Sarek; Rahbari, Kate; Ireland, Joanna; Zou, Zhongcheng; Sun, Peter D.
2014-01-01
We have previously developed a glutamine synthetase (GS)-based mammalian recombinant protein expression system that is capable of producing 5 to 30 mg/L recombinant proteins. The over expression is based on multiple rounds of target gene amplification driven by methionine sulfoximine (MSX), an inhibitor of glutamine synthetase. However, like other stable mammalian over expression systems, a major shortcoming of the GS-based expression system is its lengthy turn-around time, typically taking 4–6 months to produce. To shorten the construction time, we replaced the muti-round target gene amplifications with single-round in situ amplifications, thereby shortening the cell line construction to 2 months. The single-round in situ amplification method resulted in highest recombinant CD62L expressing CHO cell lines producing ~5mg/L soluble CD62L, similar to those derived from the multi-round amplification and selection method. In addition, we developed a MSX resistance assay as an alternative to utilizing ELISA for evaluating the expression level of stable recombinant CHO cell lines. PMID:25286402
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Shakhbazau, Antos; Shcharbin, Dzmitry; Seviaryn, Ihar; Goncharova, Natalya; Kosmacheva, Svetlana; Potapnev, Mihail; Bryszewska, Maria; Kumar, Ranjan; Biernaskie, Jeffrey; Midha, Rajiv
2012-05-07
This study reports the use of a nonviral expression system based on polyamidoamine dendrimers for time-restricted neurotrophin overproduction in mesenchymal stem cells and skin precursor-derived Schwann cells. The dendrimers were used to deliver plasmids for brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3) expression in both rodent and human stem cells, and the timelines of expression were studied. We have found that, despite the fact that transfection efficiencies and protein expression levels were comparable, dendrimer-driven expression in human mesenchymal stem cells was characterized by a more rapid decline compared to rodent cells. Transient expression systems can be beneficial for some neurotrophins, which were earlier reported to cause unwanted side effects in virus-based long-term expression models. Nonviral neurotrophin expression is a biologically safe and accessible alternative to increase the therapeutic potential of autologous adult stem cells and stem cell-derived functional differentiated cells.
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A new maltose-inducible high-performance heterologous expression system in Bacillus subtilis.
Yue, Jie; Fu, Gang; Zhang, Dawei; Wen, Jianping
2017-08-01
To improve heterologous proteins production, we constructed a maltose-inducible expression system in Bacillus subtilis. An expression system based on the promoter for maltose utilization constructed in B. subtilis. Successively, to improve the performance of the P malA -derived system, mutagenesis was employed by gradually shortening the length of P malA promoter and altering the spacing between the predicted MalR binding site and the -35 region. Furthermore, deletion of the maltose utilization genes (malL and yvdK) improved the P malA promoter activity. Finally, using this efficient maltose-inducible expression system, we enhanced the production of luciferase and D-aminoacylase, compared with the P hpaII system. A maltose-inducible expression system was constructed and evaluated. It could be used for high level expression of heterologous proteins production.
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Angius, Federica; Ilioaia, Oana; Amrani, Amira; Suisse, Annabelle; Rosset, Lindsay; Legrand, Amélie; Abou-Hamdan, Abbas; Uzan, Marc; Zito, Francesca; Miroux, Bruno
2018-06-05
Membrane protein (MP) overproduction is one of the major bottlenecks in structural genomics and biotechnology. Despite the emergence of eukaryotic expression systems, bacteria remain a cost effective and powerful tool for protein production. The T7 RNA polymerase (T7RNAP)-based expression system is a successful and efficient expression system, which achieves high-level production of proteins. However some foreign MPs require a fine-tuning of their expression to minimize the toxicity associated with their production. Here we report a novel regulation mechanism for the T7 expression system. We have isolated two bacterial hosts, namely C44(DE3) and C45(DE3), harboring a stop codon in the T7RNAP gene, whose translation is under the control of the basal nonsense suppressive activity of the BL21(DE3) host. Evaluation of hosts with superfolder green fluorescent protein (sfGFP) revealed an unprecedented tighter control of transgene expression with a marked accumulation of the recombinant protein during stationary phase. Analysis of a collection of twenty MP fused to GFP showed an improved production yield and quality of several bacterial MPs and of one human monotopic MP. These mutant hosts are complementary to the other existing T7 hosts and will increase the versatility of the T7 expression system.
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The LIKE system, a novel protein expression toolbox for Bacillus subtilis based on the liaI promoter
2012-01-01
Background Bacillus subtilis is a very important Gram-positive model organism of high biotechnological relevance, which is widely used as a host for the production of both secreted and cytoplasmic proteins. We developed a novel and efficient expression system, based on the liaI promoter (PliaI) from B. subtilis, which is under control of the LiaRS antibiotic-inducible two-component system. In the absence of a stimulus, this promoter is kept tightly inactive. Upon induction by cell wall antibiotics, it shows an over 100-fold increase in activity within 10 min. Results Based on these traits of PliaI, we developed a novel LiaRS-controlled gene expression system for B. subtilis (the “LIKE" system). Two expression vectors, the integrative pLIKE-int and the replicative pLIKE-rep, were constructed. To enhance the performance of the PliaI-derived system, site-directed mutagenesis was employed to optimize the ribosome binding site and alter its spacing to the initiation codon used for the translational fusion. The impact of these genetic modifications on protein production yield was measured using GFP as a model protein. Moreover, a number of tailored B. subtilis expression strains containing different markerless chromosomal deletions of the liaIH region were constructed to circumvent undesired protein production, enhance the positive autoregulation of the LiaRS system and thereby increase target gene expression strength from the PliaI promoter. Conclusions The LIKE protein expression system is a novel protein expression system, which offers a number of advantages over existing systems. Its major advantages are (i) a tightly switched-off promoter during exponential growth in the absence of a stimulus, (ii) a concentration-dependent activation of PliaI in the presence of suitable inducers, (iii) a very fast but transient response with a very high dynamic range of over 100-fold (up to 1,000-fold) induction, (iv) a choice from a range of well-defined, commercially available, and affordable inducers and (v) the convenient conversion of LIKE-derived inducible expression strains into strong constitutive protein production factories. PMID:23110498
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Ito, Yoichiro; Yamanishi, Mamoru; Ikeuchi, Akinori; Matsuyama, Takashi
2015-01-16
Control of the expression levels of multiple enzymes in transgenic yeasts is essential for the effective production of complex molecules through fermentation. Here, we propose a tunable strategy for the control of expression levels based on the design of terminator regions and other gene-expression control elements in Saccharomyces cerevisiae. Our genome-integrated system, which is capable of producing high expression levels over a wide dynamic range, will broadly enable metabolic engineering and synthetic biology. We demonstrated that the activities of multiple cellulases and the production of ethanol were doubled in a transgenic yeast constructed with our system compared with those achieved with a standard expression system.
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Li, Zuofeng; Wen, Jingran; Zhang, Xiaoyan; Wu, Chunxiao; Li, Zuogao; Liu, Lei
2012-01-01
Aim to ease the secondary use of clinical data in clinical research, we introduce a metadata driven web-based clinical data management system named ClinData Express. ClinData Express is made up of two parts: 1) m-designer, a standalone software for metadata definition; 2) a web based data warehouse system for data management. With ClinData Express, what the researchers need to do is to define the metadata and data model in the m-designer. The web interface for data collection and specific database for data storage will be automatically generated. The standards used in the system and the data export modular make sure of the data reuse. The system has been tested on seven disease-data collection in Chinese and one form from dbGap. The flexibility of system makes its great potential usage in clinical research. The system is available at http://code.google.com/p/clindataexpress. PMID:23304327
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In vitro protein expression: an emerging alternative to cell-based approaches.
He, Mingyue
2011-04-30
Protein expression remains a bottleneck in the production of proteins. Owing to several advantages, cell-free translation is emerging as an alternative to cell-based methods for the generation of proteins. Recent advances have led to many novel applications of cell-free systems in biotechnology, proteomics and fundamental biological research. This special issue of New Biotechnology describes recent advances in cell-free protein expression systems and their applications. Copyright © 2010 Elsevier B.V. All rights reserved.
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Vettath, Sunitha Kodengil; Shivashankar, Gaganashree; Menon, Krishnakumar N; Vijayachandran, Lakshmi S
2018-04-15
Epidermal Growth Factor Receptor variant III (EGFRvIII) is a tumor specific antigen detected in various tumors including gliomas, breast cancer, lung cancer, head and neck squamous cell carcinoma (HNSCC). Screening of EGFRvIII targeting drug molecules can be accelerated by developing drug screening platforms using recombinantly expressed protein. Choice of expression system is one of the major factors deciding the success of recombinant expression of a protein. In our study, we have tried to express and purify the extracellular domain (ECD) of this highly unstable protein using bacterial and baculovirus expression systems to select the expression system suited for our purpose. Even though the protein was successfully expressed in prokaryotic system, purification could be done only under denaturing conditions. But in the baculovirus expression system, the protein was expressed in soluble form and could be purified under native conditions, with single step of purification. Based on our results, we conclude that insect cells are better choice over E. coli cells for expressing EGFRvIII ECD in soluble form. This study provides insights for other researchers involved in expression of similar unstable membrane proteins, on selecting the best expression system and challenges involved. Copyright © 2018 Elsevier B.V. All rights reserved.
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Semantic technologies in a decision support system
NASA Astrophysics Data System (ADS)
Wasielewska, K.; Ganzha, M.; Paprzycki, M.; Bǎdicǎ, C.; Ivanovic, M.; Lirkov, I.
2015-10-01
The aim of our work is to design a decision support system based on ontological representation of domain(s) and semantic technologies. Specifically, we consider the case when Grid / Cloud user describes his/her requirements regarding a "resource" as a class expression from an ontology, while the instances of (the same) ontology represent available resources. The goal is to help the user to find the best option with respect to his/her requirements, while remembering that user's knowledge may be "limited." In this context, we discuss multiple approaches based on semantic data processing, which involve different "forms" of user interaction with the system. Specifically, we consider: (a) ontological matchmaking based on SPARQL queries and class expression, (b) graph-based semantic closeness of instances representing user requirements (constructed from the class expression) and available resources, and (c) multicriterial analysis based on the AHP method, which utilizes expert domain knowledge (also ontologically represented).
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Dickey, Alexia; Wang, Nan; Cooper, Edwin; Tull, Lauren; Breedlove, Drew; Mason, Hugh; Liu, Dehu; Wang, Kevin Yueju
2017-01-01
Lumbrokinases, a group of fibrinolytic enzymes extracted from earthworm, have been widely used to prevent and treat various cardiovascular diseases. They specifically target fibrin to effectively degrade thrombi without major side effects. Plant expression systems are becoming potential alternative expression platforms for producing pharmaceutical proteins. In this work, a lumbrokinase (PI239) was produced from a plant system. Both wild-type (WT) and plant codon-optimized (OP) PI239 gene sequences were synthesized and cloned into a geminivirus-based single-vector DNA replicon system. Both vectors were independently expressed in tobacco (Nicotiana tabacum) leaves transiently by agroinfiltration. Overexpressed PI239 resulted in sudden tissue necrosis 3 days after infiltration. Remaining proteins were purified through His-tag affinity chromatography and analyzed with SDS-PAGE and Western blot methods. Purified PI239 successfully degraded artificial fibrin with relative activity of 13,400 U/mg when compared with commercial lumbrokinase product. In vitro tests demonstrated that plant-derived PI239 dissolved human blood clots and that the plant expression system is capable of producing functional PI239.
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NASA Astrophysics Data System (ADS)
Sakata, Katsumi; Ohyanagi, Hajime; Sato, Shinji; Nobori, Hiroya; Hayashi, Akiko; Ishii, Hideshi; Daub, Carsten O.; Kawai, Jun; Suzuki, Harukazu; Saito, Toshiyuki
2015-02-01
We present a system-wide transcriptional network structure that controls cell types in the context of expression pattern transitions that correspond to cell type transitions. Co-expression based analyses uncovered a system-wide, ladder-like transcription factor cluster structure composed of nearly 1,600 transcription factors in a human transcriptional network. Computer simulations based on a transcriptional regulatory model deduced from the system-wide, ladder-like transcription factor cluster structure reproduced expression pattern transitions when human THP-1 myelomonocytic leukaemia cells cease proliferation and differentiate under phorbol myristate acetate stimulation. The behaviour of MYC, a reprogramming Yamanaka factor that was suggested to be essential for induced pluripotent stem cells during dedifferentiation, could be interpreted based on the transcriptional regulation predicted by the system-wide, ladder-like transcription factor cluster structure. This study introduces a novel system-wide structure to transcriptional networks that provides new insights into network topology.
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Quantitative Magnetic Separation of Particles and Cells using Gradient Magnetic Ratcheting
Murray, Coleman; Pao, Edward; Tseng, Peter; Aftab, Shayan; Kulkarni, Rajan; Rettig, Matthew; Di Carlo, Dino
2016-01-01
Extraction of rare target cells from biosamples is enabling for life science research. Traditional rare cell separation techniques, such as magnetic activated cell sorting (MACS), are robust but perform coarse, qualitative separations based on surface antigen expression. We report a quantitative magnetic separation technology using high-force magnetic ratcheting over arrays of magnetically soft micro-pillars with gradient spacing, and use the system to separate and concentrate magnetic beads based on iron oxide content (IOC) and cells based on surface expression. The system consists of a microchip of permalloy micro-pillar arrays with increasing lateral pitch and a mechatronic device to generate a cycling magnetic-field. Particles with higher IOC separate and equilibrate along the miro-pillar array at larger pitches. We develop a semi-analytical model that predicts behavior for particles and cells. Using the system, LNCaP cells were separated based on the bound quantity of 1μm anti-EpCAM particles as a metric for expression. The ratcheting cytometry system was able to resolve a ±13 bound particle differential, successfully distinguishing LNCaP from PC3 populations based on EpCAM expression, correlating with flow cytometry analysis. As a proof of concept, EpCAM-labeled cells from patient blood were isolated with 74% purity, demonstrating potential towards a quantitative magnetic separation instrument. PMID:26890496
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Cho, InSook; Park, Hyeoun-Ae
2006-01-01
This study evaluated the domain completeness and expressiveness issues of the International Classification for Nursing Practice-based (ICNP) nursing data dictionary (NDD) through its application in an enterprise electronic medical record (EMR) system as a standard vocabulary at a single tertiary hospital in Korea. Data from 2,262 inpatients obtained over a period of 9 weeks (May to July 2003) were extracted from the EMR system for analysis. Among the 530,218 data-input events, 401,190 (75.7%) were entered from the NDD, 20,550 (3.9%) used only free text, and 108,478 (20.4%) used a combination of coded data and free text. A content analysis of the free-text events showed that 80.3% of the expressions could be found in the NDD, whereas 10.9% were context-specific expressions such as direct quotations of patient complaints and responses, and references to the care plan or orders of physicians. A total of 7.8% of the expressions was used for a supplementary purpose such as adding a conjunction or end verb to make an expression appear as natural language. Only 1.0% of the expressions were identified as not being covered by the NDD. This evaluation study demonstrates that the ICNP-based NDD has sufficient power to cover most of the expressions used in a clinical nursing setting. PMID:16622170
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Federal Register 2010, 2011, 2012, 2013, 2014
2013-09-11
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Ruppert, Martin; Woll, Jörn; Giritch, Anatoli; Genady, Ezzat; Ma, Xueyan; Stöckigt, Joachim
2005-11-01
Acetylajmalan esterase (AAE) plays an essential role in the late stage of ajmaline biosynthesis. Based on the partial peptide sequences of AAE isolated and purified from Rauvolfia cell suspensions, a full-length AAE cDNA clone was isolated. The amino acid sequence of AAE has the highest level of identity of 40% to putative lipases known from the Arabidopsis thaliana genome project. Based on the primary structure AAE is a new member of the GDSL lipase superfamily. The expression in Escherichia coli failed although a wide range of conditions were tested. With a novel virus-based plant expression system, it was possible to express AAE functionally in leaves of Nicotiana benthamiana Domin. An extraordinarily high enzyme activity was detected in the Nicotiana tissue, which exceeded that in Rauvolfia serpentina (L.) Benth. ex Kurz cell suspension cultures about 20-fold. This expression allowed molecular analysis of AAE for the first time and increased the number of functionally expressed alkaloid genes from Rauvolfia now to eight, and the number of ajmaline pathway-specific cDNAs to a total of six.
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NASA Astrophysics Data System (ADS)
Qiao, Yaojun; Li, Ming; Yang, Qiuhong; Xu, Yanfei; Ji, Yuefeng
2015-01-01
Closed-form expressions of nonlinear interference of dense wavelength-division-multiplexed (WDM) systems with dispersion managed transmission (DMT) are derived. We carry out a simulative validation by addressing an ample and significant set of the Nyquist-WDM systems based on polarization multiplexed quadrature phase-shift keying (PM-QPSK) subcarriers at a baud rate of 32 Gbaud per channel. Simulation results show the simple closed-form analytical expressions can provide an effective tool for the quick and accurate prediction of system performance in DMT coherent optical systems.
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Rezaie, F; Davami, F; Mansouri, K; Agha Amiri, S; Fazel, R; Mahdian, R; Davoudi, N; Enayati, S; Azizi, M; Khalaj, V
2017-05-08
The Escherichia coli expression system is highly effective in producing recombinant proteins. However, there are some limitations in this system, especially in obtaining correctly folded forms of some complex proteins such as Fab fragments. To improve the solubility and folding quality of Fab fragments, we have examined the effect of simultaneous application of a SUMO fusion tag, EnBase ® cultivation mode and a redox mutant strain in the E. coli expression system. A bicistronic gene construct was designed to express an antivascular endothelial growth factor (VEGF) Fab fragment as a model system. The construct contained a dual SUMO fusion gene fragment to encode SUMO-tagged heavy and light chains. While the expression of the construct in batch cultures of BL21 or SHuffle ® transformants produced insoluble and unfolded products, the induction of the transformants in EnBase ® medium resulted in soluble and correctly folded Fab fragment, reaching as high as 19% of the total protein in shuffle strain. The functional assays indicated that the biological activity of the target Fab is similar to the commercial anti-VEGF, Lucentis ® . This study demonstrated that the combination of SUMO fusion technology, EnBase ® cultivation system and recruiting a redox mutant of E. coli can efficiently enhance the solubility and productivity of recombinant Fab fragments. The presented strategy provides not only a novel method to produce soluble and active form of an anti-VEGF Fab but also may use in the efficient production of other antibody fragments. © 2017 The Society for Applied Microbiology.
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Takala, T M; Saris, P E J; Tynkkynen, S S H
2003-01-01
A new food-grade host/vector system for Lactobacillus casei based on lactose selection was constructed. The wild-type non-starter host Lb. casei strain E utilizes lactose via a plasmid-encoded phosphotransferase system. For food-grade cloning, a stable lactose-deficient mutant was constructed by deleting a 141-bp fragment from the phospho-beta-galactosidase gene lacG via gene replacement. The deletion resulted in an inactive phospho-beta-galactosidase enzyme with an internal in-frame deletion of 47 amino acids. A complementation plasmid was constructed containing a replicon from Lactococcus lactis, the lacG gene from Lb. casei, and the constitutive promoter of pepR for lacG expression from Lb. rhamnosus. The expression of the lacG gene from the resulting food-grade plasmid pLEB600 restored the ability of the lactose-negative mutant strain to grow on lactose to the wild-type level. The vector pLEB600 was used for expression of the proline iminopeptidase gene pepI from Lb. helveticus in Lb. casei. The results show that the food-grade expression system reported in this paper can be used for expression of foreign genes in Lb. casei.
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A simple and robust vector-based shRNA expression system used for RNA interference.
Wang, Xue-jun; Li, Ying; Huang, Hai; Zhang, Xiu-juan; Xie, Pei-wen; Hu, Wei; Li, Dan-dan; Wang, Sheng-qi
2013-01-01
RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful genetic tool for conducting functional studies. Previously, vector-based shRNA-expression strategies capable of inducing RNAi in viable cells have been developed, however, these vector systems have some disadvantages, either because they were error-prone or cost prohibitive. In this report we described the development of a simple, robust shRNA expression system utilizing 1 long oligonucleotide or 2 short oligonucleotides for half the cost of conventional shRNA construction methods and with a >95% cloning success rate. The shRNA loop sequence and stem structure were also compared and carefully selected for better RNAi efficiency. Furthermore, an easier strategy was developed based on isocaudomers which permit rapid combination of the most efficient promoter-shRNA cassettes. Finally, using this method, the conservative target sites for hepatitis B virus (HBV) knockdown were systemically screened and HBV antigen expression shown to be successfully suppressed in the presence of connected multiple shRNAs both in vitro and in vivo. This novel design describes an inexpensive and effective way to clone and express single or multiple shRNAs from the same vector with the capacity for potent and effective silencing of target genes.
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Chatelle, Claire; Ochoa-Fernandez, Rocio; Engesser, Raphael; Schneider, Nils; Beyer, Hannes M; Jones, Alex R; Timmer, Jens; Zurbriggen, Matias D; Weber, Wilfried
2018-05-18
The ever-increasing complexity of synthetic gene networks and applications of synthetic biology requires precise and orthogonal gene expression systems. Of particular interest are systems responsive to light as they enable the control of gene expression dynamics with unprecedented resolution in space and time. While broadly used in mammalian backgrounds, however, optogenetic approaches in plant cells are still limited due to interference of the activating light with endogenous photoreceptors. Here, we describe the development of the first synthetic light-responsive system for the targeted control of gene expression in mammalian and plant cells that responds to the green range of the light spectrum in which plant photoreceptors have minimal activity. We first engineered a system based on the light-sensitive bacterial transcription factor CarH and its cognate DNA operator sequence CarO from Thermus thermophilus to control gene expression in mammalian cells. The system was functional in various mammalian cell lines, showing high induction (up to 350-fold) along with low leakiness, as well as high reversibility. We quantitatively described the systems characteristics by the development and experimental validation of a mathematical model. Finally, we transferred the system into A. thaliana protoplasts and demonstrated gene repression in response to green light. We expect that this system will provide new opportunities in applications based on synthetic gene networks and will open up perspectives for optogenetic studies in mammalian and plant cells.
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Wang, Jian; Liu, Yuan; Zhang, Junhua; Han, Zhengzheng; Wang, Wei; Liu, Yang; Wei, Dong; Huang, Wei
2017-11-01
Large-scale expression of β 2 -adrenergic receptor (β 2 -AR) in functional form is necessary for establishment of receptor assays for detecting illegally abused β-adrenergic agonists (β-agonists). Cell-based heterologous expression systems have manycritical difficulties in synthesizing this membrane protein, such as low protein yields and aberrant folding. To overcome these challenges, the main objective of the present work was to synthesize large amounts of functional β 2 -AR in a cell-free system based on Escherichia coli extracts. A codon-optimized porcine β 2 -AR gene (codon adaptation index: 0.96) suitable for high expression in E. coli was synthesized and transcribed to the cell-free system, which contributed to increase the expression up to 1.1 mg/ml. After purification using Ni-affinity chromatography, the bioactivity of the purified receptor was measured by novel enzyme-linked receptor assays. It was determined that the relative affinities of the purified β 2 -AR for β-agonists in descending order were as follows: clenbuterol > salbutamol > ractopamine. Moreover, their IC 50 values were 45.99, 60.38, and 78.02 µg/liter, respectively. Although activity of the cell-free system was slightly lower than activity of systems based on insect and mammalian cells, this system should allow production of β 2 -AR in bulk amounts sufficient for the development of multianalyte screening methods for detecting β-agonist residues.
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NASA Astrophysics Data System (ADS)
Vallino, J. J.; Huber, J. A.
2016-02-01
Marine biogeochemistry is orchestrated by a complex and dynamic community of microorganisms that attempt to maximize their own fecundity through a combination of competition and cooperation. At a systems level, the community can be described as a distributed metabolic network, where different species contribute their own unique set of metabolic capabilities. Our current project attempts to understand the governing principles that describe amplification or attenuation of metabolic pathways within the network through a combination of modeling and metagenomic, metatranscriptomic and biogeochemical observations. We will describe and present results from our thermodynamic-based model that determines optimal pathway expression from available resources based on the principle of maximum entropy production (MEP); that is, based on the hypothesis that non-equilibrium systems organize to maximize energy dissipation. The MEP model currently predicts metabolic pathway expression over time, and one spatial dimension. Model predictions will be compared to biogeochemical observations and gene presence and expression from samples collected over time and space from a costal meromictic basin (Siders Pond) located in Falmouth MA, US. Siders Pond permanent stratification, caused by occasional seawater intrusion, results in steep chemoclines and redox gradients, which supports both aerobic and anaerobic phototrophs as well as sulfur, Fe and Mn redox cycles. The diversity of metabolic capability and expression we have observed over depth makes it an ideal system to test our thermodynamic-based model.
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Singhal, Dinesh K; Singhal, Raxita; Malik, Hruda N; Kumar, Surender; Kumar, Sudarshan; Mohanty, Ashok K; Kaushik, Jai K; Malakar, Dhruba
2014-01-01
Nanog is a homeodomain containing protein which plays important roles in regulation of signaling pathways for maintenance and induction of pluripotency in stem cells. Because of its unique expression in stem cells it is also regarded as pluripotency marker. In this study goat Nanog (gNanog) gene has been amplified, cloned and characterized at sequence level with successful over-expression in CHO-K1 cell line using a lentiviral based system. gNanog ORF is 903 bp long which codes for Nanog protein of size 300 amino acids (aas). Complete nucleotide sequence shows some evolutionary mutation in goat in comparision to other species. Protein sequence of goat is highly similar to other species. Overall, gNanog nucleotide sequence and predicted protein sequence showed high similarity and minimum divergence with cattle (96 % identity/4 % divergence) and buffalo (94/5 %) while low similarity and high divergence with pig (84/15 %), human (81/23 %) and mouse (69/40 %) indicating evolutionary closeness of gNanog to cattle and buffalo. gNanog lentiviral expression construct was prepared for over-expression of Nanog gene in adult goat fibroblast cells. Lentiviral expression construct of Nanog enabled continuous protein expression for induction and maintenance of pluripotency. Western blotting revealed the expression of Nanog gene at protein level which supported that the lentiviral expression system is highly promising for Nanog protein expression in differentiated goat cell.
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Comparisons of Robustness and Sensitivity between Cancer and Normal Cells by Microarray Data
Chu, Liang-Hui; Chen, Bor-Sen
2008-01-01
Robustness is defined as the ability to uphold performance in face of perturbations and uncertainties, and sensitivity is a measure of the system deviations generated by perturbations to the system. While cancer appears as a robust but fragile system, few computational and quantitative evidences demonstrate robustness tradeoffs in cancer. Microarrays have been widely applied to decipher gene expression signatures in human cancer research, and quantification of global gene expression profiles facilitates precise prediction and modeling of cancer in systems biology. We provide several efficient computational methods based on system and control theory to compare robustness and sensitivity between cancer and normal cells by microarray data. Measurement of robustness and sensitivity by linear stochastic model is introduced in this study, which shows oscillations in feedback loops of p53 and demonstrates robustness tradeoffs that cancer is a robust system with some extreme fragilities. In addition, we measure sensitivity of gene expression to perturbations in other gene expression and kinetic parameters, discuss nonlinear effects in feedback loops of p53 and extend our method to robustness-based cancer drug design. PMID:19259409
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Schwessinger, Benjamin; Li, Xiang; Ellinghaus, Thomas L; Chan, Leanne Jade G; Wei, Tong; Joe, Anna; Thomas, Nicholas; Pruitt, Rory; Adams, Paul D; Chern, Maw Sheng; Petzold, Christopher J; Liu, Chang C; Ronald, Pamela C
2016-04-18
Posttranslational modification (PTM) of proteins and peptides is important for diverse biological processes in plants and animals. The paucity of heterologous expression systems for PTMs and the technical challenges associated with chemical synthesis of these modified proteins has limited detailed molecular characterization and therapeutic applications. Here we describe an optimized system for expression of tyrosine-sulfated proteins in Escherichia coli and its application in a bio-based crop protection strategy in rice.
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Schwessinger, Benjamin; Li, Xiang; Ellinghaus, Thomas L.; ...
2015-11-27
Posttranslational modification (PTM) of proteins and peptides is important for diverse biological processes in plants and animals. The paucity of heterologous expression systems for PTMs and the technical challenges associated with chemical synthesis of these modified proteins has limited detailed molecular characterization and therapeutic applications. Here we describe an optimized system for expression of tyrosine-sulfated proteins in Escherichia coli and its application in a bio-based crop protection strategy in rice.
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Transient, Inducible, Placenta-Specific Gene Expression in Mice
Fan, Xiujun; Petitt, Matthew; Gamboa, Matthew; Huang, Mei; Dhal, Sabita; Druzin, Maurice L.; Wu, Joseph C.
2012-01-01
Molecular understanding of placental functions and pregnancy disorders is limited by the absence of methods for placenta-specific gene manipulation. Although persistent placenta-specific gene expression has been achieved by lentivirus-based gene delivery methods, developmentally and physiologically important placental genes have highly stage-specific functions, requiring controllable, transient expression systems for functional analysis. Here, we describe an inducible, placenta-specific gene expression system that enables high-level, transient transgene expression and monitoring of gene expression by live bioluminescence imaging in mouse placenta at different stages of pregnancy. We used the third generation tetracycline-responsive tranactivator protein Tet-On 3G, with 10- to 100-fold increased sensitivity to doxycycline (Dox) compared with previous versions, enabling unusually sensitive on-off control of gene expression in vivo. Transgenic mice expressing Tet-On 3G were created using a new integrase-based, site-specific approach, yielding high-level transgene expression driven by a ubiquitous promoter. Blastocysts from these mice were transduced with the Tet-On 3G-response element promoter-driving firefly luciferase using lentivirus-mediated placenta-specific gene delivery and transferred into wild-type pseudopregnant recipients for placenta-specific, Dox-inducible gene expression. Systemic Dox administration at various time points during pregnancy led to transient, placenta-specific firefly luciferase expression as early as d 5 of pregnancy in a Dox dose-dependent manner. This system enables, for the first time, reliable pregnancy stage-specific induction of gene expression in the placenta and live monitoring of gene expression during pregnancy. It will be widely applicable to studies of both placental development and pregnancy, and the site-specific Tet-On G3 mouse will be valuable for studies in a broad range of tissues. PMID:23011919
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An all-in-one, Tet-On 3G inducible PiggyBac system for human pluripotent stem cells and derivatives.
Randolph, Lauren N; Bao, Xiaoping; Zhou, Chikai; Lian, Xiaojun
2017-05-08
Human pluripotent stem cells (hPSCs) offer tremendous promise in tissue engineering and cell-based therapies due to their unique combination of two properties: pluripotency and unlimited proliferative capacity. However, directed differentiation of hPSCs to clinically relevant cell lineages is needed to achieve the goal of hPSC-based therapies. This requires a deep understanding of how cell signaling pathways converge on the nucleus to control differentiation and the ability to dissect gene function in a temporal manner. Here, we report the use of the PiggyBac transposon and a Tet-On 3G drug-inducible gene expression system to achieve versatile inducible gene expression in hPSC lines. Our new system, XLone, offers improvement over previous Tet-On systems with significantly reduced background expression and increased sensitivity to doxycycline. Transgene expression in hPSCs is tightly regulated in response to doxycycline treatment. In addition, the PiggyBac elements in our XLone construct provide a rapid and efficient strategy for generating stable transgenic hPSCs. Our inducible gene expression PiggyBac transposon system should facilitate the study of gene function and directed differentiation in human stem cells.
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Dual transcriptional-translational cascade permits cellular level tuneable expression control
Morra, Rosa; Shankar, Jayendra; Robinson, Christopher J.; Halliwell, Samantha; Butler, Lisa; Upton, Mathew; Hay, Sam; Micklefield, Jason; Dixon, Neil
2016-01-01
The ability to induce gene expression in a small molecule dependent manner has led to many applications in target discovery, functional elucidation and bio-production. To date these applications have relied on a limited set of protein-based control mechanisms operating at the level of transcription initiation. The discovery, design and reengineering of riboswitches offer an alternative means by which to control gene expression. Here we report the development and characterization of a novel tunable recombinant expression system, termed RiboTite, which operates at both the transcriptional and translational level. Using standard inducible promoters and orthogonal riboswitches, a multi-layered modular genetic control circuit was developed to control the expression of both bacteriophage T7 RNA polymerase and recombinant gene(s) of interest. The system was benchmarked against a number of commonly used E. coli expression systems, and shows tight basal control, precise analogue tunability of gene expression at the cellular level, dose-dependent regulation of protein production rates over extended growth periods and enhanced cell viability. This novel system expands the number of E. coli expression systems for use in recombinant protein production and represents a major performance enhancement over and above the most widely used expression systems. PMID:26405200
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Shirahata, Mitsuaki; Iwao-Koizumi, Kyoko; Saito, Sakae; Ueno, Noriko; Oda, Masashi; Hashimoto, Nobuo; Takahashi, Jun A; Kato, Kikuya
2007-12-15
Current morphology-based glioma classification methods do not adequately reflect the complex biology of gliomas, thus limiting their prognostic ability. In this study, we focused on anaplastic oligodendroglioma and glioblastoma, which typically follow distinct clinical courses. Our goal was to construct a clinically useful molecular diagnostic system based on gene expression profiling. The expression of 3,456 genes in 32 patients, 12 and 20 of whom had prognostically distinct anaplastic oligodendroglioma and glioblastoma, respectively, was measured by PCR array. Next to unsupervised methods, we did supervised analysis using a weighted voting algorithm to construct a diagnostic system discriminating anaplastic oligodendroglioma from glioblastoma. The diagnostic accuracy of this system was evaluated by leave-one-out cross-validation. The clinical utility was tested on a microarray-based data set of 50 malignant gliomas from a previous study. Unsupervised analysis showed divergent global gene expression patterns between the two tumor classes. A supervised binary classification model showed 100% (95% confidence interval, 89.4-100%) diagnostic accuracy by leave-one-out cross-validation using 168 diagnostic genes. Applied to a gene expression data set from a previous study, our model correlated better with outcome than histologic diagnosis, and also displayed 96.6% (28 of 29) consistency with the molecular classification scheme used for these histologically controversial gliomas in the original article. Furthermore, we observed that histologically diagnosed glioblastoma samples that shared anaplastic oligodendroglioma molecular characteristics tended to be associated with longer survival. Our molecular diagnostic system showed reproducible clinical utility and prognostic ability superior to traditional histopathologic diagnosis for malignant glioma.
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Performance benchmarking of four cell-free protein expression systems.
Gagoski, Dejan; Polinkovsky, Mark E; Mureev, Sergey; Kunert, Anne; Johnston, Wayne; Gambin, Yann; Alexandrov, Kirill
2016-02-01
Over the last half century, a range of cell-free protein expression systems based on pro- and eukaryotic organisms have been developed and have found a range of applications, from structural biology to directed protein evolution. While it is generally accepted that significant differences in performance among systems exist, there is a paucity of systematic experimental studies supporting this notion. Here, we took advantage of the species-independent translation initiation sequence to express and characterize 87 N-terminally GFP-tagged human cytosolic proteins of different sizes in E. coli, wheat germ (WGE), HeLa, and Leishmania-based (LTE) cell-free systems. Using a combination of single-molecule fluorescence spectroscopy, SDS-PAGE, and Western blot analysis, we assessed the expression yields, the fraction of full-length translation product, and aggregation propensity for each of these systems. Our results demonstrate that the E. coli system has the highest expression yields. However, we observe that high expression levels are accompanied by production of truncated species-particularly pronounced in the case of proteins larger than 70 kDa. Furthermore, proteins produced in the E. coli system display high aggregation propensity, with only 10% of tested proteins being produced in predominantly monodispersed form. The WGE system was the most productive among eukaryotic systems tested. Finally, HeLa and LTE show comparable protein yields that are considerably lower than the ones achieved in the E. coli and WGE systems. The protein products produced in the HeLa system display slightly higher integrity, whereas the LTE-produced proteins have the lowest aggregation propensity among the systems analyzed. The high quality of HeLa- and LTE-produced proteins enable their analysis without purification and make them suitable for analysis of multi-domain eukaryotic proteins. © 2015 Wiley Periodicals, Inc.
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Expressive facial animation synthesis by learning speech coarticulation and expression spaces.
Deng, Zhigang; Neumann, Ulrich; Lewis, J P; Kim, Tae-Yong; Bulut, Murtaza; Narayanan, Shrikanth
2006-01-01
Synthesizing expressive facial animation is a very challenging topic within the graphics community. In this paper, we present an expressive facial animation synthesis system enabled by automated learning from facial motion capture data. Accurate 3D motions of the markers on the face of a human subject are captured while he/she recites a predesigned corpus, with specific spoken and visual expressions. We present a novel motion capture mining technique that "learns" speech coarticulation models for diphones and triphones from the recorded data. A Phoneme-Independent Expression Eigenspace (PIEES) that encloses the dynamic expression signals is constructed by motion signal processing (phoneme-based time-warping and subtraction) and Principal Component Analysis (PCA) reduction. New expressive facial animations are synthesized as follows: First, the learned coarticulation models are concatenated to synthesize neutral visual speech according to novel speech input, then a texture-synthesis-based approach is used to generate a novel dynamic expression signal from the PIEES model, and finally the synthesized expression signal is blended with the synthesized neutral visual speech to create the final expressive facial animation. Our experiments demonstrate that the system can effectively synthesize realistic expressive facial animation.
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Quantitative Magnetic Separation of Particles and Cells Using Gradient Magnetic Ratcheting.
Murray, Coleman; Pao, Edward; Tseng, Peter; Aftab, Shayan; Kulkarni, Rajan; Rettig, Matthew; Di Carlo, Dino
2016-04-13
Extraction of rare target cells from biosamples is enabling for life science research. Traditional rare cell separation techniques, such as magnetic activated cell sorting, are robust but perform coarse, qualitative separations based on surface antigen expression. A quantitative magnetic separation technology is reported using high-force magnetic ratcheting over arrays of magnetically soft micropillars with gradient spacing, and the system is used to separate and concentrate magnetic beads based on iron oxide content (IOC) and cells based on surface expression. The system consists of a microchip of permalloy micropillar arrays with increasing lateral pitch and a mechatronic device to generate a cycling magnetic field. Particles with higher IOC separate and equilibrate along the miropillar array at larger pitches. A semi-analytical model is developed that predicts behavior for particles and cells. Using the system, LNCaP cells are separated based on the bound quantity of 1 μm anti-epithelial cell adhesion molecule (EpCAM) particles as a metric for expression. The ratcheting cytometry system is able to resolve a ±13 bound particle differential, successfully distinguishing LNCaP from PC3 populations based on EpCAM expression, correlating with flow cytometry analysis. As a proof-of-concept, EpCAM-labeled cells from patient blood are isolated with 74% purity, demonstrating potential toward a quantitative magnetic separation instrument. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Gottschamel, Johanna; Lössl, Andreas; Ruf, Stephanie; Wang, Yanliang; Skaugen, Morten; Bock, Ralph; Clarke, Jihong Liu
2016-07-01
Dengue fever is a disease in many parts of the tropics and subtropics and about half the world's population is at risk of infection according to the World Health Organization. Dengue is caused by any of the four related dengue virus serotypes DEN-1, -2, -3 and -4, which are transmitted to people by Aedes aegypti mosquitoes. Currently there is only one vaccine (Dengvaxia(®)) available (limited to a few countries) on the market since 2015 after half a century's intensive efforts. Affordable and accessible vaccines against dengue are hence still urgently needed. The dengue envelop protein domain III (EDIII), which is capable of eliciting serotype-specific neutralizing antibodies, has become the focus for subunit vaccine development. To contribute to the development of an accessible and affordable dengue vaccine, in the current study we have used plant-based vaccine production systems to generate a dengue subunit vaccine candidate in tobacco. Chloroplast genome engineering was applied to express serotype-specific recombinant EDIII proteins in tobacco chloroplasts using both constitutive and ethanol-inducible expression systems. Expression of a tetravalent antigen fusion construct combining EDIII polypeptides from all four serotypes was also attempted. Transplastomic EDIII-expressing tobacco lines were obtained and homoplasmy was verified by Southern blot analysis. Northern blot analyses showed expression of EDIII antigen-encoding genes. EDIII protein accumulation levels varied for the different recombinant EDIII proteins and the different expression systems, and reached between 0.8 and 1.6 % of total cellular protein. Our study demonstrates the suitability of the chloroplast compartment as a production site for an EDIII-based vaccine candidate against dengue fever and presents a Gateway(®) plastid transformation vector for inducible transgene expression.
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Zess, Erin K; Begemann, Matthew B; Pfleger, Brian F
2016-02-01
Predictive control of gene expression is an essential tool for developing synthetic biological systems. The current toolbox for controlling gene expression in cyanobacteria is a barrier to more in-depth genetic analysis and manipulation. Towards relieving this bottleneck, this work describes the use of synthetic biology to construct an anhydrotetracycline-based induction system and adapt a trans-acting small RNA (sRNA) system for use in the cyanobacterium Synechococcus sp. strain PCC 7002. An anhydrotetracycline-inducible promoter was developed to maximize intrinsic strength and dynamic range. The resulting construct, PEZtet , exhibited tight repression and a maximum 32-fold induction upon addition of anhydrotetracycline. Additionally, a sRNA system based on the Escherichia coli IS10 RNA-IN/OUT regulator was adapted for use in Synechococcus sp. strain PCC 7002. This system exhibited 70% attenuation of target gene expression, providing a demonstration of the use of sRNAs for differential gene expression in cyanobacteria. These systems were combined to produce an inducible sRNA system, which demonstrated 59% attenuation of target gene expression. Lastly, the role of Hfq, a critical component of sRNA systems in E. coli, was investigated. Genetic studies showed that the Hfq homolog in Synechococcus sp. strain PCC 7002 did not impact repression by the engineered sRNA system. In summary, this work describes new synthetic biology tools that can be applied to physiological studies, metabolic engineering, or sRNA platforms in Synechococcus sp. strain PCC 7002. © 2015 Wiley Periodicals, Inc.
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Math expression retrieval using an inverted index over symbol pairs
NASA Astrophysics Data System (ADS)
Stalnaker, David; Zanibbi, Richard
2015-01-01
We introduce a new method for indexing and retrieving mathematical expressions, and a new protocol for evaluating math formula retrieval systems. The Tangent search engine uses an inverted index over pairs of symbols in math expressions. Each key in the index is a pair of symbols along with their relative distance and vertical displacement within an expression. Matched expressions are ranked by the harmonic mean of the percentage of symbol pairs matched in the query, and the percentage of symbol pairs matched in the candidate expression. We have found that our method is fast enough for use in real time and finds partial matches well, such as when subexpressions are re-arranged (e.g. expressions moved from the left to the right of an equals sign) or when individual symbols (e.g. variables) differ from a query expression. In an experiment using expressions from English Wikipedia, student and faculty participants (N=20) found expressions returned by Tangent significantly more similar than those from a text-based retrieval system (Lucene) adapted for mathematical expressions. Participants provided similarity ratings using a 5-point Likert scale, evaluating expressions from both algorithms one-at-a-time in a randomized order to avoid bias from the position of hits in search result lists. For the Lucene-based system, precision for the top 1 and 10 hits averaged 60% and 39% across queries respectively, while for Tangent mean precision at 1 and 10 were 99% and 60%. A demonstration and source code are publicly available.
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[Prokaryotic expression systems].
Porowińska, Dorota; Wujak, Magdalena; Roszek, Katarzyna; Komoszyński, Michał
2013-03-01
For overproduction of recombinant proteins both eukaryotic and prokaryotic expression systems are used. Choosing the right system depends, among other things, on the growth rate and culture of host cells, level of the target gene expression and posttranslational processing of the synthesized protein. Regardless of the type of expression system, its basic elements are the vector and the expression host. The most widely used system for protein overproduction, both on a laboratory and industrial scale, is the prokaryotic system. This system is based primarily on the bacteria E. coli, although increasingly often Bacillus species are used. The prokaryotic system allows one to obtain large quantities of recombinant proteins in a short time. A simple and inexpensive bacterial cell culture and well-known mechanisms of transcription and translation facilitate the use of these microorganisms. The simplicity of genetic modifications and the availability of many bacterial mutants are additional advantages of the prokaryotic system. In this article we characterize the structural elements of prokaryotic expression vectors. Also strategies for preparation of the target protein gene that increase productivity, facilitate detection and purification of recombinant protein and provide its activity are discussed. Bacterial strains often used as host cells in expression systems as well as the potential location of heterologous proteins are characterized. Knowledge of the basic elements of the prokaryotic expression system allows for production of biologically active proteins in a short time and in satisfactory quantities.
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A triplex ribozyme expression system based on a single hairpin ribozyme.
Aquino-Jarquin, Guillermo; Benítez-Hess, María Luisa; DiPaolo, Joseph A; Alvarez-Salas, Luis M
2008-09-01
Triplex ribozyme (RZ) configurations allow for the individual activity of trans-acting RZs in multiple expression cassettes (multiplex), thereby increasing target cleavage relative to conventionally expressed RZs. Although hairpin RZs have been advantageously compared to hammerhead RZs, their longer size and structural features complicated triplex design. We present a triplex expression system based on a single hairpin RZ with transcleavage capability and simple engineering. The system was tested in vitro using cis- and trans-cleavage kinetic assays against a known target RNA from HPV-16 E6/E7 mRNA. Single and multiplex triplex RZ constructs were more efficient in cleaving the target than tandem-cloned hairpin RZs, suggesting that the release of individual RZs enhanced trans-cleavage kinetics. Multiplex systems constructed with two different hairpin RZs resulted in better trans-cleavage compared to standard double-RZ constructs. In addition, the triplex RZ performed cis- and trans-cleavage in cervical cancer cells. The use of triplex configurations with multiplex RZs permit differential targeting of the same or different RNA, thus improving potential use against unstable targets. This prototype will provide the basis for the development of future RZ-based therapies and technologies.
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NASA Technical Reports Server (NTRS)
Yam, Y.; Lang, J. H.; Johnson, T. L.; Shih, S.; Staelin, D. H.
1983-01-01
A model reduction procedure based on aggregation with respect to sensor and actuator influences rather than modes is presented for large systems of coupled second-order differential equations. Perturbation expressions which can predict the effects of spillover on both the aggregated and residual states are derived. These expressions lead to the development of control system design constraints which are sufficient to guarantee, to within the validity of the perturbations, that the residual states are not destabilized by control systems designed from the reduced model. A numerical example is provided to illustrate the application of the aggregation and control system design method.
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Adult mouse brain gene expression patterns bear an embryologic imprint
Zapala, Matthew A.; Hovatta, Iiris; Ellison, Julie A.; Wodicka, Lisa; Del Rio, Jo A.; Tennant, Richard; Tynan, Wendy; Broide, Ron S.; Helton, Rob; Stoveken, Barbara S.; Winrow, Christopher; Lockhart, Daniel J.; Reilly, John F.; Young, Warren G.; Bloom, Floyd E.; Lockhart, David J.; Barlow, Carrolee
2005-01-01
The current model to explain the organization of the mammalian nervous system is based on studies of anatomy, embryology, and evolution. To further investigate the molecular organization of the adult mammalian brain, we have built a gene expression-based brain map. We measured gene expression patterns for 24 neural tissues covering the mouse central nervous system and found, surprisingly, that the adult brain bears a transcriptional “imprint” consistent with both embryological origins and classic evolutionary relationships. Embryonic cellular position along the anterior–posterior axis of the neural tube was shown to be closely associated with, and possibly a determinant of, the gene expression patterns in adult structures. We also observed a significant number of embryonic patterning and homeobox genes with region-specific expression in the adult nervous system. The relationships between global expression patterns for different anatomical regions and the nature of the observed region-specific genes suggest that the adult brain retains a degree of overall gene expression established during embryogenesis that is important for regional specificity and the functional relationships between regions in the adult. The complete collection of extensively annotated gene expression data along with data mining and visualization tools have been made available on a publicly accessible web site (www.barlow-lockhart-brainmapnimhgrant.org). PMID:16002470
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Liang, Xun; Sun, Leqiang; Yu, Teng; Pan, Yongfei; Wang, Dongdong; Hu, Xueying; Fu, Zhenfang; He, Qigai; Cao, Gang
2016-01-18
Virus evolves rapidly to escape vaccine-induced immunity, posing a desperate demand for efficient vaccine development biotechnologies. Here we present an express vaccine development strategy based on CRISPR/Cas9 and Cre/Lox system against re-emerging Pseudorabies virus, which caused the recent devastating swine pseudorabies outbreak in China. By CRISPR/Cas9 system, the virulent genes of the newly isolated strain were simultaneously substituted by marker genes, which were subsequently excised using Cre/Lox system for vaccine safety concern. Notably, single cell FACS technology was applied to further promote virus purification efficiency. The combination of these state-of-art technologies greatly accelerated vaccine development. Finally, vaccination and challenge experiments proved this vaccine candidate's protective efficacy in pigs and the promise to control current pseudorabies outbreak. This is, to our knowledge, the first successful vaccine development based on gene edit technologies, demonstrating these technologies leap from laboratory to industry. It may pave the way for future express antiviral vaccine development.
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Liang, Xun; Sun, Leqiang; Yu, Teng; Pan, Yongfei; Wang, Dongdong; Hu, Xueying; Fu, Zhenfang; He, Qigai; Cao, Gang
2016-01-01
Virus evolves rapidly to escape vaccine-induced immunity, posing a desperate demand for efficient vaccine development biotechnologies. Here we present an express vaccine development strategy based on CRISPR/Cas9 and Cre/Lox system against re-emerging Pseudorabies virus, which caused the recent devastating swine pseudorabies outbreak in China. By CRISPR/Cas9 system, the virulent genes of the newly isolated strain were simultaneously substituted by marker genes, which were subsequently excised using Cre/Lox system for vaccine safety concern. Notably, single cell FACS technology was applied to further promote virus purification efficiency. The combination of these state-of-art technologies greatly accelerated vaccine development. Finally, vaccination and challenge experiments proved this vaccine candidate’s protective efficacy in pigs and the promise to control current pseudorabies outbreak. This is, to our knowledge, the first successful vaccine development based on gene edit technologies, demonstrating these technologies leap from laboratory to industry. It may pave the way for future express antiviral vaccine development. PMID:26777545
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Wang, Xia; Yang, Jian-Guo; Chen, Li; Wang, Ji-Long; Cheng, Qi; Dixon, Ray; Wang, Yi-Ping
2013-01-01
Biological nitrogen fixation is a complex process requiring multiple genes working in concert. To date, the Klebsiella pneumoniae nif gene cluster, divided into seven operons, is one of the most studied systems. Its nitrogen fixation capacity is subject to complex cascade regulation and physiological limitations. In this report, the entire K. pneumoniae nif gene cluster was reassembled as operon-based BioBrick parts in Escherichia coli. It provided ∼100% activity of native K. pneumoniae system. Based on the expression levels of these BioBrick parts, a T7 RNA polymerase–LacI expression system was used to replace the σ54-dependent promoters located upstream of nif operons. Expression patterns of nif operons were critical for the maximum activity of the recombinant system. By mimicking these expression levels with variable-strength T7-dependent promoters, ∼42% of the nitrogenase activity of the σ54-dependent nif system was achieved in E. coli. When the newly constructed T7-dependent nif system was challenged with different genetic and physiological conditions, it bypassed the original complex regulatory circuits, with minor physiological limitations. Therefore, we have successfully replaced the nif regulatory elements with a simple expression system that may provide the first step for further research of introducing nif genes into eukaryotic organelles, which has considerable potentials in agro-biotechnology. PMID:23935879
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Fox, Christopher J; Barton, Jason J S
2007-01-05
The neural representation of facial expression within the human visual system is not well defined. Using an adaptation paradigm, we examined aftereffects on expression perception produced by various stimuli. Adapting to a face, which was used to create morphs between two expressions, substantially biased expression perception within the morphed faces away from the adapting expression. This adaptation was not based on low-level image properties, as a different image of the same person displaying that expression produced equally robust aftereffects. Smaller but significant aftereffects were generated by images of different individuals, irrespective of gender. Non-face visual, auditory, or verbal representations of emotion did not generate significant aftereffects. These results suggest that adaptation affects at least two neural representations of expression: one specific to the individual (not the image), and one that represents expression across different facial identities. The identity-independent aftereffect suggests the existence of a 'visual semantic' for facial expression in the human visual system.
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Geib, Elena; Brock, Matthias
2017-01-01
Fungi are treasure chests for yet unexplored natural products. However, exploitation of their real potential remains difficult as a significant proportion of biosynthetic gene clusters appears silent under standard laboratory conditions. Therefore, elucidation of novel products requires gene activation or heterologous expression. For heterologous gene expression, we previously developed an expression platform in Aspergillus niger that is based on the transcriptional regulator TerR and its target promoter P terA . In this study, we extended this system by regulating expression of terR by the doxycycline inducible Tet-on system. Reporter genes cloned under the control of the target promoter P terA remained silent in the absence of doxycycline, but were strongly expressed when doxycycline was added. Reporter quantification revealed that the coupled system results in about five times higher expression rates compared to gene expression under direct control of the Tet-on system. As production of secondary metabolites generally requires the expression of several biosynthetic genes, the suitability of the self-cleaving viral peptide sequence P2A was tested in this optimised expression system. P2A allowed polycistronic expression of genes required for Asp-melanin formation in combination with the gene coding for the red fluorescent protein tdTomato. Gene expression and Asp-melanin formation was prevented in the absence of doxycycline and strongly induced by addition of doxycycline. Fluorescence studies confirmed the correct subcellular localisation of the respective enzymes. This tightly regulated but strongly inducible expression system enables high level production of secondary metabolites most likely even those with toxic potential. Furthermore, this system is compatible with polycistronic gene expression and, thus, suitable for the discovery of novel natural products.
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Saijo, Takanori; Nagasawa, Akitsu
2014-01-01
A newly developed copper-inducible gene expression system overcame the mixed results reported earlier, worked well both in cultured cells and a whole plant, and enabled to control flowering timing. Copper is one of the essential microelements and is readily taken up by plants. However, to date, it has rarely been used to control the expression of genes of interest, probably due to the inefficiency of the gene expression systems. In this study, we successfully developed a copper-inducible gene expression system that is based on the regulation of the yeast metallothionein gene. This system can be applied in the field and regulated at approximately one-hundredth of the rate used for registered copper-based fungicides. In the presence of copper, a translational fusion of the ACE1 transcription factor with the VP16 activation domain (VP16AD) of herpes simplex virus strongly activated transcription of the GFP gene in transgenic Arabidopsis. Interestingly, insertion of the To71 sequence, a 5'-untranslated region of the 130k/180k gene of tomato mosaic virus, upstream of the GFP gene reduced the basal expression of GFP in the absence of copper to almost negligible levels, even in soil-grown plants that were supplemented with ordinary liquid nutrients. Exposure of plants to 100 μM copper resulted in an over 1,000-fold induction ratio at the transcriptional level of GFP. This induction was copper-specific and dose-dependent with rapid and reversible responses. Using this expression system, we also succeeded in regulating floral transition by copper treatment. These results indicate that our newly developed copper-inducible system can accelerate gene functional analysis in model plants and can be used to generate novel agronomic traits in crop species.
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2014-01-01
Background The Sterile Insect Technique (SIT) is an accepted species-specific genetic control approach that acts as an insect birth control measure, which can be improved by biotechnological engineering to facilitate its use and widen its applicability. First transgenic insects carrying a single killing system have already been released in small scale trials. However, to evade resistance development to such transgenic approaches, completely independent ways of transgenic killing should be established and combined. Perspective Most established transgenic sexing and reproductive sterility systems are based on the binary tTA expression system that can be suppressed by adding tetracycline to the food. However, to create 'redundant killing' an additional independent conditional expression system is required. Here we present a perspective on the use of a second food-controllable binary expression system - the inducible Q system - that could be used in combination with site-specific recombinases to generate independent transgenic killing systems. We propose the combination of an already established transgenic embryonic sexing system to meet the SIT requirement of male-only releases based on the repressible tTA system together with a redundant male-specific reproductive sterility system, which is activated by Q-system controlled site-specific recombination and is based on a spermatogenesis-specifically expressed endonuclease acting on several species-specific target sites leading to chromosome shredding. Conclusion A combination of a completely independent transgenic sexing and a redundant reproductive male sterility system, which do not share any active components and mediate the induced lethality by completely independent processes, would meet the 'redundant killing' criteria for suppression of resistance development and could therefore be employed in large scale long-term suppression programs using biotechnologically enhanced SIT. PMID:25471733
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RGSS-ID: an approach to new radiologic reporting system.
Ikeda, M; Sakuma, S; Maruyama, K
1990-01-01
RGSS-ID is a developmental computer system that applies artificial intelligence (AI) methods to a reporting system. The representation scheme called Generalized Finding Representation (GFR) is proposed to bridge the gap between natural language expressions in the radiology report and AI methods. The entry process of RGSS-ID is made mainly by selecting items; our system allows a radiologist to compose a sentence which can be completely parsed by the computer. Further RGSS-ID encodes findings into the expression corresponding to GFR, and stores this expression into the knowledge data base. The final printed report is made in the natural language.
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Hierarchical Recognition Scheme for Human Facial Expression Recognition Systems
Siddiqi, Muhammad Hameed; Lee, Sungyoung; Lee, Young-Koo; Khan, Adil Mehmood; Truc, Phan Tran Ho
2013-01-01
Over the last decade, human facial expressions recognition (FER) has emerged as an important research area. Several factors make FER a challenging research problem. These include varying light conditions in training and test images; need for automatic and accurate face detection before feature extraction; and high similarity among different expressions that makes it difficult to distinguish these expressions with a high accuracy. This work implements a hierarchical linear discriminant analysis-based facial expressions recognition (HL-FER) system to tackle these problems. Unlike the previous systems, the HL-FER uses a pre-processing step to eliminate light effects, incorporates a new automatic face detection scheme, employs methods to extract both global and local features, and utilizes a HL-FER to overcome the problem of high similarity among different expressions. Unlike most of the previous works that were evaluated using a single dataset, the performance of the HL-FER is assessed using three publicly available datasets under three different experimental settings: n-fold cross validation based on subjects for each dataset separately; n-fold cross validation rule based on datasets; and, finally, a last set of experiments to assess the effectiveness of each module of the HL-FER separately. Weighted average recognition accuracy of 98.7% across three different datasets, using three classifiers, indicates the success of employing the HL-FER for human FER. PMID:24316568
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Lee, Chia-Ju; Devine, Beth; Tarczy-Hornoch, Peter
2017-01-01
Pharmacogenomics holds promise as a critical component of precision medicine. Yet, the use of pharmacogenomics in routine clinical care is minimal, partly due to the lack of efficient and effective use of existing evidence. This paper describes the design, development, implementation and evaluation of a knowledge-based system that fulfills three critical features: a) providing clinically relevant evidence, b) applying an evidence-based approach, and c) using semantically computable formalism, to facilitate efficient evidence assessment to support timely decisions on adoption of pharmacogenomics in clinical care. To illustrate functionality, the system was piloted in the context of clopidogrel and warfarin pharmacogenomics. In contrast to existing pharmacogenomics knowledge bases, the developed system is the first to exploit the expressivity and reasoning power of logic-based representation formalism to enable unambiguous expression and automatic retrieval of pharmacogenomics evidence to support systematic review with meta-analysis.
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AMTEC radioisotope power system design and analysis for Pluto Express Fly-By
DOE Office of Scientific and Technical Information (OSTI.GOV)
Hendricks, T.J.; Huang, C.; Sievers, R.K.
1997-12-31
The Pluto Express Fly-By program requires a Radioisotope Power System (RPS) to supply spacecraft power for various internal functions and mission instruments and experiments. AMTEC (Alkali-Metal Thermal-Electric Conversion) power conversion is the DOE-selected technology for an advanced, high-efficiency RPS to power the Pluto Express Fly-By spacecraft. An AMTEC-based RPS using the General Purpose Heat Source (GPHS) has been conceptually designed to satisfy the Pluto Express power requirements. Integrated AMTEC cell and system thermal/electrical design analyses, structural design analyses, and mass analyses were performed to define an optimum system design. Using fresh radioisotope fuel at beginning of mission, the RPS producesmore » 102 watts of power, has a mass of 8.35 kg (specific power density = 12.2 watts/kg), with a system conversion efficiency of 20.3%. Mass/power scale-up estimates have also been generated, indicating that a 150-watt version of this RPS would weigh approximately 11.3 kg. This paper presents and discusses the key features of this RPS design, the design and analysis methodology, and the numerous system and AMTEC cell tradeoff studies establishing the optimum AMTEC-based RPS.« less
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Braun, Martin; Kirsten, Robert; Rupp, Niels J; Moch, Holger; Fend, Falko; Wernert, Nicolas; Kristiansen, Glen; Perner, Sven
2013-05-01
Quantification of protein expression based on immunohistochemistry (IHC) is an important step for translational research and clinical routine. Several manual ('eyeballing') scoring systems are used in order to semi-quantify protein expression based on chromogenic intensities and distribution patterns. However, manual scoring systems are time-consuming and subject to significant intra- and interobserver variability. The aim of our study was to explore, whether new image analysis software proves to be sufficient as an alternative tool to quantify protein expression. For IHC experiments, one nucleus specific marker (i.e., ERG antibody), one cytoplasmic specific marker (i.e., SLC45A3 antibody), and one marker expressed in both compartments (i.e., TMPRSS2 antibody) were chosen. Stainings were applied on TMAs, containing tumor material of 630 prostate cancer patients. A pathologist visually quantified all IHC stainings in a blinded manner, applying a four-step scoring system. For digital quantification, image analysis software (Tissue Studio v.2.1, Definiens AG, Munich, Germany) was applied to obtain a continuous spectrum of average staining intensity. For each of the three antibodies we found a strong correlation of the manual protein expression score and the score of the image analysis software. Spearman's rank correlation coefficient was 0.94, 0.92, and 0.90 for ERG, SLC45A3, and TMPRSS2, respectively (p⟨0.01). Our data suggest that the image analysis software Tissue Studio is a powerful tool for quantification of protein expression in IHC stainings. Further, since the digital analysis is precise and reproducible, computer supported protein quantification might help to overcome intra- and interobserver variability and increase objectivity of IHC based protein assessment.
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Bleckmann, Maren; Schürig, Margitta; Chen, Fang-Fang; Yen, Zen-Zen; Lindemann, Nils; Meyer, Steffen; Spehr, Johannes; van den Heuvel, Joop
2016-01-01
The Baculovirus Expression Vector System (BEVS) is widely used to produce high amounts of recombinant proteins. Nevertheless, generating recombinant baculovirus in high quality is rather time-consuming and labor-intensive. Alternatively, virus-free expression in insect cells did not achieve similar expression levels for most proteins so far. The transactivation method is a promising approach for protein expression in Sf21 cells. It combines advantages of BEVS and plasmid-based expression by activating strong virus-dependent promoters on a transfected plasmid by baculoviral coinfection. Here, we identified expression elements required for transactivation. Therefore, we designed several vectors comprising different viral promoters or promoter combinations and tested them for eGFP expression using the automated BioLector microcultivation system. Remarkably, only the combination of the very late promoter p10 together with the homologous region 5 (hr5) could boost expression during transactivation. Other elements, like p10 alone or the late viral promoter polH, did not respond to transactivation. A new combination of hr5 and p10 with the strongest immediate early OpMNPV viral promoter OpIE2 improved the yield of eGFP by ~25% in comparison to the previous applied hr5-IE1-p10 expression cassette. Furthermore, we observed a strong influence of the transcription termination sequence and vector backbone on the level of expression. Finally, the expression levels for transactivation, BEVS and solely plasmid-based expression were compared for the marker protein eGFP, underlining the potential of transactivation for fast recombinant protein expression in Sf21 cells. In conclusion, essential elements for transactivation could be identified. The optimal elements were applied to generate an improved vector applicable in virus-free plasmid-based expression, transactivation and BEVS.
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Hong, Y K; Kim, D H; Beletskii, A; Lee, C; Memili, E; Strauss, W M
2001-04-01
Most conditional expression vectors designed for mammalian cells have been valuable systems for studying genes of interest by regulating their expressions. The available vectors, however, are reliable for the short-length cDNA clones and not optimal for relatively long fragments of genomic DNA or long cDNAs. Here, we report the construction of two bacterial artificial chromosome (BAC) vectors, capable of harboring large inserts and shuttling among Escherichia coli, yeast, and mammalian cells. These two vectors, pEYMT and pEYMI, contain conditional expression systems which are designed to be regulated by tetracycline and mouse interferons, respectively. To test the properties of the vectors, we cloned in both vectors the green fluorescence protein (GFP) through an in vitro ligation reaction and the 17.8-kb-long X-inactive-specific transcript (Xist) cDNA through homologous recombination in yeast. Subsequently, we characterized their regulated expression properties using real-time quantitative RT-PCR (TaqMan) and RNA-fluorescent in situ hybridization (FISH). We demonstrate that these two BAC vectors are good systems for recombination-based cloning and regulated expression of large genes in mammalian cells. Copyright 2001 Academic Press.
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Multi-layer sparse representation for weighted LBP-patches based facial expression recognition.
Jia, Qi; Gao, Xinkai; Guo, He; Luo, Zhongxuan; Wang, Yi
2015-03-19
In this paper, a novel facial expression recognition method based on sparse representation is proposed. Most contemporary facial expression recognition systems suffer from limited ability to handle image nuisances such as low resolution and noise. Especially for low intensity expression, most of the existing training methods have quite low recognition rates. Motivated by sparse representation, the problem can be solved by finding sparse coefficients of the test image by the whole training set. Deriving an effective facial representation from original face images is a vital step for successful facial expression recognition. We evaluate facial representation based on weighted local binary patterns, and Fisher separation criterion is used to calculate the weighs of patches. A multi-layer sparse representation framework is proposed for multi-intensity facial expression recognition, especially for low-intensity expressions and noisy expressions in reality, which is a critical problem but seldom addressed in the existing works. To this end, several experiments based on low-resolution and multi-intensity expressions are carried out. Promising results on publicly available databases demonstrate the potential of the proposed approach.
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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.
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Wei, Junhong; Tian, Jinjin; Pan, Guoqing; Xie, Jie; Bao, Jialing; Zhou, Zeyang
2017-06-01
To develop a reliable and easy to use expression system for antibiotic production improvement of Streptomyces. A two-compound T7 RNA polymerase-dependent gene expression system was developed to fulfill this demand. In this system, the T7 RNA polymerase coding sequence was optimized based on the codon usage of Streptomyces coelicolor. To evaluate the functionality of this system, we constructed an activator gene overexpression strain for enhancement of actinorhodin production. By overexpression of the positive regulator actII-ORF4 with this system, the maximum actinorhodin yield of engineered strain was 15-fold higher and the fermentation time was decreased by 48 h. The modified two-compound T7 expression system improves both antibiotic production and accelerates the fermentation process in Streptomyces. This provides a general and useful strategy for strain improvement of important antibiotic producing Streptomyces strains.
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Nine-year-old children use norm-based coding to visually represent facial expression.
Burton, Nichola; Jeffery, Linda; Skinner, Andrew L; Benton, Christopher P; Rhodes, Gillian
2013-10-01
Children are less skilled than adults at making judgments about facial expression. This could be because they have not yet developed adult-like mechanisms for visually representing faces. Adults are thought to represent faces in a multidimensional face-space, and have been shown to code the expression of a face relative to the norm or average face in face-space. Norm-based coding is economical and adaptive, and may be what makes adults more sensitive to facial expression than children. This study investigated the coding system that children use to represent facial expression. An adaptation aftereffect paradigm was used to test 24 adults and 18 children (9 years 2 months to 9 years 11 months old). Participants adapted to weak and strong antiexpressions. They then judged the expression of an average expression. Adaptation created aftereffects that made the test face look like the expression opposite that of the adaptor. Consistent with the predictions of norm-based but not exemplar-based coding, aftereffects were larger for strong than weak adaptors for both age groups. Results indicate that, like adults, children's coding of facial expressions is norm-based. PsycINFO Database Record (c) 2013 APA, all rights reserved.
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Production of recombinant allergens in plants.
Schmidt, Georg; Gadermaier, Gabriele; Pertl, Heidi; Siegert, Marc; Oksman-Caldentey, Kirsi-Marja; Ritala, Anneli; Himly, Martin; Obermeyer, Gerhard; Ferreira, Fatima
2008-10-01
A large percentage of allergenic proteins are of plant origin. Hence, plant-based expression systems are considered ideal for the recombinant production of certain allergens. First attempts to establish production of plant-derived allergens in plants focused on transient expression in Nicotiana benthamiana infected with recombinant viral vectors. Accordingly, allergens from birch and mugwort pollen, as well as from apple have been expressed in plants. Production of house dust mite allergens has been achieved by Agrobacterium-mediated transformation of tobacco plants. Beside the use of plants as production systems, other approaches have focused on the development of edible vaccines expressing allergens or epitopes thereof, which bypasses the need of allergen purification. The potential of this approach has been convincingly demonstrated for transgenic rice seeds expressing seven dominant human T cell epitopes derived from Japanese cedar pollen allergens. Parallel to efforts in developing recombinant-based diagnostic and therapeutic reagents, different gene-silencing approaches have been used to decrease the expression of allergenic proteins in allergen sources. In this way hypoallergenic ryegrass, soybean, rice, apple, and tomato were developed.
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A multiplexable TALE-based binary expression system for in vivo cellular interaction studies.
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.
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Unified modeling language and design of a case-based retrieval system in medical imaging.
LeBozec, C.; Jaulent, M. C.; Zapletal, E.; Degoulet, P.
1998-01-01
One goal of artificial intelligence research into case-based reasoning (CBR) systems is to develop approaches for designing useful and practical interactive case-based environments. Explaining each step of the design of the case-base and of the retrieval process is critical for the application of case-based systems to the real world. We describe herein our approach to the design of IDEM--Images and Diagnosis from Examples in Medicine--a medical image case-based retrieval system for pathologists. Our approach is based on the expressiveness of an object-oriented modeling language standard: the Unified Modeling Language (UML). We created a set of diagrams in UML notation illustrating the steps of the CBR methodology we used. The key aspect of this approach was selecting the relevant objects of the system according to user requirements and making visualization of cases and of the components of the case retrieval process. Further evaluation of the expressiveness of the design document is required but UML seems to be a promising formalism, improving the communication between the developers and users. Images Figure 6 Figure 7 PMID:9929346
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Unified modeling language and design of a case-based retrieval system in medical imaging.
LeBozec, C; Jaulent, M C; Zapletal, E; Degoulet, P
1998-01-01
One goal of artificial intelligence research into case-based reasoning (CBR) systems is to develop approaches for designing useful and practical interactive case-based environments. Explaining each step of the design of the case-base and of the retrieval process is critical for the application of case-based systems to the real world. We describe herein our approach to the design of IDEM--Images and Diagnosis from Examples in Medicine--a medical image case-based retrieval system for pathologists. Our approach is based on the expressiveness of an object-oriented modeling language standard: the Unified Modeling Language (UML). We created a set of diagrams in UML notation illustrating the steps of the CBR methodology we used. The key aspect of this approach was selecting the relevant objects of the system according to user requirements and making visualization of cases and of the components of the case retrieval process. Further evaluation of the expressiveness of the design document is required but UML seems to be a promising formalism, improving the communication between the developers and users.
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Zichel, R; Mimran, A; Keren, A; Barnea, A; Steinberger-Levy, I; Marcus, D; Turgeman, A; Reuveny, S
2010-05-01
Botulinum toxins produced by the anaerobic bacterium Clostridium botulinum are the most potent biological toxins in nature. Traditionally, people at risk are immunized with a formaldehyde-inactivated toxin complex. Second generation vaccines are based on the recombinant carboxy-terminal heavy-chain (Hc) fragment of the neurotoxin. However, the materialization of this approach is challenging, mainly due to the high AT content of clostridial genes. Herein, we present an alternative strategy in which the native genes encoding Hc proteins of botulinum toxins A, B, and E were used to express the recombinant Hc fragments in a cell-free expression system. We used the unique property of this open system to introduce different combinations of chaperone systems, protein disulfide isomerase (PDI), and reducing/oxidizing environments directly to the expression reaction. Optimized expression conditions led to increased production of soluble Hc protein, which was successfully scaled up using a continuous exchange (CE) cell-free system. Hc proteins were produced at a concentration of more than 1 mg/ml and purified by one-step Ni(+) affinity chromatography. Mice immunized with three injections containing 5 microg of any of the in vitro-expressed, alum-absorbed, Hc vaccines generated a serum enzyme-linked immunosorbent assay (ELISA) titer of 10(5) against the native toxin complex, which enabled protection against a high-dose toxin challenge (10(3) to 10(6) mouse 50% lethal dose [MsLD(50)]). Finally, immunization with a trivalent HcA, HcB, and HcE vaccine protected mice against the corresponding trivalent 10(5) MsLD(50) toxin challenge. Our results together with the latest developments in scalability of the in vitro protein expression systems offer alternative routes for the preparation of botulinum vaccine.
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Muecas: A Multi-Sensor Robotic Head for Affective Human Robot Interaction and Imitation
Cid, Felipe; Moreno, Jose; Bustos, Pablo; Núñez, Pedro
2014-01-01
This paper presents a multi-sensor humanoid robotic head for human robot interaction. The design of the robotic head, Muecas, is based on ongoing research on the mechanisms of perception and imitation of human expressions and emotions. These mechanisms allow direct interaction between the robot and its human companion through the different natural language modalities: speech, body language and facial expressions. The robotic head has 12 degrees of freedom, in a human-like configuration, including eyes, eyebrows, mouth and neck, and has been designed and built entirely by IADeX (Engineering, Automation and Design of Extremadura) and RoboLab. A detailed description of its kinematics is provided along with the design of the most complex controllers. Muecas can be directly controlled by FACS (Facial Action Coding System), the de facto standard for facial expression recognition and synthesis. This feature facilitates its use by third party platforms and encourages the development of imitation and of goal-based systems. Imitation systems learn from the user, while goal-based ones use planning techniques to drive the user towards a final desired state. To show the flexibility and reliability of the robotic head, the paper presents a software architecture that is able to detect, recognize, classify and generate facial expressions in real time using FACS. This system has been implemented using the robotics framework, RoboComp, which provides hardware-independent access to the sensors in the head. Finally, the paper presents experimental results showing the real-time functioning of the whole system, including recognition and imitation of human facial expressions. PMID:24787636
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Kobayashi, Takehito; Yagi, Yusuke; Nakamura, Takahiro
2016-01-01
The pentatricopeptide repeat (PPR) motif is a sequence-specific RNA/DNA-binding module. Elucidation of the RNA/DNA recognition mechanism has enabled engineering of PPR motifs as new RNA/DNA manipulation tools in living cells, including for genome editing. However, the biochemical characteristics of PPR proteins remain unknown, mostly due to the instability and/or unfolding propensities of PPR proteins in heterologous expression systems such as bacteria and yeast. To overcome this issue, we constructed reporter systems using animal cultured cells. The cell-based system has highly attractive features for PPR engineering: robust eukaryotic gene expression; availability of various vectors, reagents, and antibodies; highly efficient DNA delivery ratio (>80 %); and rapid, high-throughput data production. In this chapter, we introduce an example of such reporter systems: a PPR-based sequence-specific translational activation system. The cell-based reporter system can be applied to characterize plant genes of interested and to PPR engineering.
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Research issues of geometry-based visual languages and some solutions
NASA Astrophysics Data System (ADS)
Green, Thorn G.
This dissertation addresses the problem of how to design visual language systems that are based upon Geometric Algebra, and provide a visual coupling of algebraic expressions and geometric depictions. This coupling of algebraic expressions and geometric depictions provides a new means for expressing both mathematical and geometric relationships present in mathematics, physics, and Computer-Aided Geometric Design (CAGD). Another significant feature of such a system is that the result of changing a parameter (by dragging the mouse) can be seen immediately in the depiction(s) of all expressions that use that parameter. This greatly aides the cognition of the relationships between variables. Systems for representing such a coupling of algebra and geometry have characteristics of both visual language systems, and systems for scientific visualization. Instead of using a parsing or dataflow paradigm for the visual language representation, the systems instead represent equations as manipulatible constrained diagrams for their visualization. This requires that the design of such a system have (but is not limited to) a means for parsing equations entered by the user, a scheme for producing a visual representation of these equations; techniques for maintaining the coupling between the expressions entered and the diagrams displayed; algorithms for maintaining the consistency of the diagrams; and, indexing capabilities that are efficient enough to allow diagrams to be created, and manipulated in a short enough period of time. The author proposes solutions for how such a design can be realized.
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Population-expression models of immune response
NASA Astrophysics Data System (ADS)
Stromberg, Sean P.; Antia, Rustom; Nemenman, Ilya
2013-06-01
The immune response to a pathogen has two basic features. The first is the expansion of a few pathogen-specific cells to form a population large enough to control the pathogen. The second is the process of differentiation of cells from an initial naive phenotype to an effector phenotype which controls the pathogen, and subsequently to a memory phenotype that is maintained and responsible for long-term protection. The expansion and the differentiation have been considered largely independently. Changes in cell populations are typically described using ecologically based ordinary differential equation models. In contrast, differentiation of single cells is studied within systems biology and is frequently modeled by considering changes in gene and protein expression in individual cells. Recent advances in experimental systems biology make available for the first time data to allow the coupling of population and high dimensional expression data of immune cells during infections. Here we describe and develop population-expression models which integrate these two processes into systems biology on the multicellular level. When translated into mathematical equations, these models result in non-conservative, non-local advection-diffusion equations. We describe situations where the population-expression approach can make correct inference from data while previous modeling approaches based on common simplifying assumptions would fail. We also explore how model reduction techniques can be used to build population-expression models, minimizing the complexity of the model while keeping the essential features of the system. While we consider problems in immunology in this paper, we expect population-expression models to be more broadly applicable.
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Identifying novel glioma associated pathways based on systems biology level meta-analysis.
Hu, Yangfan; Li, Jinquan; Yan, Wenying; Chen, Jiajia; Li, Yin; Hu, Guang; Shen, Bairong
2013-01-01
With recent advances in microarray technology, including genomics, proteomics, and metabolomics, it brings a great challenge for integrating this "-omics" data to analysis complex disease. Glioma is an extremely aggressive and lethal form of brain tumor, and thus the study of the molecule mechanism underlying glioma remains very important. To date, most studies focus on detecting the differentially expressed genes in glioma. However, the meta-analysis for pathway analysis based on multiple microarray datasets has not been systematically pursued. In this study, we therefore developed a systems biology based approach by integrating three types of omics data to identify common pathways in glioma. Firstly, the meta-analysis has been performed to study the overlapping of signatures at different levels based on the microarray gene expression data of glioma. Among these gene expression datasets, 12 pathways were found in GeneGO database that shared by four stages. Then, microRNA expression profiles and ChIP-seq data were integrated for the further pathway enrichment analysis. As a result, we suggest 5 of these pathways could be served as putative pathways in glioma. Among them, the pathway of TGF-beta-dependent induction of EMT via SMAD is of particular importance. Our results demonstrate that the meta-analysis based on systems biology level provide a more useful approach to study the molecule mechanism of complex disease. The integration of different types of omics data, including gene expression microarrays, microRNA and ChIP-seq data, suggest some common pathways correlated with glioma. These findings will offer useful potential candidates for targeted therapeutic intervention of glioma.
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An Overview and History of Glyco-Engineering in Insect Expression Systems.
Geisler, Christoph; Mabashi-Asazuma, Hideaki; Jarvis, Donald L
2015-01-01
Insect systems, including the baculovirus-insect cell and Drosophila S2 cell systems are widely used as recombinant protein production platforms. Historically, however, no insect-based system has been able to produce glycoproteins with human-type glycans, which often influence the clinical efficacy of therapeutic glycoproteins and the overall structures and functions of other recombinant glycoprotein products. In addition, some insect cell systems produce N-glycans with immunogenic epitopes. Over the past 20 years, these problems have been addressed by efforts to glyco-engineer insect-based expression systems. These efforts have focused on introducing the capacity to produce complex-type, terminally sialylated N-glycans and eliminating the capacity to produce immunogenic N-glycans. Various glyco-engineering approaches have included genetically engineering insect cells, baculoviral vectors, and/or insects with heterologous genes encoding the enzymes required to produce various glycosyltransferases, sugars, nucleotide sugars, and nucleotide sugar transporters, as well as an enzyme that can deplete GDP-fucose. In this chapter, we present an overview and history of glyco-engineering in insect expression systems as a prelude to subsequent chapters, which will highlight various methods used for this purpose.
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Jani, Saurin D; Argraves, Gary L; Barth, Jeremy L; Argraves, W Scott
2010-04-01
An important objective of DNA microarray-based gene expression experimentation is determining inter-relationships that exist between differentially expressed genes and biological processes, molecular functions, cellular components, signaling pathways, physiologic processes and diseases. Here we describe GeneMesh, a web-based program that facilitates analysis of DNA microarray gene expression data. GeneMesh relates genes in a query set to categories available in the Medical Subject Headings (MeSH) hierarchical index. The interface enables hypothesis driven relational analysis to a specific MeSH subcategory (e.g., Cardiovascular System, Genetic Processes, Immune System Diseases etc.) or unbiased relational analysis to broader MeSH categories (e.g., Anatomy, Biological Sciences, Disease etc.). Genes found associated with a given MeSH category are dynamically linked to facilitate tabular and graphical depiction of Entrez Gene information, Gene Ontology information, KEGG metabolic pathway diagrams and intermolecular interaction information. Expression intensity values of groups of genes that cluster in relation to a given MeSH category, gene ontology or pathway can be displayed as heat maps of Z score-normalized values. GeneMesh operates on gene expression data derived from a number of commercial microarray platforms including Affymetrix, Agilent and Illumina. GeneMesh is a versatile web-based tool for testing and developing new hypotheses through relating genes in a query set (e.g., differentially expressed genes from a DNA microarray experiment) to descriptors making up the hierarchical structure of the National Library of Medicine controlled vocabulary thesaurus, MeSH. The system further enhances the discovery process by providing links between sets of genes associated with a given MeSH category to a rich set of html linked tabular and graphic information including Entrez Gene summaries, gene ontologies, intermolecular interactions, overlays of genes onto KEGG pathway diagrams and heatmaps of expression intensity values. GeneMesh is freely available online at http://proteogenomics.musc.edu/genemesh/.
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Exploiting Nanotechnology for the Development of MicroRNA-Based Cancer Therapeutics.
Tyagi, Nikhil; Arora, Sumit; Deshmukh, Sachin K; Singh, Seema; Marimuthu, Saravanakumar; Singh, Ajay P
2016-01-01
MicroRNAs (miRNAs/miRs) represent a novel class of small non-coding RNAs that post-transcriptionally regulate gene expression by base pairing with complementary sequences in the 3' untranslated region (UTR) of target mRNAs. Functional studies suggest that miRNAs control almost every biological process, and their aberrant expression leads to a disease state, such as cancer. Differential expression of miRNAs in cancerous versus normal cells have generated enormous interest for the development of miRNA-based cancer cell-targeted therapeutics. Depending on the miRNA function and expression in cancer, two types of miRNA-based therapeutic strategies can be utilized that either restore or inhibit miRNA function through exogenous delivery of miRNAs mimics or inhibitors (anti-miRs). However, hydrophilic nature of miRNA mimics/anti-miRs, sensitivity to nuclease degradation in serum, poor penetration and reduced uptake by the tumor cells are chief hurdles in accomplishing their efficient in vivo delivery. To overcome these barriers, several nanotechnology-based systems are being developed and tested for delivery efficacy. This review summarizes the importance of miRNAs-based therapeutics in cancer, associated translational challenges and novel nanotechnology-assisted delivery systems that hold potential for next-generation miRNA-based cancer therapeutics.
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Comparing Acquisition of Exchange-Based and Signed Mands with Children with Autism
ERIC Educational Resources Information Center
Barlow, Kathryn E.; Tiger, Jeffrey H.; Slocum, Sarah K.; Miller, Sarah J.
2013-01-01
Therapists and educators frequently teach alternative-communication systems, such as picture exchanges or manual signs, to individuals with developmental disabilities who present with expressive language deficits. Michael (1985) recommended a taxonomy for alternative communication systems that differentiated between selection-based systems in…
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Spontaneous Facial Mimicry in Response to Dynamic Facial Expressions
ERIC Educational Resources Information Center
Sato, Wataru; Yoshikawa, Sakiko
2007-01-01
Based on previous neuroscientific evidence indicating activation of the mirror neuron system in response to dynamic facial actions, we hypothesized that facial mimicry would occur while subjects viewed dynamic facial expressions. To test this hypothesis, dynamic/static facial expressions of anger/happiness were presented using computer-morphing…
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Production of Emotional Facial Expressions in European American, Japanese, and Chinese Infants.
ERIC Educational Resources Information Center
Camras, Linda A.; And Others
1998-01-01
European American, Japanese, and Chinese 11-month-olds participated in emotion-inducing laboratory procedures. Facial responses were scored with BabyFACS, an anatomically based coding system. Overall, Chinese infants were less expressive than European American and Japanese infants, suggesting that differences in expressivity between European…
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Mastering algebra retrains the visual system to perceive hierarchical structure in equations.
Marghetis, Tyler; Landy, David; Goldstone, Robert L
2016-01-01
Formal mathematics is a paragon of abstractness. It thus seems natural to assume that the mathematical expert should rely more on symbolic or conceptual processes, and less on perception and action. We argue instead that mathematical proficiency relies on perceptual systems that have been retrained to implement mathematical skills. Specifically, we investigated whether the visual system-in particular, object-based attention-is retrained so that parsing algebraic expressions and evaluating algebraic validity are accomplished by visual processing. Object-based attention occurs when the visual system organizes the world into discrete objects, which then guide the deployment of attention. One classic signature of object-based attention is better perceptual discrimination within, rather than between, visual objects. The current study reports that object-based attention occurs not only for simple shapes but also for symbolic mathematical elements within algebraic expressions-but only among individuals who have mastered the hierarchical syntax of algebra. Moreover, among these individuals, increased object-based attention within algebraic expressions is associated with a better ability to evaluate algebraic validity. These results suggest that, in mastering the rules of algebra, people retrain their visual system to represent and evaluate abstract mathematical structure. We thus argue that algebraic expertise involves the regimentation and reuse of evolutionarily ancient perceptual processes. Our findings implicate the visual system as central to learning and reasoning in mathematics, leading us to favor educational approaches to mathematics and related STEM fields that encourage students to adapt, not abandon, their use of perception.
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EXPRESS Rack Technology for Space Station
NASA Technical Reports Server (NTRS)
Davis, Ted B.; Adams, J. Brian; Fisher, Edward M., Jr.; Prickett, Guy B.; Smith, Timothy G.
1999-01-01
The EXPRESS rack provides accommodations for standard Mid-deck Locker and ISIS drawer payloads on the International Space Station. A design overview of the basic EXPRESS rack and two derivatives, the Human Research Facility and the Habitat Holding Rack, is given in Part I. In Part II, the design of the Solid State Power Control Module (SSPCM) is reviewed. The SSPCM is a programmable and remotely controllable power switching and voltage conversion unit which distributes and protects up to 3kW of 12OVDC and 28VDC power to payloads and rack subsystem components. Part III details the development and testing of a new data storage device, the BRP EXPRESS Memory Unit (BEMU). The BEMU is a conduction-cooled device which operates on 28VDC and is based on Boeing-modified 9GB commercial disk-drive technology. In Part IV results of a preliminary design effort for a rack Passive Damping System (PDS) are reported. The PDS is intended to isolate ISPR-based experiment racks from on-orbit vibration. System performance predictions based on component developmental testing indicate that such a system can provide effective isolation at frequencies of 1 Hz and above.
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Kim, Pyung-Hwan; Lee, Minhyung; Nam, Kihoon; Kim, Sung Wan
2014-01-01
Glucagon-like peptide 1 (GLP-1) agonist, exenxdin-4, is currently being advanced as a promising diabetes remedy via a variety of incretin actions similar with GLP-1. In this study, we investigated an effective anti-diabetic therapy via exendin-4 expressing chimeric plasmid based on two-step transcription amplification (TSTA) system with dendrimer-type bioreducible polymer for more improved incretin-based gene therapy. Arginine-grafted poly (cystaminebisacrylamide-diaminohexane) (ABP)-conjugated poly (amido amine) (PAMAM) dendrimer (PAM-ABP) was used as gene carrier. PAM-ABP/chimeric DNA polyplex was markedly elevated exendin-4 expression in ectopic cells as well as increased insulin production through an enhanced activation of protein kinase K (PKA) induced by up-regulation of exendin-4-stimulated cyclic adenosine monophosphate (cAMP) in pancreatic β-cell. Consistent with these results, intravenous administration of PAM-ABP/chimeric DNA polyplex improved glucoregulotory effects, as well as increased insulin secretion by high expression of exendin-4 in blood in type 2 diabetic mice with no any toxicity. Our exendin-4 system can be attributed to provide a potential diabetes therapeutic agent for improved incretin gene therapy. PMID:24839613
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Holaskova, Edita; Galuszka, Petr; Frebort, Ivo; Oz, M Tufan
2015-11-01
Antimicrobial peptides (AMPs) are vital components of the innate immune system of nearly all living organisms. They generally act in the first line of defense against various pathogenic bacteria, parasites, enveloped viruses and fungi. These low molecular mass peptides are considered prospective therapeutic agents due to their broad-spectrum rapid activity, low cytotoxicity to mammalian cells and unique mode of action which hinders emergence of pathogen resistance. In addition to medical use, AMPs can also be employed for development of innovative approaches for plant protection in agriculture. Conferred disease resistance by AMPs might help us surmount losses in yield, quality and safety of agricultural products due to plant pathogens. Heterologous expression in plant-based systems, also called plant molecular farming, offers cost-effective large-scale production which is regarded as one of the most important factors for clinical or agricultural use of AMPs. This review presents various types of AMPs as well as plant-based platforms ranging from cell suspensions to whole plants employed for peptide production. Although AMP production in plants holds great promises for medicine and agriculture, specific technical limitations regarding product yield, function and stability still remain. Additionally, establishment of particular stable expression systems employing plants or plant tissues generally requires extended time scale for platform development compared to certain other heterologous systems. Therefore, fast and promising tools for evaluation of plant-based expression strategies and assessment of function and stability of the heterologously produced AMPs are critical for molecular farming and plant protection. Copyright © 2015 Elsevier Inc. All rights reserved.
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Establishing a high yielding streptomyces-based cell-free protein synthesis system.
Li, Jian; Wang, He; Kwon, Yong-Chan; Jewett, Michael C
2017-06-01
Cell-free protein synthesis (CFPS) has emerged as a powerful platform for applied biotechnology and synthetic biology, with a range of applications in synthesizing proteins, evolving proteins, and prototyping genetic circuits. To expand the current CFPS repertoire, we report here the development and optimization of a Streptomyces-based CFPS system for the expression of GC-rich genes. By developing a streamlined crude extract preparation protocol and optimizing reaction conditions, we were able to achieve active enhanced green fluorescent protein (EGFP) yields of greater than 50 μg/mL with batch reactions lasting up to 3 h. By adopting a semi-continuous reaction format, the EGFP yield could be increased to 282 ± 8 μg/mL and the reaction time was extended to 48 h. Notably, our extract preparation procedures were robust to multiple Streptomyces lividans and Streptomyces coelicolor strains, although expression yields varied. We show that our optimized Streptomyces lividans system provides benefits when compared to an Escherichia coli-based CFPS system for increasing percent soluble protein expression for four Streptomyces-originated high GC-content genes that are involved in biosynthesis of the nonribosomal peptides tambromycin and valinomycin. Looking forward, we believe that our Streptomyces-based CFPS system will contribute significantly towards efforts to express complex natural product gene clusters (e.g., nonribosomal peptides and polyketides), providing a new avenue for obtaining and studying natural product biosynthesis pathways. Biotechnol. Bioeng. 2017;114: 1343-1353. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.
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NASA Technical Reports Server (NTRS)
Pelfrey, Joseph J.; Jordan, Lee P.
2008-01-01
The EXpedite the PRocessing of Experiments to Space Station or EXPRESS Rack System has provided accommodations and facilitated operations for microgravity-based research payloads for over 6 years on the International Space Station (ISS). The EXPRESS Rack accepts Space Shuttle middeck type lockers and International Subrack Interface Standard (ISIS) drawers, providing a modular-type interface on the ISS. The EXPRESS Rack provides 28Vdc power, Ethernet and RS-422 data interfaces, thermal conditioning, vacuum exhaust, and Nitrogen supply for payload use. The EXPRESS Rack system also includes payload checkout capability with a flight rack or flight rack emulator prior to launch, providing a high degree of confidence in successful operations once an-orbit. In addition, EXPRESS trainer racks are provided to support crew training of both rack systems and subrack operations. Standard hardware and software interfaces provided by the EXPRESS Rack simplify the integration processes for ISS payload development. The EXPRESS Rack is designed to accommodate multidiscipline research, allowing for the independent operation of each subrack payload within a single rack. On-orbit operations began for the EXPRESS Rack Project on April 24, 2001, with one rack operating continuously to support high-priority payloads. The other on-orbit EXPRESS Racks operate based on payload need and resource availability. Over 50 multi-discipline payloads have now been supported on-orbit by the EXPRESS Rack Program. Sustaining engineering, logistics, and maintenance functions are in place to maintain hardware, operations and provide software upgrades. Additional EXPRESS Racks are planned for launch prior to ISS completion in support of long-term operations and the planned transition of the U.S. Segment to a National Laboratory.
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Recognizing Action Units for Facial Expression Analysis
Tian, Ying-li; Kanade, Takeo; Cohn, Jeffrey F.
2010-01-01
Most automatic expression analysis systems attempt to recognize a small set of prototypic expressions, such as happiness, anger, surprise, and fear. Such prototypic expressions, however, occur rather infrequently. Human emotions and intentions are more often communicated by changes in one or a few discrete facial features. In this paper, we develop an Automatic Face Analysis (AFA) system to analyze facial expressions based on both permanent facial features (brows, eyes, mouth) and transient facial features (deepening of facial furrows) in a nearly frontal-view face image sequence. The AFA system recognizes fine-grained changes in facial expression into action units (AUs) of the Facial Action Coding System (FACS), instead of a few prototypic expressions. Multistate face and facial component models are proposed for tracking and modeling the various facial features, including lips, eyes, brows, cheeks, and furrows. During tracking, detailed parametric descriptions of the facial features are extracted. With these parameters as the inputs, a group of action units (neutral expression, six upper face AUs and 10 lower face AUs) are recognized whether they occur alone or in combinations. The system has achieved average recognition rates of 96.4 percent (95.4 percent if neutral expressions are excluded) for upper face AUs and 96.7 percent (95.6 percent with neutral expressions excluded) for lower face AUs. The generalizability of the system has been tested by using independent image databases collected and FACS-coded for ground-truth by different research teams. PMID:25210210
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Gering, Kevin L.
2013-01-01
A system includes an electrochemical cell, monitoring hardware, and a computing system. The monitoring hardware samples performance characteristics of the electrochemical cell. The computing system determines cell information from the performance characteristics. The computing system also analyzes the cell information of the electrochemical cell with a Butler-Volmer (BV) expression modified to determine exchange current density of the electrochemical cell by including kinetic performance information related to pulse-time dependence, electrode surface availability, or a combination thereof. A set of sigmoid-based expressions may be included with the modified-BV expression to determine kinetic performance as a function of pulse time. The determined exchange current density may be used with the modified-BV expression, with or without the sigmoid expressions, to analyze other characteristics of the electrochemical cell. Model parameters can be defined in terms of cell aging, making the overall kinetics model amenable to predictive estimates of cell kinetic performance along the aging timeline.
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Noncoherent Detection of Coherent Optical Heterodyne Signals Corrupted by Laser Phase Noise
1991-03-01
replicated speech at the receiving end through the photoelectric effect . Bell’s photophone was the first practical use of light as a transmission...source dominates system performance. An analyti- cal expression representing the effect of laser phase noise on system performance is derived based on a...decision threshold analysis illustrates which noise source dominates system performance. An analytical expression representing the effect of laser phase
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Hennessy, Rosanna C; Christiansen, Line; Olsson, Stefan; Stougaard, Peter
2018-01-01
Fluorescence-based reporter systems are valuable tools for studying gene expression dynamics in living cells. Here we describe a dual-fluorescence reporter system carrying the red fluorescent marker mCherry and the blue fluorescent protein EBFP2 enabling the simultaneous analysis of two promoters in broad-host range autofluorescent Gram-negative bacteria. Copyright © 2017 Elsevier B.V. All rights reserved.
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[Expression of acylamidase gene in Rhodococcus erythropolis strains].
Lavrov, K V; Novikov, A D; Riabchenko, L E; Ianenko, A S
2014-09-01
The expression of a new acylamidase gene from R. erythropolis 37 was studied in Rhodococcus erythropolis strains. This acylamidase, as a result of its unique substrate specificity, can hydrolyse N-substituted amides (4'-nitroacetanilide, N-isopropylacrylamide, N'N-dimethylaminopropylacrylamide). A new expression system based on the use of the promoter region of nitrilhydratase genes from R. rhodochrous M8 was created to achieve constitutive synthesis of acylamidase in R. erythropolis cells. A fourfold improvement in the acylamidase activity of recombinant R. erythropolis cells as compared with the parent wild-type strain was obtained through the use of the new expression system.
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Simple method for assembly of CRISPR synergistic activation mediator gRNA expression array.
Vad-Nielsen, Johan; Nielsen, Anders Lade; Luo, Yonglun
2018-05-20
When studying complex interconnected regulatory networks, effective methods for simultaneously manipulating multiple genes expression are paramount. Previously, we have developed a simple method for generation of an all-in-one CRISPR gRNA expression array. We here present a Golden Gate Assembly-based system of synergistic activation mediator (SAM) compatible CRISPR/dCas9 gRNA expression array for the simultaneous activation of multiple genes. Using this system, we demonstrated the simultaneous activation of the transcription factors, TWIST, SNAIL, SLUG, and ZEB1 a human breast cancer cell line. Copyright © 2018 Elsevier B.V. All rights reserved.
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Dendritic Cell-Based Genetic Immunotherapy for Ovarian Cancer
2007-12-01
CAR. CD40 is a surface marker expressed by DCs that plays a crucial role in their maturation and subsequent stimulation of T cells. DC infection with... surface . CD40 is a cell surface marker expressed by DCs, is crucial for their maturation and the subsequent activation of the immune system by the DCs...cell surface . CD40 is a cell surface marker expressed by DCs, is crucial for their maturation and the subsequent activation of the immune system by the
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Expression of monellin in a food-grade delivery system in Saccharomyces cerevisiae.
Liu, Jun; Yan, Da-zhong; Zhao, Sheng-jun
2015-10-01
Genetically modified (GM) foods have caused much controversy. Construction of a food-grade delivery system is a desirable technique with presumptive impact on industrial applications from the perspective of bio-safety. The aim of this study was to construct a food-grade delivery system for Saccharomyces cerevisiae and to study the expression of monellin from the berries of the West African forest plant Dioscoreophyllum cumminsii in this system. A food-grade system for S. cerevisiae was constructed based on ribosomal DNA (rDNA)-mediated homologous recombination to enable high-copy-number integration of the expression cassette inserted into the rDNA locus. A copper resistance gene (CUP1) was used as the selection marker for yeast transformation. Because variants of transformants containing different copy numbers at the CUP1 locus can be readily selected after growth in the presence of elevated copper levels, we suggest that this system would prove useful in the generation of tandemly iterated gene clusters. Using this food-grade system, a single-chain monellin gene was heterologously expressed. The yield of monellin reached a maximum of 675 mg L(-1) . This system harbors exclusively S. cerevisiae DNA with no antibiotic resistance genes, and it should therefore be appropriate for safe use in the food industry. Monellin was shown to be expressed in this food-grade delivery system. To our knowledge, this is the first report so far on expression of monellin in a food-grade expression system in S. cerevisiae. © 2014 Society of Chemical Industry.
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NASA Technical Reports Server (NTRS)
Yam, Yeung; Johnson, Timothy L.; Lang, Jeffrey H.
1987-01-01
A model reduction technique based on aggregation with respect to sensor and actuator influence functions rather than modes is presented for large systems of coupled second-order differential equations. Perturbation expressions which can predict the effects of spillover on both the reduced-order plant model and the neglected plant model are derived. For the special case of collocated actuators and sensors, these expressions lead to the derivation of constraints on the controller gains that are, given the validity of the perturbation technique, sufficient to guarantee the stability of the closed-loop system. A case study demonstrates the derivation of stabilizing controllers based on the present technique. The use of control and observation synthesis in modifying the dimension of the reduced-order plant model is also discussed. A numerical example is provided for illustration.
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A bio-inspired structural health monitoring system based on ambient vibration
NASA Astrophysics Data System (ADS)
Lin, Tzu-Kang; Kiremidjian, Anne; Lei, Chi-Yang
2010-11-01
A structural health monitoring (SHM) system based on naïve Bayesian (NB) damage classification and DNA-like expression data was developed in this research. Adapted from the deoxyribonucleic acid (DNA) array concept in molecular biology, the proposed structural health monitoring system is constructed utilizing a double-tier regression process to extract the expression array from the structural time history recorded during external excitations. The extracted array is symbolized as the various genes of the structure from the viewpoint of molecular biology and reflects the possible damage conditions prevalent in the structure. A scaled down, six-story steel building mounted on the shaking table of the National Center for Research on Earthquake Engineering (NCREE) was used as the benchmark. The structural response at different damage levels and locations under ambient vibration was collected to support the database for the proposed SHM system. To improve the precision of detection in practical applications, the system was enhanced by an optimization process using the likelihood selection method. The obtained array representing the DNA array of the health condition of the structure was first evaluated and ranked. A total of 12 groups of expression arrays were regenerated from a combination of four damage conditions. To keep the length of the array unchanged, the best 16 coefficients from every expression array were selected to form the optimized SHM system. Test results from the ambient vibrations showed that the detection accuracy of the structural damage could be greatly enhanced by the optimized expression array, when compared to the original system. Practical verification also demonstrated that a rapid and reliable result could be given by the final system within 1 min. The proposed system implements the idea of transplanting the DNA array concept from molecular biology into the field of SHM.
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Generalization of the Activated Complex Theory of Reaction Rates. II. Classical Mechanical Treatment
DOE R&D Accomplishments Database
Marcus, R. A.
1964-01-01
In its usual classical form activated complex theory assumes a particular expression for the kinetic energy of the reacting system -- one associated with a rectilinear motion along the reaction coordinate. The derivation of the rate expression given in the present paper is based on the general kinetic energy expression.
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Production of recombinant allergens in plants
2010-01-01
A large percentage of allergenic proteins are of plant origin. Hence, plant-based expression systems are considered ideal for the recombinant production of certain allergens. First attempts to establish production of plant-derived allergens in plants focused on transient expression in Nicotiana benthamiana infected with recombinant viral vectors. Accordingly, allergens from birch and mugwort pollen, as well as from apple have been expressed in plants. Production of house dust mite allergens has been achieved by Agrobacterium-mediated transformation of tobacco plants. Beside the use of plants as production systems, other approaches have focused on the development of edible vaccines expressing allergens or epitopes thereof, which bypasses the need of allergen purification. The potential of this approach has been convincingly demonstrated for transgenic rice seeds expressing seven dominant human T cell epitopes derived from Japanese cedar pollen allergens. Parallel to efforts in developing recombinant-based diagnostic and therapeutic reagents, different gene-silencing approaches have been used to decrease the expression of allergenic proteins in allergen sources. In this way hypoallergenic ryegrass, soybean, rice, apple, and tomato were developed. PMID:21258627
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Genome-wide expression profiling in pediatric septic shock
Wong, Hector R.
2013-01-01
For nearly a decade, our research group has had the privilege of developing and mining a multi-center, microarray-based, genome-wide expression database of critically ill children (≤ 10 years of age) with septic shock. Using bioinformatic and systems biology approaches, the expression data generated through this discovery-oriented, exploratory approach have been leveraged for a variety of objectives, which will be reviewed. Fundamental observations include wide spread repression of gene programs corresponding to the adaptive immune system, and biologically significant differential patterns of gene expression across developmental age groups. The data have also identified gene expression-based subclasses of pediatric septic shock having clinically relevant phenotypic differences. The data have also been leveraged for the discovery of novel therapeutic targets, and for the discovery and development of novel stratification and diagnostic biomarkers. Almost a decade of genome-wide expression profiling in pediatric septic shock is now demonstrating tangible results. The studies have progressed from an initial discovery-oriented and exploratory phase, to a new phase where the data are being translated and applied to address several areas of clinical need. PMID:23329198
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Endoribonuclease-Based Two-Component Repressor Systems for Tight Gene Expression Control in Plants
Liang, Yan; Richardson, Sarah; Yan, Jingwei; ...
2017-01-17
Tight control and multifactorial regulation of gene expression are important challenges in genetic engineering and are critical for the development of regulatory circuits. In meeting these challenges we will facilitate transgene expression regulation and support the fine-tuning of metabolic pathways to avoid the accumulation of undesired intermediates. By employing the endoribonuclease Csy4 and its recognition sequence from Pseudomonas aeruginosa and manipulating 5'UTR of mRNA, we developed a two-component expression–repression system to tightly control synthesis of transgene products. We demonstrated that this regulatory device was functional in monocotyledonous and dicotyledonous plant species, and showed that it can be used to repressmore » transgene expression by >400-fold and to synchronize transgene repression. In addition to tissue-specific transgene repression, this system offers stimuli-dependent expression control. Here, we identified 54 orthologous systems from various bacteria, using a bioinformatics approach and then validated in planta the activity for a few of those systems, demonstrating the potential diversity of such a two-component repressor system.« less
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Endoribonuclease-Based Two-Component Repressor Systems for Tight Gene Expression Control in Plants
DOE Office of Scientific and Technical Information (OSTI.GOV)
Liang, Yan; Richardson, Sarah; Yan, Jingwei
Tight control and multifactorial regulation of gene expression are important challenges in genetic engineering and are critical for the development of regulatory circuits. In meeting these challenges we will facilitate transgene expression regulation and support the fine-tuning of metabolic pathways to avoid the accumulation of undesired intermediates. By employing the endoribonuclease Csy4 and its recognition sequence from Pseudomonas aeruginosa and manipulating 5'UTR of mRNA, we developed a two-component expression–repression system to tightly control synthesis of transgene products. We demonstrated that this regulatory device was functional in monocotyledonous and dicotyledonous plant species, and showed that it can be used to repressmore » transgene expression by >400-fold and to synchronize transgene repression. In addition to tissue-specific transgene repression, this system offers stimuli-dependent expression control. Here, we identified 54 orthologous systems from various bacteria, using a bioinformatics approach and then validated in planta the activity for a few of those systems, demonstrating the potential diversity of such a two-component repressor system.« less
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Support vector machine-based facial-expression recognition method combining shape and appearance
NASA Astrophysics Data System (ADS)
Han, Eun Jung; Kang, Byung Jun; Park, Kang Ryoung; Lee, Sangyoun
2010-11-01
Facial expression recognition can be widely used for various applications, such as emotion-based human-machine interaction, intelligent robot interfaces, face recognition robust to expression variation, etc. Previous studies have been classified as either shape- or appearance-based recognition. The shape-based method has the disadvantage that the individual variance of facial feature points exists irrespective of similar expressions, which can cause a reduction of the recognition accuracy. The appearance-based method has a limitation in that the textural information of the face is very sensitive to variations in illumination. To overcome these problems, a new facial-expression recognition method is proposed, which combines both shape and appearance information, based on the support vector machine (SVM). This research is novel in the following three ways as compared to previous works. First, the facial feature points are automatically detected by using an active appearance model. From these, the shape-based recognition is performed by using the ratios between the facial feature points based on the facial-action coding system. Second, the SVM, which is trained to recognize the same and different expression classes, is proposed to combine two matching scores obtained from the shape- and appearance-based recognitions. Finally, a single SVM is trained to discriminate four different expressions, such as neutral, a smile, anger, and a scream. By determining the expression of the input facial image whose SVM output is at a minimum, the accuracy of the expression recognition is much enhanced. The experimental results showed that the recognition accuracy of the proposed method was better than previous researches and other fusion methods.
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Evolving fuzzy rules in a learning classifier system
NASA Technical Reports Server (NTRS)
Valenzuela-Rendon, Manuel
1993-01-01
The fuzzy classifier system (FCS) combines the ideas of fuzzy logic controllers (FLC's) and learning classifier systems (LCS's). It brings together the expressive powers of fuzzy logic as it has been applied in fuzzy controllers to express relations between continuous variables, and the ability of LCS's to evolve co-adapted sets of rules. The goal of the FCS is to develop a rule-based system capable of learning in a reinforcement regime, and that can potentially be used for process control.
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Bockamp, Ernesto; Sprengel, Rolf; Eshkind, Leonid; Lehmann, Thomas; Braun, Jan M; Emmrich, Frank; Hengstler, Jan G
2008-03-01
Many mouse models are currently available, providing avenues to elucidate gene function and to recapitulate specific pathological conditions. To a large extent, successful translation of clinical evidence or analytical data into appropriate mouse models is possible through progress in transgenic or gene-targeting technology. Beginning with a review of standard mouse transgenics and conventional gene targeting, this article will move on to discussing the basics of conditional gene expression: the tetracycline (tet)-off and tet-on systems based on the transactivators tet-controlled transactivator (Tta) and reverse tet-on transactivator (rtTA) that allow downregulation or induction of gene expression; Cre or Flp recombinase-mediated modifications, including excision, inversion, insertion and interchromosomal translocation; combination of the tet and Cre systems, permitting inducible knockout, reporter gene activation or activation of point mutations; the avian retroviral system based on delivery of rtTA specifically into cells expressing the avian retroviral receptor, which enables cell type-specific, inducible gene expression; the tamoxifen system, one of the most frequently applied steroid receptor-based systems, allows rapid activation of a fusion protein between the gene of interest and a mutant domain of the estrogen receptor, whereby activation does not depend on transcription; and techniques for cell type-specific ablation. The diphtheria toxin receptor system offers the advantage that it can be combined with the 'zoo' of Cre recombinase driver mice. Having described the basics we move on to the cutting edge: generation of genome-wide sets of conditional knockout mice. To this end, large ongoing projects apply two strategies: gene trapping based on random integration of trapping vectors into introns leading to truncation of the transcript, and gene targeting, representing the directed approach using homologous recombination. It can be expected that in the near future genome-wide sets of such mice will be available. Finally, the possibilities of conditional expression systems for investigating gene function in tissue regeneration will be illustrated by examples for neurodegenerative disease, liver regeneration and wound healing of the skin.
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A DNA replicon system for rapid high-level production of virus-like particles in plants.
Huang, Zhong; Chen, Qiang; Hjelm, Brooke; Arntzen, Charles; Mason, Hugh
2009-07-01
Recombinant virus-like particles (VLPs) represent a safe and effective vaccine strategy. We previously described a stable transgenic plant system for inexpensive production and oral delivery of VLP vaccines. However, the relatively low-level antigen accumulation and long-time frame to produce transgenic plants are the two major roadblocks in the practical development of plant-based VLP production. In this article, we describe the optimization of geminivirus-derived DNA replicon vectors for rapid, high-yield plant-based production of VLPs. Co-delivery of bean yellow dwarf virus (BeYDV)-derived vector and Rep/RepA-supplying vector by agroinfiltration of Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within 5 days. Co-expression of the P19 protein of tomato bush stunt virus, a gene silencing inhibitor, further enhanced VLP accumulation by stabilizing the mRNA. With this system, hepatitis B core antigen (HBc) and Norwalk virus capsid protein (NVCP) were produced at 0.80 and 0.34 mg/g leaf fresh weight, respectively. Sedimentation analysis and electron microscopy of transiently expressed antigens verified the efficient assembly of VLPs. Furthermore, a single replicon vector containing a built-in Rep/RepA cassette without P19 drove protein expression at similar levels as the three-component system. These results demonstrate the advantages of fast and high-level production of VLP-based vaccines using the BeYDV-derived DNA replicon system for transient expression in plants. (c) 2009 Wiley Periodicals, Inc.
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A DNA replicon system for rapid high-level production of virus-like particles in plants
Huang, Zhong; Chen, Qiang; Hjelm, Brooke; Arntzen, Charles
2009-01-01
Recombinant virus-like particles (VLPs) represent a safe and effective vaccine strategy. We previously described a stable transgenic plant system for inexpensive production and oral delivery of VLP vaccines. However, the relatively low level antigen accumulation and long time frame to produce transgenic plants are the two major roadblocks in the practical development of plant-based VLP production. In this paper, we describe the optimization of geminivirus-derived DNA replicon vectors for rapid, high-yield plant-based production of VLPs. Co-delivery of bean yellow dwarf virus (BeYDV)-derived vector and Rep/RepA-supplying vector by agroinfiltration of Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within five days. Co-expression of the P19 protein of tomato bush stunt virus, a gene silencing inhibitor, further enhanced VLP accumulation by stabilizing the mRNA. With this system, hepatitis B core antigen (HBc) and Norwalk virus capsid protein (NVCP) were produced at 0.80 and 0.34 mg/g leaf fresh weight, respectively. Sedimentation analysis and electron microscopy of transiently expressed antigens verified the efficient assembly of VLPs. Furthermore, a single replicon vector containing a built-in Rep/RepA cassette without p19 drove protein expression at similar levels as the three-component system. These results demonstrate the advantages of fast and high-level production of VLP-based vaccines using the BeYDV-derived DNA replicon system for transient expression in plants. PMID:19309755
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MultiSite Gateway-Compatible Cell Type-Specific Gene-Inducible System for Plants1[OPEN
Siligato, Riccardo; Wang, Xin; Yadav, Shri Ram; Lehesranta, Satu; Ma, Guojie; Ursache, Robertas; Sevilem, Iris; Zhang, Jing; Gorte, Maartje; Prasad, Kalika; Heidstra, Renze
2016-01-01
A powerful method to study gene function is expression or overexpression in an inducible, cell type-specific system followed by observation of consequent phenotypic changes and visualization of linked reporters in the target tissue. Multiple inducible gene overexpression systems have been developed for plants, but very few of these combine plant selection markers, control of expression domains, access to multiple promoters and protein fusion reporters, chemical induction, and high-throughput cloning capabilities. Here, we introduce a MultiSite Gateway-compatible inducible system for Arabidopsis (Arabidopsis thaliana) plants that provides the capability to generate such constructs in a single cloning step. The system is based on the tightly controlled, estrogen-inducible XVE system. We demonstrate that the transformants generated with this system exhibit the expected cell type-specific expression, similar to what is observed with constitutively expressed native promoters. With this new system, cloning of inducible constructs is no longer limited to a few special cases but can be used as a standard approach when gene function is studied. In addition, we present a set of entry clones consisting of histochemical and fluorescent reporter variants designed for gene and promoter expression studies. PMID:26644504
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Butt, Omer I.; Buehler, Paul W.; D'Agnillo, Felice
2011-01-01
Systemic exposure to cell-free hemoglobin (Hb) or its breakdown products after hemolysis or with the use of Hb-based oxygen therapeutics may alter the function and integrity of the blood-brain barrier. Using a guinea pig exchange transfusion model, we investigated the effect of a polymerized cell-free Hb (HbG) on the expression of endothelial tight junction proteins (zonula occludens 1, claudin-5, and occludin), astrocyte activation, IgG extravasation, heme oxygenase (HO), iron deposition, oxidative end products (4-hydroxynonenal adducts and 8-hydroxydeoxyguanosine), and apoptosis (cleaved caspase 3). Reduced zonula occludens 1 expression was observed after HbG transfusion as evidenced by Western blot and confocal microscopy. Claudin-5 distribution was altered in small- to medium-sized vessels. However, total expression of claudin-5 and occludin remained unchanged except for a notable increase in occludin 72 hours after HbG transfusion. HbG-transfused animals also showed increased astrocytic glial fibrillary acidic protein expression and IgG extravasation after 72 hours. Increased HO activity and HO-1 expression with prominent enhancement of HO-1 immunoreactivity in CD163-expressing perivascular cells and infiltrating monocytes/macrophages were also observed. Consistent with oxidative stress, HbG increased iron deposition, 4-hydroxynonenal and 8-hydroxydeoxyguanosine immunoreactivity, and cleaved caspase-3 expression. Systemic exposure to an extracellular Hb triggers blood-brain barrier disruption and oxidative stress, which may have important implications for the use of Hb-based therapeutics and may provide indirect insight on the central nervous system vasculopathies associated with excessive hemolysis. PMID:21356382
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Support vector machine for automatic pain recognition
NASA Astrophysics Data System (ADS)
Monwar, Md Maruf; Rezaei, Siamak
2009-02-01
Facial expressions are a key index of emotion and the interpretation of such expressions of emotion is critical to everyday social functioning. In this paper, we present an efficient video analysis technique for recognition of a specific expression, pain, from human faces. We employ an automatic face detector which detects face from the stored video frame using skin color modeling technique. For pain recognition, location and shape features of the detected faces are computed. These features are then used as inputs to a support vector machine (SVM) for classification. We compare the results with neural network based and eigenimage based automatic pain recognition systems. The experiment results indicate that using support vector machine as classifier can certainly improve the performance of automatic pain recognition system.
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Genomic Analysis and Isolation of RNA Polymerase II Dependent Promoters from Spodoptera frugiperda.
Bleckmann, Maren; Fritz, Markus H-Y; Bhuju, Sabin; Jarek, Michael; Schürig, Margitta; Geffers, Robert; Benes, Vladimir; Besir, Hüseyin; van den Heuvel, Joop
2015-01-01
The Baculoviral Expression Vector System (BEVS) is the most commonly used method for high expression of recombinant protein in insect cells. Nevertheless, expression of some target proteins--especially those entering the secretory pathway--provides a severe challenge for the baculovirus infected insect cells, due to the reorganisation of intracellular compounds upon viral infection. Therefore, alternative strategies for recombinant protein production in insect cells like transient plasmid-based expression or stable expression cell lines are becoming more popular. However, the major bottleneck of these systems is the lack of strong endogenous polymerase II dependent promoters, as the strong baculoviral p10 and polH promoters used in BEVS are only functional in presence of the viral transcription machinery during the late phase of infection. In this work we present a draft genome and a transcriptome analysis of Sf21 cells for the identification of the first known endogenous Spodoptera frugiperda promoters. Therefore, putative promoter sequences were identified and selected because of high mRNA level or in analogy to other strong promoters in other eukaryotic organism. The chosen endogenous Sf21 promoters were compared to early viral promoters for their efficiency to trigger eGFP expression using transient plasmid based transfection in a BioLector Microfermentation system. Furthermore, promoter activity was not only shown in Sf21 cells but also in Hi5 cells. The novel endogenous Sf21 promoters were ranked according to their activity and expand the small pool of available promoters for stable insect cell line development and transient plasmid expression in insect cells. The best promoter was used to improve plasmid based transient transfection in insect cells substantially.
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Lacombe, Séverine; Bangratz, Martine; Brizard, Jean-Paul; Petitdidier, Elodie; Pagniez, Julie; Sérémé, Drissa; Lemesre, Jean-Loup; Brugidou, Christophe
2018-01-01
In recent years, plants have been shown to be an efficient alternative expression system for high-value pharmaceuticals such as vaccines. However, constitutive expression of recombinant protein remains uncertain on their level of production and biological activity. To overcome these problems, transitory expression systems have been developed. Here, a series of experiments were performed to determine the most effective conditions to enhance vaccine antigen transient accumulation in Nicotiana benthamiana leaves using the promastigote surface antigen (PSA) from the parasitic protozoan Leishmania infantum. This protein has been previously identified as the major antigen of a licensed canine anti-leishmaniasis vaccine. The classical prokaryote Escherichia coli biosystem failed in accumulating PSA. Consequently, the standard plant system based on N. benthamiana has been optimized for the production of putatively active PSA. First, the RNA silencing defense mechanism set up by the plant against PSA ectopic expression was abolished by using three viral suppressors acting at different steps of the RNA silencing pathway. Then, we demonstrated that the signal peptide at the N-terminal side of the PSA is required for its accumulation. The PSA ER signaling and retention with the PSA signal peptide and the KDEL motif, respectively were optimized to significantly increase its accumulation. Finally, we demonstrate that the production of recombinant PSA in N. benthamiana leaves allows the conservation of its immunogenic property. These approaches demonstrate that based on these optimizations, plant based systems can be used to effectively produce the biological active PSA protein. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
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Biodegradable Bioadherent Microcapsules for Orally Administered Sustained Release Vaccines. Phase 1.
1995-10-23
based on homology with the yeast alcohol oxidase (AOX) or histidine (his 4) loci and also upon the site of linearization of the plasmid since DNA ends...and Quantitation of Recombinant Fasciola Protein from the Pichia Yeast Expression System ...... 9 B. Production of Microcarriers...Recombinant Fasciola Protein from the Pichia Yeast Expression System ..... 15 B. Production of Microcarriers
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Iyer, Sukanya
2013-01-01
Realizing the potential of cell free systems will require development of ligand sensitive gene promoters that control gene expression in response to a ligand of interest. Here, we describe an approach to designing ligand sensitive transcriptional control in cell free systems that is based on the combination of a DNA aptamer that binds thrombin and the T7 bacteriophage promoter. Placement of the aptamer near the T7 promoter, and using a primarily single stranded template, results in up to a five-fold change in gene expression in a ligand concentration dependent manner. We further demonstrate that the sensitivity to thrombin concentration and the fold change in expression can be tuned by altering the position of the aptamer. The results described here pave the way for the use of DNA aptamers to achieve modular regulation of transcription in response to a wide variety of ligands in cell free systems. PMID:24059754
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A novel gene expression-based prognostic scoring system to predict survival in gastric cancer
Wang, Pin; Wang, Yunshan; Hang, Bo; ...
2016-07-11
Analysis of gene expression patterns in gastric cancer (GC) can help to identify a comprehensive panel of gene biomarkers for predicting clinical outcomes and to discover potential new therapeutic targets. Here, a multi-step bioinformatics analytic approach was developed to establish a novel prognostic scoring system for GC. We first identified 276 genes that were robustly differentially expressed between normal and GC tissues, of which, 249 were found to be significantly associated with overall survival (OS) by univariate Cox regression analysis. The biological functions of 249 genes are related to cell cycle, RNA/ncRNA process, acetylation and extracellular matrix organization. A networkmore » was generated for view of the gene expression architecture of 249 genes in 265 GCs. Finally, we applied a canonical discriminant analysis approach to identify a 53-gene signature and a prognostic scoring system was established based on a canonical discriminant function of 53 genes. The prognostic scores strongly predicted patients with GC to have either a poor or good OS. Our study raises the prospect that the practicality of GC patient prognosis can be assessed by this prognostic scoring system.« less
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Walwyn, David Richard; Huddy, Suzanne M; Rybicki, Edward P
2015-01-01
Despite the advantages of plant-based transient expression systems relative to microbial or mammalian cell systems, the commercial production of recombinant proteins using plants has not yet been achieved to any significant extent. One of the challenges has been the lack of published data on the costs of manufacture for products other than biopharmaceuticals. In this study, we report on the techno-economic analysis of the production of a standard commercial enzyme, namely, horseradish peroxidase (HRP), using a transient expression system in Nicotiana benthamiana. Based on the proven plant yield of 240 mg HRP/kg biomass, a biomass productivity of 15-kg biomass/m(2)/year and a process yield of 54 % (mg HRP product/mg HRP in biomass), it is apparent that HRP can be manufactured economically via transient expression in plants in a large-scale facility (>5 kg HRP/year). At this level, the process is competitive versus the existing technology (extraction of the enzyme from horseradish), and the product is of comparable or improved activity, containing only the preferred isoenzyme C. Production scale, protein yield and biomass productivity are found to be the most important determinants of overall viability.
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A novel gene expression-based prognostic scoring system to predict survival in gastric cancer
DOE Office of Scientific and Technical Information (OSTI.GOV)
Wang, Pin; Wang, Yunshan; Hang, Bo
Analysis of gene expression patterns in gastric cancer (GC) can help to identify a comprehensive panel of gene biomarkers for predicting clinical outcomes and to discover potential new therapeutic targets. Here, a multi-step bioinformatics analytic approach was developed to establish a novel prognostic scoring system for GC. We first identified 276 genes that were robustly differentially expressed between normal and GC tissues, of which, 249 were found to be significantly associated with overall survival (OS) by univariate Cox regression analysis. The biological functions of 249 genes are related to cell cycle, RNA/ncRNA process, acetylation and extracellular matrix organization. A networkmore » was generated for view of the gene expression architecture of 249 genes in 265 GCs. Finally, we applied a canonical discriminant analysis approach to identify a 53-gene signature and a prognostic scoring system was established based on a canonical discriminant function of 53 genes. The prognostic scores strongly predicted patients with GC to have either a poor or good OS. Our study raises the prospect that the practicality of GC patient prognosis can be assessed by this prognostic scoring system.« less
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Sweeney, Torres; Lejeune, Alex; Moloney, Aidan P; Monahan, Frank J; Gettigan, Paul Mc; Downey, Gerard; Park, Stephen D E; Ryan, Marion T
2016-09-21
Differences between cattle production systems can influence the nutritional and sensory characteristics of beef, in particular its fatty acid (FA) composition. As beef products derived from pasture-based systems can demand a higher premium from consumers, there is a need to understand the biological characteristics of pasture produced meat and subsequently to develop methods of authentication for these products. Here, we describe an approach to authentication that focuses on differences in the transcriptomic profile of muscle from animals finished in different systems of production of practical relevance to the Irish beef industry. The objectives of this study were to identify a panel of differentially expressed (DE) genes/networks in the muscle of cattle raised outdoors on pasture compared to animals raised indoors on a concentrate based diet and to subsequently identify an optimum panel which can classify the meat based on a production system. A comparison of the muscle transcriptome of outdoor/pasture-fed and Indoor/concentrate-fed cattle resulted in the identification of 26 DE genes. Functional analysis of these genes identified two significant networks (1: Energy Production, Lipid Metabolism, Small Molecule Biochemistry; and 2: Lipid Metabolism, Molecular Transport, Small Molecule Biochemistry), both of which are involved in FA metabolism. The expression of selected up-regulated genes in the outdoor/pasture-fed animals correlated positively with the total n-3 FA content of the muscle. The pathway and network analysis of the DE genes indicate that peroxisome proliferator-activated receptor (PPAR) and FYN/AMPK could be implicit in the regulation of these alterations to the lipid profile. In terms of authentication, the expression profile of three DE genes (ALAD, EIF4EBP1 and NPNT) could almost completely separate the samples based on production system (95 % authentication for animals on pasture-based and 100 % for animals on concentrate- based diet) in this context. The majority of DE genes between muscle of the outdoor/pasture-fed and concentrate-fed cattle were related to lipid metabolism and in particular β-oxidation. In this experiment the combined expression profiles of ALAD, EIF4EBP1 and NPNT were optimal in classifying the muscle transcriptome based on production system. Given the overall lack of comparable studies and variable concordance with those that do exist, the use of transcriptomic data in authenticating production systems requires more exploration across a range of contexts and breeds.
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Shrestha, Prashanta; Smith, Mark Thomas; Bundy, Bradley Charles
2014-01-25
Site-specific incorporation of unnatural amino acids (uAAs) during protein synthesis expands the proteomic code through the addition of unique residue chemistry. This field provides a unique tool to improve pharmacokinetics, cancer treatments, vaccine development, proteomics and protein engineering. The limited ability to predict the characteristics of proteins with uAA-incorporation creates a need for a low-cost system with the potential for rapid screening. Escherichia coli-based cell-free protein synthesis is a compelling platform for uAA incorporation due to the open and accessible nature of the reaction environment. However, typical cell-free systems can be expensive due to the high cost of energizing reagents. By employing alternative energy sources, we reduce the cost of uAA-incorporation in CFPS by 55%. While alternative energy systems reduce cost, the time investment to develop gene libraries can remain cumbersome. Cell-free systems allow the direct use of PCR products known as linear expression templates, thus alleviating tedious plasmid library preparations steps. We report the specific costs of CFPS with uAA incorporation, demonstrate that LETs are suitable expression templates with uAA-incorporation, and consider the substantial reduction in labor intensity using LET-based expression for CFPS uAA incorporation. Copyright © 2013 Elsevier B.V. All rights reserved.
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Wei, Baojun; Wei, Yuxiang; Zhang, Kuo; Yang, Changmei; Wang, Jing; Xu, Ruihuan; Zhan, Sien; Lin, Guigao; Wang, Wei; Liu, Min; Wang, Lunan; Zhang, Rui; Li, Jinming
2008-01-01
To construct a one-plasmid expression system of the armored RNA containing long chimeric RNA by increasing the number and affinity of the pac site. The plasmid pET-MS2-pac was constructed with one C-variant pac site, and then the plasmid pM-CR-2C containing 1,891-bp chimeric sequences and two C-variant pac sites was produced. Meanwhile, three plasmids (pM-CR-C, pM-CR-2W and pM-CR-W) were obtained as parallel controls with a different number and affinity of the pac site. Finally, the armored RNA was expressed and purified. The armored RNA with 1,891 bases target RNA was expressed successfully by the one-plasmid expression system with two C-variant pac sites, while for one pac site, no matter whether the affinity was changed or not, only the 1,200 bases target RNA was packaged. It was also found that the C-variant pac site could increase the expression efficiency of the armored RNA. The armored RNA with 1,891-bp exogenous RNA in our study showed the characterization of ribonuclease resistance and stability at different time points and temperature conditions. The armored RNA with 1,891 bases exogenous RNA was constructed and the expression system can be used as a platform for preparation of the armored RNA containing long RNA sequences. Copyright 2008 S. Karger AG, Basel.
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Meyer, Vera; Wanka, Franziska; van Gent, Janneke; Arentshorst, Mark; van den Hondel, Cees A. M. J. J.; Ram, Arthur F. J.
2011-01-01
Filamentous fungi are the cause of serious human and plant diseases but are also exploited in biotechnology as production platforms. Comparative genomics has documented their genetic diversity, and functional genomics and systems biology approaches are under way to understand the functions and interaction of fungal genes and proteins. In these approaches, gene functions are usually inferred from deletion or overexpression mutants. However, studies at these extreme points give only limited information. Moreover, many overexpression studies use metabolism-dependent promoters, often causing pleiotropic effects and thus limitations in their significance. We therefore established and systematically evaluated a tunable expression system for Aspergillus niger that is independent of carbon and nitrogen metabolism and silent under noninduced conditions. The system consists of two expression modules jointly targeted to a defined genomic locus. One module ensures constitutive expression of the tetracycline-dependent transactivator rtTA2S-M2, and one module harbors the rtTA2S-M2-dependent promoter that controls expression of the gene of interest (the Tet-on system). We show here that the system is tight, responds within minutes after inducer addition, and allows fine-tuning based on the inducer concentration or gene copy number up to expression levels higher than the expression levels of the gpdA promoter. We also validate the Tet-on system for the generation of conditional overexpression mutants and demonstrate its power when combined with a gene deletion approach. Finally, we show that the system is especially suitable when the functions of essential genes must be examined. PMID:21378046
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Pinto, Rita; Hansen, Lars; Hintze, John; Almeida, Raquel; Larsen, Sylvester; Coskun, Mehmet; Davidsen, Johanne; Mitchelmore, Cathy; David, Leonor; Troelsen, Jesper Thorvald; Bennett, Eric Paul
2017-07-27
Tetracycline-based inducible systems provide powerful methods for functional studies where gene expression can be controlled. However, the lack of tight control of the inducible system, leading to leakiness and adverse effects caused by undesirable tetracycline dosage requirements, has proven to be a limitation. Here, we report that the combined use of genome editing tools and last generation Tet-On systems can resolve these issues. Our principle is based on precise integration of inducible transcriptional elements (coined PrIITE) targeted to: (i) exons of an endogenous gene of interest (GOI) and (ii) a safe harbor locus. Using PrIITE cells harboring a GFP reporter or CDX2 transcription factor, we demonstrate discrete inducibility of gene expression with complete abrogation of leakiness. CDX2 PrIITE cells generated by this approach uncovered novel CDX2 downstream effector genes. Our results provide a strategy for characterization of dose-dependent effector functions of essential genes that require absence of endogenous gene expression. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
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Geiling, Benjamin; Vandal, Guillaume; Posner, Ada R.; de Bruyns, Angeline; Dutchak, Kendall L.; Garnett, Samantha; Dankort, David
2013-01-01
The ability to express exogenous cDNAs while suppressing endogenous genes via RNAi represents an extremely powerful research tool with the most efficient non-transient approach being accomplished through stable viral vector integration. Unfortunately, since traditional restriction enzyme based methods for constructing such vectors are sequence dependent, their construction is often difficult and not amenable to mass production. Here we describe a non-sequence dependent Gateway recombination cloning system for the rapid production of novel lentiviral (pLEG) and retroviral (pREG) vectors. Using this system to recombine 3 or 4 modular plasmid components it is possible to generate viral vectors expressing cDNAs with or without inhibitory RNAs (shRNAmirs). In addition, we demonstrate a method to rapidly produce and triage novel shRNAmirs for use with this system. Once strong candidate shRNAmirs have been identified they may be linked together in tandem to knockdown expression of multiple targets simultaneously or to improve the knockdown of a single target. Here we demonstrate that these recombinant vectors are able to express cDNA and effectively knockdown protein expression using both cell culture and animal model systems. PMID:24146852
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Kortmann, Maike; Kuhl, Vanessa; Klaffl, Simon; Bott, Michael
2015-01-01
Corynebacterium glutamicum has become a favourite model organism in white biotechnology. Nevertheless, only few systems for the regulatable (over)expression of homologous and heterologous genes are currently available, all of which are based on the endogenous RNA polymerase. In this study, we developed an isopropyl-β-d-1-thiogalactopyranosid (IPTG)-inducible T7 expression system in the prophage-free strain C. glutamicum MB001. For this purpose, part of the DE3 region of Escherichia coli BL21(DE3) including the T7 RNA polymerase gene 1 under control of the lacUV5 promoter was integrated into the chromosome, resulting in strain MB001(DE3). Furthermore, the expression vector pMKEx2 was constructed allowing cloning of target genes under the control of the T7lac promoter. The properties of the system were evaluated using eyfp as heterologous target gene. Without induction, the system was tightly repressed, resulting in a very low specific eYFP fluorescence (= fluorescence per cell density). After maximal induction with IPTG, the specific fluorescence increased 450-fold compared with the uninduced state and was about 3.5 times higher than in control strains expressing eyfp under control of the IPTG-induced tac promoter with the endogenous RNA polymerase. Flow cytometry revealed that T7-based eyfp expression resulted in a highly uniform population, with 99% of all cells showing high fluorescence. Besides eyfp, the functionality of the corynebacterial T7 expression system was also successfully demonstrated by overexpression of the C. glutamicum pyk gene for pyruvate kinase, which led to an increase of the specific activity from 2.6 to 135 U mg−1. It thus presents an efficient new tool for protein overproduction, metabolic engineering and synthetic biology approaches with C. glutamicum. PMID:25488698
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A plasmid-based reverse genetics system for influenza A virus.
Pleschka, S; Jaskunas, R; Engelhardt, O G; Zürcher, T; Palese, P; García-Sastre, A
1996-01-01
A reverse genetics system for negative-strand RNA viruses was first successfully developed for influenza viruses. This technology involved the transfection of in vitro-reconstituted ribonucleoprotein (RNP) complexes into influenza virus-infected cells. We have now developed a method that allows intracellular reconstitution of RNP complexes from plasmid-based expression vectors. Expression of a viral RNA-like transcript is achieved from a plasmid containing a truncated human polymerase I (polI) promoter and a ribozyme sequence that generates the desired 3' end by autocatalytic cleavage. The polI-driven plasmid is cotransfected into human 293 cells with polII-responsive plasmids that express the viral PB1, PB2, PA, and NP proteins. This exclusively plasmid-driven system results in the efficient transcription and replication of the viral RNA-like reporter and allows the study of cis- and trans-acting signals involved in the transcription and replication of influenza virus RNAs. Using this system, we have also been able to rescue a synthetic neuraminidase gene into a recombinant influenza virus. This method represents a convenient alternative to the previously established RNP transfection system. PMID:8648766
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Zhelyabovskaya, Olga B.; Berlin, Yuri A.; Birikh, Klara R.
2004-01-01
In bacterial expression systems, translation initiation is usually the rate limiting and the least predictable stage of protein synthesis. Efficiency of a translation initiation site can vary dramatically depending on the sequence context. This is why many standard expression vectors provide very poor expression levels of some genes. This notion persuaded us to develop an artificial genetic selection protocol, which allows one to find for a given target gene an individual efficient ribosome binding site from a random pool. In order to create Darwinian pressure necessary for the genetic selection, we designed a system based on translational coupling, in which microorganism survival in the presence of antibiotic depends on expression of the target gene, while putting no special requirements on this gene. Using this system we obtained superproducing constructs for the human protein RACK1 (receptor for activated C kinase). PMID:15034151
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Mevaere, Jimmy; Goulard, Christophe; Schneider, Olha; Sekurova, Olga N; Ma, Haiyan; Zirah, Séverine; Afonso, Carlos; Rebuffat, Sylvie; Zotchev, Sergey B; Li, Yanyan
2018-05-29
Lasso peptides are ribosomally synthesized and post-translationally modified peptides produced by bacteria. They are characterized by an unusual lariat-knot structure. Targeted genome scanning revealed a wide diversity of lasso peptides encoded in actinobacterial genomes, but cloning and heterologous expression of these clusters turned out to be problematic. To circumvent this, we developed an orthogonal expression system for heterologous production of actinobacterial lasso peptides in Streptomyces hosts based on a newly-identified regulatory circuit from Actinoalloteichus fjordicus. Six lasso peptide gene clusters, mainly originating from marine Actinobacteria, were chosen for proof-of-concept studies. By varying the Streptomyces expression hosts and a small set of culture conditions, three new lasso peptides were successfully produced and characterized by tandem MS. The newly developed expression system thus sets the stage to uncover and bioengineer the chemo-diversity of actinobacterial lasso peptides. Moreover, our data provide some considerations for future bioprospecting efforts for such peptides.
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iFER: facial expression recognition using automatically selected geometric eye and eyebrow features
NASA Astrophysics Data System (ADS)
Oztel, Ismail; Yolcu, Gozde; Oz, Cemil; Kazan, Serap; Bunyak, Filiz
2018-03-01
Facial expressions have an important role in interpersonal communications and estimation of emotional states or intentions. Automatic recognition of facial expressions has led to many practical applications and became one of the important topics in computer vision. We present a facial expression recognition system that relies on geometry-based features extracted from eye and eyebrow regions of the face. The proposed system detects keypoints on frontal face images and forms a feature set using geometric relationships among groups of detected keypoints. Obtained feature set is refined and reduced using the sequential forward selection (SFS) algorithm and fed to a support vector machine classifier to recognize five facial expression classes. The proposed system, iFER (eye-eyebrow only facial expression recognition), is robust to lower face occlusions that may be caused by beards, mustaches, scarves, etc. and lower face motion during speech production. Preliminary experiments on benchmark datasets produced promising results outperforming previous facial expression recognition studies using partial face features, and comparable results to studies using whole face information, only slightly lower by ˜ 2.5 % compared to the best whole face facial recognition system while using only ˜ 1 / 3 of the facial region.
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Forest fire autonomous decision system based on fuzzy logic
NASA Astrophysics Data System (ADS)
Lei, Z.; Lu, Jianhua
2010-11-01
The proposed system integrates GPS / pseudolite / IMU and thermal camera in order to autonomously process the graphs by identification, extraction, tracking of forest fire or hot spots. The airborne detection platform, the graph-based algorithms and the signal processing frame are analyzed detailed; especially the rules of the decision function are expressed in terms of fuzzy logic, which is an appropriate method to express imprecise knowledge. The membership function and weights of the rules are fixed through a supervised learning process. The perception system in this paper is based on a network of sensorial stations and central stations. The sensorial stations collect data including infrared and visual images and meteorological information. The central stations exchange data to perform distributed analysis. The experiment results show that working procedure of detection system is reasonable and can accurately output the detection alarm and the computation of infrared oscillations.
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NASA Technical Reports Server (NTRS)
Sitnikova, N.; Volkov, D.; Maximov, I.; Petrusevich, V.; Allen, D.
2003-01-01
This 12-part report documents the data obtained from various sensor measurements taken aboard the Russian Express-A2 and Express-A3 spacecraft in Geosynchronous Earth Orbit (GEO). These GEO communications satellites, which were designed and built by NPO Prikladnoy Mekhaniki (NPO PM) of Zheleznogorsk, Russia, utilize Hall thruster propulsion systems for north-south and east-west stationkeeping and as of June 2002, were still operating at 80 E. and 11 W., respectively. Express-A2 was launched on March 12, 2000, while Express-A3 was launched on June 24, 2000. The diagnostic equipment from which these data were taken includes electric field strength sensors, ion current and energy sensors, and pressure sensors. The diagnostics and the Hall thruster propulsion systems are described in detail along with lists of tabular data from those diagnostics and propulsion system and other satellite systems. Space Power, Inc., now part of Pratt & Whitney's Chemical Systems Division, to the NASA Glenn Research Center, obtained these data over several periods from March 12, 2000, through September 30, 2001. Each of the 12 individual reports describe, in detail, the propulsion systems as well as the diagnostic sensors utilized. Finally, parts 11 and 12 include the requirements to which NPO PM prepared and delivered these data.
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A novel intranuclear RNA vector system for long-term stem cell modification
Ikeda, Yasuhiro; Makino, Akiko; Matchett, William E.; Holditch, Sara J.; Lu, Brian; Dietz, Allan B.; Tomonaga, Keizo
2015-01-01
Genetically modified stem and progenitor cells have emerged as a promising regenerative platform in the treatment of genetic and degenerative disorders, highlighted by their successful therapeutic use in inherent immunodeficiencies. However, biosafety concerns over insertional mutagenesis resulting from integrating recombinant viral vectors have overshadowed the widespread clinical applications of genetically modified stem cells. Here, we report an RNA-based episomal vector system, amenable for long-term transgene expression in stem cells. Specifically, we used a unique intranuclear RNA virus, Borna disease virus (BDV), as the gene transfer vehicle, capable of persistent infections in various cell types. BDV-based vectors allowed for long-term transgene expression in mesenchymal stem cells (MSCs) without affecting cellular morphology, cell surface CD105 expression, or the adipogenicity of MSCs. Similarly, replication-defective BDV vectors achieved long-term transduction of human induced pluripotent stem cells (iPSCs), while maintaining the ability to differentiate into three embryonic germ layers. Thus, the BDV-based vectors offer a genomic modification-free, episomal RNA delivery system for sustained stem cell transduction. PMID:26632671
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A gene expression system offering multiple levels of regulation: the Dual Drug Control (DDC) system.
Sudomoina, Marina; Latypova, Ekaterina; Favorova, Olga O; Golemis, Erica A; Serebriiskii, Ilya G
2004-04-29
Whether for cell culture studies of protein function, construction of mouse models to enable in vivo analysis of disease epidemiology, or ultimately gene therapy of human diseases, a critical enabling step is the ability to achieve finely controlled regulation of gene expression. Previous efforts to achieve this goal have explored inducible drug regulation of gene expression, and construction of synthetic promoters based on two-hybrid paradigms, among others. In this report, we describe the combination of dimerizer-regulated two-hybrid and tetracycline regulatory elements in an ordered cascade, placing expression of endpoint reporters under the control of two distinct drugs. In this Dual Drug Control (DDC) system, a first plasmid expresses fusion proteins to DBD and AD, which interact only in the presence of a small molecule dimerizer; a second plasmid encodes a cassette transcriptionally responsive to the first DBD, directing expression of the Tet-OFF protein; and a third plasmid encodes a reporter gene transcriptionally responsive to binding by Tet-OFF. We evaluate the dynamic range and specificity of this system in comparison to other available systems. This study demonstrates the feasibility of combining two discrete drug-regulated expression systems in a temporally sequential cascade, without loss of dynamic range of signal induction. The efficient layering of control levels allowed by this combination of elements provides the potential for the generation of complex control circuitry that may advance ability to regulate gene expression in vivo.
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NASA Astrophysics Data System (ADS)
Harrison, Tyler; Paproski, Robert J.; Zemp, Roger J.
2012-02-01
Tyrosinase, a key enzyme in the production of melanin, has shown promise as a reporter of genetic activity. While green fluorescent protein has been used extensively in this capacity, it is limited in its ability to provide information deep in tissue at a reasonable resolution. As melanin is a strong absorber of light, it is possible to image gene expression using tyrosinase with photoacoustic imaging technologies, resulting in excellent resolutions at multiple-centimeter depths. While our previous work has focused on creating and imaging MCF-7 cells with doxycycline-controlled tyrosinase expression, we have now established the viability of these cells in a murine model. Using an array-based photoacoustic imaging system with 5 MHz center frequency, we capture interleaved ultrasound and photoacoustic images of tyrosinase-expressing MCF-7 tumors both in a tissue mimicking phantom, and in vivo. Images of both the tyrosinase-expressing tumor and a control tumor are presented as both coregistered ultrasound-photoacoustic B-scan images and 3-dimensional photoacoustic volumes created by mechanically scanning the transducer. We find that the tyrosinase-expressing tumor is visible with a signal level 12dB greater than that of the control tumor in vivo. Phantom studies with excised tumors show that the tyrosinase-expressing tumor is visible at depths in excess of 2cm, and have suggested that our imaging system is sensitive to a transfection rate of less than 1%.
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Marbach, Daniel; Roy, Sushmita; Ay, Ferhat; Meyer, Patrick E.; Candeias, Rogerio; Kahveci, Tamer; Bristow, Christopher A.; Kellis, Manolis
2012-01-01
Gaining insights on gene regulation from large-scale functional data sets is a grand challenge in systems biology. In this article, we develop and apply methods for transcriptional regulatory network inference from diverse functional genomics data sets and demonstrate their value for gene function and gene expression prediction. We formulate the network inference problem in a machine-learning framework and use both supervised and unsupervised methods to predict regulatory edges by integrating transcription factor (TF) binding, evolutionarily conserved sequence motifs, gene expression, and chromatin modification data sets as input features. Applying these methods to Drosophila melanogaster, we predict ∼300,000 regulatory edges in a network of ∼600 TFs and 12,000 target genes. We validate our predictions using known regulatory interactions, gene functional annotations, tissue-specific expression, protein–protein interactions, and three-dimensional maps of chromosome conformation. We use the inferred network to identify putative functions for hundreds of previously uncharacterized genes, including many in nervous system development, which are independently confirmed based on their tissue-specific expression patterns. Last, we use the regulatory network to predict target gene expression levels as a function of TF expression, and find significantly higher predictive power for integrative networks than for motif or ChIP-based networks. Our work reveals the complementarity between physical evidence of regulatory interactions (TF binding, motif conservation) and functional evidence (coordinated expression or chromatin patterns) and demonstrates the power of data integration for network inference and studies of gene regulation at the systems level. PMID:22456606
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Mifepristone-inducible transgene expression in neural progenitor cells in vitro and in vivo
Hjelm, BE; Grunseich, C; Gowing, G; Avalos, P; Tian, J; Shelley, BC; Mooney, M; Narwani, K; Shi, Y; Svendsen, CN; Wolfe, JH; Fischbeck, KH; Pierson, TM
2016-01-01
Numerous gene and cell therapy strategies are being developed for the treatment of neurodegenerative disorders. Many of these strategies use constitutive expression of therapeutic transgenic proteins, and although functional in animal models of disease, this method is less likely to provide adequate flexibility for delivering therapy to humans. Ligand-inducible gene expression systems may be more appropriate for these conditions, especially within the central nervous system (CNS). Mifepristone’s ability to cross the blood–brain barrier makes it an especially attractive ligand for this purpose. We describe the production of a mifepristone-inducible vector system for regulated expression of transgenes within the CNS. Our inducible system used a lentivirus-based vector platform for the ex vivo production of mifepristone-inducible murine neural progenitor cells that express our transgenes of interest. These cells were processed through a series of selection steps to ensure that the cells exhibited appropriate transgene expression in a dose-dependent and temporally controlled manner with minimal background activity. Inducible cells were then transplanted into the brains of rodents, where they exhibited appropriate mifepristone-inducible expression. These studies detail a strategy for regulated expression in the CNS for use in the development of safe and efficient gene therapy for neurological disorders. PMID:26863047
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Thuenemann, Eva C; Meyers, Ann E; Verwey, Jeanette; Rybicki, Edward P; Lomonossoff, George P
2013-09-01
Plant expression systems based on nonreplicating virus-based vectors can be used for the simultaneous expression of multiple genes within the same cell. They therefore have great potential for the production of heteromultimeric protein complexes. This work describes the efficient plant-based production and assembly of Bluetongue virus-like particles (VLPs), requiring the simultaneous expression of four distinct proteins in varying amounts. Such particles have the potential to serve as a safe and effective vaccine against Bluetongue virus (BTV), which causes high mortality rates in ruminants and thus has a severe effect on the livestock trade. Here, VLPs produced and assembled in Nicotiana benthamiana using the cowpea mosaic virus-based HyperTrans (CPMV-HT) and associated pEAQ plant transient expression vector system were shown to elicit a strong antibody response in sheep. Furthermore, they provided protective immunity against a challenge with a South African BTV-8 field isolate. The results show that transient expression can be used to produce immunologically relevant complex heteromultimeric structures in plants in a matter of days. The results have implications beyond the realm of veterinary vaccines and could be applied to the production of VLPs for human use or the coexpression of multiple enzymes for the manipulation of metabolic pathways. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.
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A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants[OPEN
Gil-Humanes, Javier; Čegan, Radim; Kono, Thomas J.Y.; Konečná, Eva; Belanto, Joseph J.; Starker, Colby G.
2017-01-01
We report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches. Our reagents, based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression. Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions. Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination. Regulation of transcription is also possible. A Web-based tool streamlines vector selection and construction. One advantage of our platform is the use of the Csy-type (CRISPR system yersinia) ribonuclease 4 (Csy4) and tRNA processing enzymes to simultaneously express multiple guide RNAs (gRNAs). For example, we demonstrate targeted deletions in up to six genes by expressing 12 gRNAs from a single transcript. Csy4 and tRNA expression systems are almost twice as effective in inducing mutations as gRNAs expressed from individual RNA polymerase III promoters. Mutagenesis can be further enhanced 2.5-fold by incorporating the Trex2 exonuclease. Finally, we demonstrate that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons. The reagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Medicago truncatula, wheat (Triticum aestivum), and barley (Hordeum vulgare). PMID:28522548
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A multi-purpose toolkit to enable advanced genome engineering in plants
Cermak, Tomas; Curtin, Shaun J.; Gil-Humanes, Javier; ...
2017-05-18
Here, we report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches. Our reagents, based on Transcription Activator-Like Effector Nucleases TALENs and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression. Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions. Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination. Regulation of transcription is also possible. A web-based tool streamlines vector selection and construction. One advantagemore » of our platform is the use of the Csy-type (CRISPR system yersinia) ribonuclease 4 Csy4 and tRNA processing enzymes to simultaneously express multiple guide RNAs (gRNAs). For example, we demonstrate targeted deletions in up to six genes by expressing twelve gRNAs from a single transcript. Csy4 and tRNA expression systems are almost twice as effective in inducing mutations as gRNAs expressed from individual RNA polymerase III promoters. Mutagenesis can be further enhanced 2.5-fold by incorporating the Trex2 exonuclease. Finally, we demonstrate that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons. The reagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Medicago truncatula, wheat (Triticum aestivum), and barley (Hordeum vulgare).« less
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A multi-purpose toolkit to enable advanced genome engineering in plants
DOE Office of Scientific and Technical Information (OSTI.GOV)
Cermak, Tomas; Curtin, Shaun J.; Gil-Humanes, Javier
Here, we report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches. Our reagents, based on Transcription Activator-Like Effector Nucleases TALENs and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression. Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions. Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination. Regulation of transcription is also possible. A web-based tool streamlines vector selection and construction. One advantagemore » of our platform is the use of the Csy-type (CRISPR system yersinia) ribonuclease 4 Csy4 and tRNA processing enzymes to simultaneously express multiple guide RNAs (gRNAs). For example, we demonstrate targeted deletions in up to six genes by expressing twelve gRNAs from a single transcript. Csy4 and tRNA expression systems are almost twice as effective in inducing mutations as gRNAs expressed from individual RNA polymerase III promoters. Mutagenesis can be further enhanced 2.5-fold by incorporating the Trex2 exonuclease. Finally, we demonstrate that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons. The reagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Medicago truncatula, wheat (Triticum aestivum), and barley (Hordeum vulgare).« less
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Pathway-Based Concentration Response Profiles from Toxicogenomics Data
Microarray analysis of gene expression of in vitro systems could be a powerful tool for assessing chemical hazard. Differentially expressed genes specific to cells, chemicals, and concentrations can be organized into molecular pathways that inform mode of action. An important par...
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Improvement of emotional healthcare system with stress detection from ECG signal.
Tivatansakul, S; Ohkura, M
2015-01-01
Our emotional healthcare system is designed to cope with users' negative emotions in daily life. To make the system more intelligent, we integrated emotion recognition by facial expression to provide appropriate services based on user's current emotional state. Our emotion recognition by facial expression has confusion issue to recognize some positive, neutral and negative emotions that make the emotional healthcare system provide a relaxation service even though users don't have negative emotions. Therefore, to increase the effectiveness of the system to provide the relaxation service, we integrate stress detection from ECG signal. The stress detection might be able to address the confusion issue of emotion recognition by facial expression to provide the service. Indeed, our results show that integration of stress detection increases the effectiveness and efficiency of the emotional healthcare system to provide services.
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Retrovirus-based vectors for transient and permanent cell modification.
Schott, Juliane W; Hoffmann, Dirk; Schambach, Axel
2015-10-01
Retroviral vectors are commonly employed for long-term transgene expression via integrating vector technology. However, three alternative retrovirus-based platforms are currently available that allow transient cell modification. Gene expression can be mediated from either episomal DNA or RNA templates, or selected proteins can be directly transferred through retroviral nanoparticles. The different technologies are functionally graded with respect to safety, expression magnitude and expression duration. Improvement of the initial technologies, including modification of vector designs, targeted increase in expression strength and duration as well as improved safety characteristics, has allowed maturation of retroviral systems into efficient and promising tools that meet the technological demands of a wide variety of potential application areas. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Propagation of flat-topped multi-Gaussian beams through a double-lens system with apertures.
Gao, Yanqi; Zhu, Baoqiang; Liu, Daizhong; Lin, Zunqi
2009-07-20
A general model for different apertures and flat-topped laser beams based on the multi-Gaussian function is developed. The general analytical expression for the propagation of a flat-topped beam through a general double-lens system with apertures is derived using the above model. Then, the propagation characteristics of the flat-topped beam through a spatial filter are investigated by using a simplified analytical expression. Based on the Fluence beam contrast and the Fill factor, the influences of a pinhole size on the propagation of the flat-topped multi-Gaussian beam (FMGB) through the spatial filter are illustrated. An analytical expression for the propagation of the FMGB through the spatial filter with a misaligned pinhole is presented, and the influences of the pinhole offset are evaluated.
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Halbmayr, Elisabeth; Mathiesen, Geir; Nguyen, Thu-Ha; Maischberger, Thomas; Peterbauer, Clemens K; Eijsink, Vincent G H; Haltrich, Dietmar
2008-06-25
This work presents the cloning and expression of the genes encoding heterodimeric beta-galactosidases from Lactobacillus reuteri L103, Lactobacillus acidophilus R22, Lactobacillus plantarum WCFS1, and Lactobacillus sakei Lb790. These enzymes consist of two subunits of approximately 73 and 35 kDa, which are encoded by two overlapping genes, lacL and lacM, respectively. We have cloned these genes into the lactobacillal expression vectors pSIP403 and pSIP409, which are based on the sakacin P operon of L. sakei ( Sørvig et al. Microbiology 2005, 151, 2439- 2449 ), and expressed them in the host strains L. plantarum WCFS1 and L. sakei Lb790. Results varied considerably, ranging from 2.23 to 61.1 U/mg of beta-galactosidase activity, depending on the origin of the lacLM genes, the host strain, and the expression vector used. Highest expression levels were obtained in a laboratory cultivation of L. plantarum WCFS1 harboring the plasmid pEH3R containing the lacLM gene from L. reuteri L103. These cultivations yielded approximately 23 000 U of beta-galactosidase activity per liter, corresponding to the formation of roughly 100 mg of recombinant protein per liter of fermentation medium, and beta-galactosidase levels amounted to 55% of the total intracellular protein of the host organism. To further verify the suitability of this expression system, recombinant beta-galactosidase from L. reuteri was purified to apparent homogeneity. The properties of the purified enzyme were essentially identical with the properties of purified native beta-galactosidase from L. reuteri L103. The presented results lead the way to efficient overproduction of beta-galactosidase in a food-grade expression system, which is of high interest for applications in food industry.
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Towards Symbolic Model Checking for Multi-Agent Systems via OBDDs
NASA Technical Reports Server (NTRS)
Raimondi, Franco; Lomunscio, Alessio
2004-01-01
We present an algorithm for model checking temporal-epistemic properties of multi-agent systems, expressed in the formalism of interpreted systems. We first introduce a technique for the translation of interpreted systems into boolean formulae, and then present a model-checking algorithm based on this translation. The algorithm is based on OBDD's, as they offer a compact and efficient representation for boolean formulae.
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Chen, X; Li, Y; Xiong, K; Wagner, T E
1994-01-01
A novel cytoplasmic gene expression system has been developed. This system differs from other expression systems in that it relies on the co-delivery of plasmid DNA and T7 RNA polymerase (RNAP) during transfection. The plasmid contains a T7 RNAP gene driven by the T7 promoter (T7 autogene) and a functional/reporter gene driven by another T7 promoter (T7T7/T7-gene construct). Once this DNA-enzyme complex is introduced into eukaryotic cells, the transcription of the T7 RNAP and the functional/reporter genes is initiated by the co-delivered T7 RNAP. The T7 RNAP, which is responsible for the initiation and maintenance of expression of both T7 and functional/reporter genes, is replenished by translation of newly synthesized T7 mRNA. This T7 system was designed in such a manner that the expression of the functional/reporter genes can occur in the cytoplasm and does not require any nuclear involvement. When transfected by either a pT7T7/T7Luc or a pT7T7/T7hGH plasmids with the cointroduced T7 RNAP, mouse L cells were found to express high levels of luciferase immediately after transfection, apparently due to the cytoplasmic gene expression; the expression of human growth hormone (hGH) could be sustained for at least 6 days. Both T7 and hGH mRNA were expressed by the cells transfected with pT7T7/T7hGH. These results suggest that this cytoplasmic expression system may be used for certain targets of somatic gene therapy. Images PMID:8029020
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An interval logic for higher-level temporal reasoning
NASA Technical Reports Server (NTRS)
Schwartz, R. L.; Melliar-Smith, P. M.; Vogt, F. H.; Plaisted, D. A.
1983-01-01
Prior work explored temporal logics, based on classical modal logics, as a framework for specifying and reasoning about concurrent programs, distributed systems, and communications protocols, and reported on efforts using temporal reasoning primitives to express very high level abstract requirements that a program or system is to satisfy. Based on experience with those primitives, this report describes an Interval Logic that is more suitable for expressing such higher level temporal properties. The report provides a formal semantics for the Interval Logic, and several examples of its use. A description of decision procedures for the logic is also included.
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Image ratio features for facial expression recognition application.
Song, Mingli; Tao, Dacheng; Liu, Zicheng; Li, Xuelong; Zhou, Mengchu
2010-06-01
Video-based facial expression recognition is a challenging problem in computer vision and human-computer interaction. To target this problem, texture features have been extracted and widely used, because they can capture image intensity changes raised by skin deformation. However, existing texture features encounter problems with albedo and lighting variations. To solve both problems, we propose a new texture feature called image ratio features. Compared with previously proposed texture features, e.g., high gradient component features, image ratio features are more robust to albedo and lighting variations. In addition, to further improve facial expression recognition accuracy based on image ratio features, we combine image ratio features with facial animation parameters (FAPs), which describe the geometric motions of facial feature points. The performance evaluation is based on the Carnegie Mellon University Cohn-Kanade database, our own database, and the Japanese Female Facial Expression database. Experimental results show that the proposed image ratio feature is more robust to albedo and lighting variations, and the combination of image ratio features and FAPs outperforms each feature alone. In addition, we study asymmetric facial expressions based on our own facial expression database and demonstrate the superior performance of our combined expression recognition system.
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A Natural Language Interface Concordant with a Knowledge Base.
Han, Yong-Jin; Park, Seong-Bae; Park, Se-Young
2016-01-01
The discordance between expressions interpretable by a natural language interface (NLI) system and those answerable by a knowledge base is a critical problem in the field of NLIs. In order to solve this discordance problem, this paper proposes a method to translate natural language questions into formal queries that can be generated from a graph-based knowledge base. The proposed method considers a subgraph of a knowledge base as a formal query. Thus, all formal queries corresponding to a concept or a predicate in the knowledge base can be generated prior to query time and all possible natural language expressions corresponding to each formal query can also be collected in advance. A natural language expression has a one-to-one mapping with a formal query. Hence, a natural language question is translated into a formal query by matching the question with the most appropriate natural language expression. If the confidence of this matching is not sufficiently high the proposed method rejects the question and does not answer it. Multipredicate queries are processed by regarding them as a set of collected expressions. The experimental results show that the proposed method thoroughly handles answerable questions from the knowledge base and rejects unanswerable ones effectively.
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Molecular Beacon-Based MicroRNA Imaging During Neurogenesis.
Lee, Jonghwan; Kim, Soonhag
2016-01-01
The fluorescence monitoring system for examining endogenous microRNA (miRNA) activity in cellular level provides crucial information on not only understanding a critical role of miRNA involving a variety of biological processes, but also evaluating miRNA expression patterns in a noninvasive manner. In this protocol, we report the details of a new procedure for a molecular beacon-based miRNA monitoring system, which includes the illustration scheme for miRNA detection strategy, exogenous miRNA detection, and measurement of endogenous miRNA expression level during neurogenesis. The fluorescence signal of miR-124a beacon quenched by BHQ2 was gradually recovered as increasing concentration of the miR-124a in tube. The functional work of miR-124a beacon was examined in intracellular environment, allowing for the internalization of the miR-124a beacon by lipofectamine, which resulted in activated fluorescent signals of the miR-124a beacon in the HeLa cells after the addition of synthetic miR-124a. The endogenous miR-124a expression level was detected by miR-124a beacon system during neurogenesis, showing brighter fluorescence intensity in cytoplasmic area of P19 cells after induction of neuronal differentiation by retinoic acid. The molecular beacon based-miRNA detection technique could be applicable to the simultaneous visualization of a variety of miRNA expression patterns using different fluorescence dyes. For the study of examining endogenous miRNA expression level using miRNA-beacon system, if cellular differentiation step is already prepared, transfection step of miR-124a beacon into P19 cells, and acquisition of activated fluorescence signal measured by confocal microscope can be conducted approximately within 6 h.
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Bekele, E; Bian, D; Peterman, J; Park, S; Sarkar, N
2017-06-01
Schizophrenia is a life-long, debilitating psychotic disorder with poor outcome that affects about 1% of the population. Although pharmacotherapy can alleviate some of the acute psychotic symptoms, residual social impairments present a significant barrier that prevents successful rehabilitation. With limited resources and access to social skills training opportunities, innovative technology has emerged as a potentially powerful tool for intervention. In this paper, we present a novel virtual reality (VR)-based system for understanding facial emotion processing impairments that may lead to poor social outcome in schizophrenia. We henceforth call it a VR System for Affect Analysis in Facial Expressions (VR-SAAFE). This system integrates a VR-based task presentation platform that can minutely control facial expressions of an avatar with or without accompanying verbal interaction, with an eye-tracker to quantitatively measure a participants real-time gaze and a set of physiological sensors to infer his/her affective states to allow in-depth understanding of the emotion recognition mechanism of patients with schizophrenia based on quantitative metrics. A usability study with 12 patients with schizophrenia and 12 healthy controls was conducted to examine processing of the emotional faces. Preliminary results indicated that there were significant differences in the way patients with schizophrenia processed and responded towards the emotional faces presented in the VR environment compared with healthy control participants. The preliminary results underscore the utility of such a VR-based system that enables precise and quantitative assessment of social skill deficits in patients with schizophrenia.
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Possibilities of identifying cyber attack in noisy space of n-dimensional abstract system
NASA Astrophysics Data System (ADS)
Jašek, Roman; Dvořák, Jiří; Janková, Martina; Sedláček, Michal
2016-06-01
This article briefly mentions some selected options of current concept for identifying cyber attacks from the perspective of the new cyberspace of real system. In the cyberspace, there is defined n-dimensional abstract system containing elements of the spatial arrangement of partial system elements such as micro-environment of cyber systems surrounded by other suitably arranged corresponding noise space. This space is also gradually supplemented by a new image of dynamic processes in a discreet environment, and corresponding again to n-dimensional expression of time space defining existence and also the prediction for expected cyber attacksin the noise space. Noises are seen here as useful and necessary for modern information and communication technologies (e.g. in processes of applied cryptography in ICT) and then the so-called useless noises designed for initial (necessary) filtering of this highly aggressive environment and in future expectedly offensive background in cyber war (e.g. the destruction of unmanned means of an electromagnetic pulse, or for destruction of new safety barriers created on principles of electrostatic field or on other principles of modern physics, etc.). The key to these new options is the expression of abstract systems based on the models of microelements of cyber systems and their hierarchical concept in structure of n-dimensional system in given cyberspace. The aim of this article is to highlight the possible systemic expression of cyberspace of abstract system and possible identification in time-spatial expression of real environment (on microelements of cyber systems and their surroundings with noise characteristics and time dimension in dynamic of microelements' own time and externaltime defined by real environment). The article was based on a partial task of faculty specific research.
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Possibilities of identifying cyber attack in noisy space of n-dimensional abstract system
DOE Office of Scientific and Technical Information (OSTI.GOV)
Jašek, Roman; Dvořák, Jiří; Janková, Martina
This article briefly mentions some selected options of current concept for identifying cyber attacks from the perspective of the new cyberspace of real system. In the cyberspace, there is defined n-dimensional abstract system containing elements of the spatial arrangement of partial system elements such as micro-environment of cyber systems surrounded by other suitably arranged corresponding noise space. This space is also gradually supplemented by a new image of dynamic processes in a discreet environment, and corresponding again to n-dimensional expression of time space defining existence and also the prediction for expected cyber attacksin the noise space. Noises are seen heremore » as useful and necessary for modern information and communication technologies (e.g. in processes of applied cryptography in ICT) and then the so-called useless noises designed for initial (necessary) filtering of this highly aggressive environment and in future expectedly offensive background in cyber war (e.g. the destruction of unmanned means of an electromagnetic pulse, or for destruction of new safety barriers created on principles of electrostatic field or on other principles of modern physics, etc.). The key to these new options is the expression of abstract systems based on the models of microelements of cyber systems and their hierarchical concept in structure of n-dimensional system in given cyberspace. The aim of this article is to highlight the possible systemic expression of cyberspace of abstract system and possible identification in time-spatial expression of real environment (on microelements of cyber systems and their surroundings with noise characteristics and time dimension in dynamic of microelements’ own time and externaltime defined by real environment). The article was based on a partial task of faculty specific research.« less
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Vrahatis, Aristidis G; Dimitrakopoulos, Georgios N; Tsakalidis, Athanasios K; Bezerianos, Anastasios
2015-01-01
In the road for network medicine the newly emerged systems-level subpathway-based analysis methods offer new disease genes, drug targets and network-based biomarkers. In parallel, paired miRNA/mRNA expression data enable simultaneously monitoring of the micronome effect upon the signaling pathways. Towards this orientation, we present a methodological pipeline for the identification of differentially expressed subpathways along with their miRNA regulators by using KEGG signaling pathway maps, miRNA-target interactions and expression profiles from paired miRNA/mRNA experiments. Our pipeline offered new biological insights on a real application of paired miRNA/mRNA expression profiles with respect to the dynamic changes from colostrum to mature milk whey; several literature supported genes and miRNAs were recontextualized through miRNA-mediated differentially expressed subpathways.
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Ran, Tao; Liu, Yong; Li, Hengzhi; Tang, Shaoxun; He, Zhixiong; Munteanu, Cristian R; González-Díaz, Humberto; Tan, Zhiliang; Zhou, Chuanshe
2016-07-27
The management of ruminant growth yield has economic importance. The current work presents a study of the spatiotemporal dynamic expression of Ghrelin and GHR at mRNA levels throughout the gastrointestinal tract (GIT) of kid goats under housing and grazing systems. The experiments show that the feeding system and age affected the expression of either Ghrelin or GHR with different mechanisms. Furthermore, the experimental data are used to build new Machine Learning models based on the Perturbation Theory, which can predict the effects of perturbations of Ghrelin and GHR mRNA expression on the growth yield. The models consider eight longitudinal GIT segments (rumen, abomasum, duodenum, jejunum, ileum, cecum, colon and rectum), seven time points (0, 7, 14, 28, 42, 56 and 70 d) and two feeding systems (Supplemental and Grazing feeding) as perturbations from the expected values of the growth yield. The best regression model was obtained using Random Forest, with the coefficient of determination R(2) of 0.781 for the test subset. The current results indicate that the non-linear regression model can accurately predict the growth yield and the key nodes during gastrointestinal development, which is helpful to optimize the feeding management strategies in ruminant production system.
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Ran, Tao; Liu, Yong; Li, Hengzhi; Tang, Shaoxun; He, Zhixiong; Munteanu, Cristian R.; González-Díaz, Humberto; Tan, Zhiliang; Zhou, Chuanshe
2016-01-01
The management of ruminant growth yield has economic importance. The current work presents a study of the spatiotemporal dynamic expression of Ghrelin and GHR at mRNA levels throughout the gastrointestinal tract (GIT) of kid goats under housing and grazing systems. The experiments show that the feeding system and age affected the expression of either Ghrelin or GHR with different mechanisms. Furthermore, the experimental data are used to build new Machine Learning models based on the Perturbation Theory, which can predict the effects of perturbations of Ghrelin and GHR mRNA expression on the growth yield. The models consider eight longitudinal GIT segments (rumen, abomasum, duodenum, jejunum, ileum, cecum, colon and rectum), seven time points (0, 7, 14, 28, 42, 56 and 70 d) and two feeding systems (Supplemental and Grazing feeding) as perturbations from the expected values of the growth yield. The best regression model was obtained using Random Forest, with the coefficient of determination R2 of 0.781 for the test subset. The current results indicate that the non-linear regression model can accurately predict the growth yield and the key nodes during gastrointestinal development, which is helpful to optimize the feeding management strategies in ruminant production system. PMID:27460882
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Du, Lijuan; Zhou, Amy; Patel, Akshay; Rao, Mishal; Anderson, Kelsey; Roy, Sougata
2017-07-01
Fibroblast growth factors (FGF) are essential signaling proteins that regulate diverse cellular functions in developmental and metabolic processes. In Drosophila, the FGF homolog, branchless (bnl) is expressed in a dynamic and spatiotemporally restricted pattern to induce branching morphogenesis of the trachea, which expresses the Bnl-receptor, breathless (btl). Here we have developed a new strategy to determine bnl- expressing cells and study their interactions with the btl-expressing cells in the range of tissue patterning during Drosophila development. To enable targeted gene expression specifically in the bnl expressing cells, a new LexA based bnl enhancer trap line was generated using CRISPR/Cas9 based genome editing. Analyses of the spatiotemporal expression of the reporter in various embryonic stages, larval or adult tissues and in metabolic hypoxia, confirmed its target specificity and versatility. With this tool, new bnl expressing cells, their unique organization and functional interactions with the btl-expressing cells were uncovered in a larval tracheoblast niche in the leg imaginal discs, in larval photoreceptors of the developing retina, and in the embryonic central nervous system. The targeted expression system also facilitated live imaging of simultaneously labeled Bnl sources and tracheal cells, which revealed a unique morphogenetic movement of the embryonic bnl- source. Migration of bnl- expressing cells may create a dynamic spatiotemporal pattern of the signal source necessary for the directional growth of the tracheal branch. The genetic tool and the comprehensive profile of expression, organization, and activity of various types of bnl-expressing cells described in this study provided us with an important foundation for future research investigating the mechanisms underlying Bnl signaling in tissue morphogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.
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Cooperating reduction machines
DOE Office of Scientific and Technical Information (OSTI.GOV)
Kluge, W.E.
1983-11-01
This paper presents a concept and a system architecture for the concurrent execution of program expressions of a concrete reduction language based on lamda-expressions. If formulated appropriately, these expressions are well-suited for concurrent execution, following a demand-driven model of computation. In particular, recursive program expressions with nonlinear expansion may, at run time, recursively be partitioned into a hierarchy of independent subexpressions which can be reduced by a corresponding hierarchy of virtual reduction machines. This hierarchy unfolds and collapses dynamically, with virtual machines recursively assuming the role of masters that create and eventually terminate, or synchronize with, slaves. The paper alsomore » proposes a nonhierarchically organized system of reduction machines, each featuring a stack architecture, that effectively supports the allocation of virtual machines to the real machines of the system in compliance with their hierarchical order of creation and termination. 25 references.« less
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Omura, Natsumi; Yoshikawa, Tomoki; Fujii, Hikaru; Shibamura, Miho; Inagaki, Takuya; Kato, Hirofumi; Egawa, Kazutaka; Harada, Shizuko; Yamada, Souichi; Takeyama, Haruko; Saijo, Masayuki
2018-04-27
A novel system was developed for generating a highly-attenuated vaccinia virus LC16m8 (m8, third generation smallpox vaccine) that expresses foreign genes. The innovations in this system are its excisable selection marker, specificity of the integration site of a gene of interest, and easy identification of clones with the fluorescent signal. Using this system, recombinant m8s, which expressed either herpes simplex virus 2 (HSV-2) glycoprotein B (gB)-, gD-, or both gB and gD (gB+gD) were developed, and their efficacy was evaluated. First, the induction of a specific IgG against these HSV-2 glycoproteins in mice infected with each of these recombinant m8s was confirmed with an immunofluorescence assay. Next, mice pre-infected with each of the recombinant m8s were infected with HSV-2 at the lethal dose to examine the vaccine efficacy. The fatality rate in mice pre-infected with either of the recombinant gB+gD- or gD-expressing m8s significantly decreased in comparison with that of the control. The survival rate in both male and female mice pre-infected with either of the recombinant gB+gD- and gD-expressing m8s increased to 100 % and 60 %, respectively, while most of the control mice died. In summary, this new system might be applicable for generating a novel m8-based vaccine.
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A View of the Therapy for Bell's Palsy Based on Molecular Biological Analyses of Facial Muscles.
Moriyama, Hiroshi; Mitsukawa, Nobuyuki; Itoh, Masahiro; Otsuka, Naruhito
2017-12-01
Details regarding the molecular biological features of Bell's palsy have not been widely reported in textbooks. We genetically analyzed facial muscles and clarified these points. We performed genetic analysis of facial muscle specimens from Japanese patients with severe (House-Brackmann facial nerve grading system V) and moderate (House-Brackmann facial nerve grading system III) dysfunction due to Bell's palsy. Microarray analysis of gene expression was performed using specimens from the healthy and affected sides, and gene expression was compared. Changes in gene expression were defined as an affected side/healthy side ratio of >1.5 or <0.5. We observed that the gene expression in Bell's palsy changes with the degree of facial nerve palsy. Especially, muscle, neuron, and energy category genes tended to fluctuate with the degree of facial nerve palsy. It is expected that this study will aid in the development of new treatments and diagnostic/prognostic markers based on the severity of facial nerve palsy.
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Zhang, Fan; Liu, Runsheng; Zheng, Jie
2016-12-23
Linking computational models of signaling pathways to predicted cellular responses such as gene expression regulation is a major challenge in computational systems biology. In this work, we present Sig2GRN, a Cytoscape plugin that is able to simulate time-course gene expression data given the user-defined external stimuli to the signaling pathways. A generalized logical model is used in modeling the upstream signaling pathways. Then a Boolean model and a thermodynamics-based model are employed to predict the downstream changes in gene expression based on the simulated dynamics of transcription factors in signaling pathways. Our empirical case studies show that the simulation of Sig2GRN can predict changes in gene expression patterns induced by DNA damage signals and drug treatments. As a software tool for modeling cellular dynamics, Sig2GRN can facilitate studies in systems biology by hypotheses generation and wet-lab experimental design. http://histone.scse.ntu.edu.sg/Sig2GRN/.
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GCaMP expression in retinal ganglion cells characterized using a low-cost fundus imaging system
NASA Astrophysics Data System (ADS)
Chang, Yao-Chuan; Walston, Steven T.; Chow, Robert H.; Weiland, James D.
2017-10-01
Objective. Virus-transduced, intracellular-calcium indicators are effective reporters of neural activity, offering the advantage of cell-specific labeling. Due to the existence of an optimal time window for the expression of calcium indicators, a suitable tool for tracking GECI expression in vivo following transduction is highly desirable. Approach. We developed a noninvasive imaging approach based on a custom-modified, low-cost fundus viewing system that allowed us to monitor and characterize in vivo bright-field and fluorescence images of the mouse retina. AAV2-CAG-GCaMP6f was injected into a mouse eye. The fundus imaging system was used to measure fluorescence at several time points post injection. At defined time points, we prepared wholemount retina mounted on a transparent multielectrode array and used calcium imaging to evaluate the responsiveness of retinal ganglion cells (RGCs) to external electrical stimulation. Main results. The noninvasive fundus imaging system clearly resolves individual (RGCs and axons. RGC fluorescence intensity and the number of observable fluorescent cells show a similar rising trend from week 1 to week 3 after viral injection, indicating a consistent increase of GCaMP6f expression. Analysis of the in vivo fluorescence intensity trend and in vitro neurophysiological responsiveness shows that the slope of intensity versus days post injection can be used to estimate the optimal time for calcium imaging of RGCs in response to external electrical stimulation. Significance. The proposed fundus imaging system enables high-resolution digital fundus imaging in the mouse eye, based on off-the-shelf components. The long-term tracking experiment with in vitro calcium imaging validation demonstrates the system can serve as a powerful tool monitoring the level of genetically-encoded calcium indicator expression, further determining the optimal time window for following experiment.
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Recombinational Cloning Using Gateway and In-Fusion Cloning Schemes
Throop, Andrea L.; LaBaer, Joshua
2015-01-01
The comprehensive study of protein structure and function, or proteomics, depends on the obtainability of full-length cDNAs in species-specific expression vectors and subsequent functional analysis of the expressed protein. Recombinational cloning is a universal cloning technique based on site-specific recombination that is independent of the insert DNA sequence of interest, which differentiates this method from the classical restriction enzyme-based cloning methods. Recombinational cloning enables rapid and efficient parallel transfer of DNA inserts into multiple expression systems. This unit summarizes strategies for generating expression-ready clones using the most popular recombinational cloning technologies, including the commercially available Gateway® (Life Technologies) and In-Fusion® (Clontech) cloning technologies. PMID:25827088
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NASA Technical Reports Server (NTRS)
Sitnikova, N.; Volkov, D.; Maximov, I.; Petrusevich, V.; Allen, D.
2003-01-01
This 12-part report documents the data obtained from various sensor measurements taken aboard the Russian Express-A2 and Express-A3 spacecraft in Geosynchronous Earth Orbit (GEO). These GEO communications satellites, which were designed and built by NPO Prikladnoy Mekhaniki (NPO PM) of Zheleznogorsk, Russia, utilize Hall thruster propulsion systems for north-south and east-west stationkeeping and as of June 2002, were still operating at 80 E. and 11 W., respectively. Express-A2 was launched on March 12, 2000, while Express-A3 was launched on June 24, 2000. The diagnostic equipment from which these data were taken includes electric field strength sensors, ion current and energy sensors, and pressure sensors. The diagnostics and the Hall thruster propulsion systems are described in detail along with lists of tabular data from those diagnostics and propulsion system and other satellite systems. Space Power, Inc., now part of Pratt & Whitney s Chemical Systems Division, under contract NAS3 99151 to the NASA Glenn Research Center, obtained these data over several periods from March 12, 2000, through September 30, 2001. Each of the 12 individual reports describe, in detail, the propulsion systems as well as the diagnostic sensors utilized. Finally, parts 11 and 12 include the requirements to which NPO PM prepared and delivered these data.
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NASA Technical Reports Server (NTRS)
Sitnikova, N.; Volkov, D.; Maximov, I.; Petrusevich, V.; Allen, D.
2003-01-01
This 12-part report documents the data obtained from various sensor measurements taken aboard the Russian Express-A2 and Express-A3 spacecraft in Geosynchronous Earth Orbit (GEO). These GEO communications satellites, which were designed and built by NPO Prikladnoy Mekhaniki (NPO PM) of Zheleznogorsk, Russia, utilize Hall thruster propulsion systems for north-south and east-west stationkeeping and as of June 2002, were still operating at 80deg E. and 11deg W., respectively. Express-A2 was launched on March 12, 2000, while Express-A3 was launched on June 24, 2000. The diagnostic equipment from which these data were taken includes electric field strength sensors, ion current and energy sensors, and pressure sensors. The diagnostics and the Hall thruster propulsion systems are described in detail along with lists of tabular data from those diagnostics and propulsion system and other satellite systems. Space Power, Inc., now part of Pratt & Whitney's Chemical Systems Division, under contract NAS3-99151 to the NASA Glenn Research Center, obtained these data over several periods from March 12, 2000, through September 30, 2001. Each of the 12 individual reports describe, in detail, the propulsion systems as well as the diagnostic sensors utilized. Finally, parts 11 and 12 include the requirements to which NPO PM prepared and delivered these data.
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NASA Technical Reports Server (NTRS)
Sitnikova, N.; Volkov, D.; Maximov, I.; Petrusevich, V.; Allen, D.
2003-01-01
This 12-part report documents the data obtained from various sensor measurements taken aboard the Russian Express-A2 and Express-A3 spacecraft in Geosynchronous Earth Orbit (GEO). These GEO communications satellites, which were designed and built by NPO Prikladnoy Mekhaniki (NPO PM) of Zheleznogorsk, Russia, utilize Hall thruster propulsion systems for north-south and east-west stationkeeping and as of June 2002, were still operating at 80 deg E and 11 deg W, respectively. Express-A2 was launched on March 12, 2000, while Express-A3 was launched on June 24, 2000. The diagnostic equipment from which these data were taken includes electric field strength sensors, ion current and energy sensors, and pressure sensors. The diagnostics and the Hall thruster propulsion systems are described in detail along with lists of tabular data from those diagnostics and propulsion system and other satellite systems. Space Power, Inc., now part of Pratt & Whitney's Chemical Systems Division, under contract NAS3-99151 to the NASA Glenn Research Center, obtained these data over several periods from March 12, 2000, through September 30, 2001. Each of the 12 individual reports describe, in detail, the propulsion systems as well as the diagnostic sensors utilized. Finally, parts 11 and 12 include the requirements to which NPO PM prepared and delivered these data.
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Hong, Haifa; Ye, Lincai; Chen, Huiwen; Xia, Yu; Liu, Yue; Liu, Jinfen; Lu, Yanan; Zhang, Haibo
2015-08-01
We aimed to evaluate global changes in protein expression associated with patency by undertaking proteomic analysis of human constricted and patent ductus arteriosus (DA). Ten constricted and 10 patent human DAs were excised from infants with ductal-dependent heart disease during surgery. Using isobaric tags for relative and absolute quantitation-based quantitative proteomics, 132 differentially expressed proteins were identified. Of 132 proteins, voltage-gated sodium channel 1.3 (SCN3A), myosin 1d (Myo1d), Rho GTPase activating protein 26 (ARHGAP26), and retinitis pigmentosa 1 (RP1) were selected for validation by Western blot and quantitative real-time polymerase chain reaction analyses. Significant upregulation of SCN3A, Myo1d, and RP1 messenger RNA, and protein levels was observed in the patent DA group (all P ≤ 0.048). ARHGAP26 messenger RNA and protein levels were decreased in patent DA tissue (both P ≤ 0.018). Immunohistochemistry analysis revealed that Myo1d, ARHGAP26, and RP1 were specifically expressed in the subendothelial region of constricted DAs; however, diffuse expression of these proteins was noted in the patent group. Proteomic analysis revealed global changes in the expression of proteins that regulate oxygen sensing, ion channels, smooth muscle cell migration, nervous system, immune system, and metabolism, suggesting a basis for the systemic regulation of DA patency by diverse signaling pathways, which will be confirmed in further studies.
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Wang, Tiantian; Sun, Hui; Zhang, Jie; Liu, Qing; Wang, Longjiang; Chen, Peipei; Wang, Fangkun; Li, Hongmei; Xiao, Yihong; Zhao, Xiaomin
2014-03-01
In the present study, an a-agglutinin-based Saccharomyces boulardii surface display system was successfully established using a single expression vector. Based on the two protein co-expression vector pSP-G1 built by Partow et al., a S. boulardii surface display vector-pSDSb containing all the display elements was constructed. The display results of heterologous proteins were confirmed by successfully displaying enhanced green fluorescent protein (EGFP) and chicken Eimeria tenella Microneme-2 proteins (EtMic2) on the S. boulardii cell surface. The DNA sequence of AGA1 gene from S. boulardii (SbAGA1) was determined and used as the cell wall anchor partner. This is the first time heterologous proteins have been displayed on the cell surface of S. boulardii. Because S. boulardii is probiotic and eukaryotic, its surface display system would be very valuable, particularly in the development of a live vaccine against various pathogenic organisms especially eukaryotic pathogens such as protistan parasites. Copyright © 2013 Elsevier Inc. All rights reserved.
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Equations for the determination of humidity from dewpoint and psychrometric data
NASA Technical Reports Server (NTRS)
Parish, O. O.; Putnam, T. W.
1977-01-01
A general expression based on the Claperon-Clausius differential equation that relates saturation vapor pressure, absolute temperature, and the latent heat of transformation was derived that expresses saturation vapor pressure as a function of absolute temperature. This expression was then used to derive general expressions for vapor pressure, absolute humidity, and relative humidity as functions of either dewpoint and ambient temperature or psychrometric parameters. Constants for all general expressions were then evaluated to give specific expressions in both the international system of units and U.S. customary units for temperatures above and below freezing.
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DOT National Transportation Integrated Search
1973-02-01
The volume presents the models used to analyze basic features of the system, establish feasibility of techniques, and evaluate system performance. The models use analytical expressions and computer simulations to represent the relationship between sy...
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A Recessive Pollination Control System for Wheat Based on Intein-Mediated Protein Splicing.
Gils, Mario
2017-01-01
A transgene-expression system for wheat that relies on the complementation of inactive precursor protein fragments through a split-intein system is described. The N- and C-terminal fragments of a barnase gene from Bacillus amyloliquifaciens were fused to intein sequences from Synechocystis sp. and transformed into wheat plants. Upon translation, both barnase fragments are assembled by an autocatalytic intein-mediated trans-splicing reaction, thus forming a cytotoxic enzyme. This chapter focuses on the use of introns and flexible polypeptide linkers to foster the expression of a split-barnase expression system in plants. The methods and protocols that were employed with the objective to test the effects of such genetic elements on transgene expression and to find the optimal design of expression vectors for use in wheat are provided. Split-inteins can be used to form an agriculturally important trait (male sterility) in wheat plants. The use of this principle for the production of hybrid wheat seed is described. The suggested toolbox will hopefully be a valuable contribution to future optimization strategies in this commercially important crop.
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Falch, Ken Vidar; Detlefs, Carsten; Snigirev, Anatoly; Mathiesen, Ragnvald H
2018-01-01
Analytical expressions for the transmission cross-coefficients for x-ray microscopes based on compound refractive lenses are derived based on Gaussian approximations of the source shape and energy spectrum. The effects of partial coherence, defocus, beam convergence, as well as lateral and longitudinal chromatic aberrations are accounted for and discussed. Taking the incoherent limit of the transmission cross-coefficients, a compact analytical expression for the modulation transfer function of the system is obtained, and the resulting point, line and edge spread functions are presented. Finally, analytical expressions for optimal numerical aperture, coherence ratio, and bandwidth are given. Copyright © 2017 Elsevier B.V. All rights reserved.
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ERIC Educational Resources Information Center
Camras, Linda A.; Oster, Harriet; Bakeman, Roger; Meng, Zhaolan; Ujiie, Tatsuo; Campos, Joseph J.
2007-01-01
Do infants show distinct negative facial expressions for different negative emotions? To address this question, European American, Chinese, and Japanese 11-month-olds were videotaped during procedures designed to elicit mild anger or frustration and fear. Facial behavior was coded using Baby FACS, an anatomically based scoring system. Infants'…
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Ishiwata, Ryosuke R; Morioka, Masaki S; Ogishima, Soichi; Tanaka, Hiroshi
2009-02-15
BioCichlid is a 3D visualization system of time-course microarray data on molecular networks, aiming at interpretation of gene expression data by transcriptional relationships based on the central dogma with physical and genetic interactions. BioCichlid visualizes both physical (protein) and genetic (regulatory) network layers, and provides animation of time-course gene expression data on the genetic network layer. Transcriptional regulations are represented to bridge the physical network (transcription factors) and genetic network (regulated genes) layers, thus integrating promoter analysis into the pathway mapping. BioCichlid enhances the interpretation of microarray data and allows for revealing the underlying mechanisms causing differential gene expressions. BioCichlid is freely available and can be accessed at http://newton.tmd.ac.jp/. Source codes for both biocichlid server and client are also available.
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Modulation of ColE1-like Plasmid Replication for Recombinant Gene Expression
Camps, Manel
2010-01-01
ColE1-like plasmids constitute the most popular vectors for recombinant protein expression. ColE1 plasmid replication is tightly controlled by an antisense RNA mechanism that is highly dynamic, tuning plasmid metabolic burden to the physiological state of the host. Plasmid homeostasis is upset upon induction of recombinant protein expression because of non-physiological levels of expression and because of the frequently biased amino acid composition of recombinant proteins. Disregulation of plasmid replication is the main cause of collapse of plasmid-based expression systems because of a simultaneous increase in the metabolic burden (due to increased average copy number) and in the probability of generation of plasmid-free cells (due to increased copy number variation). Interference between regulatory elements of co-resident plasmids causes comparable effects on plasmid stability (plasmid incompatibility). Modulating plasmid copy number for recombinant gene expression aims at achieving a high gene dosage while preserving the stability of the expression system. Here I present strategies targeting plasmid replication for optimizing recombinant gene expression. Specifically, I review approaches aimed at modulating the antisense regulatory system (as well as their implications for plasmid incompatibility) and innovative strategies involving modulation of host factors, of R-loop formation, and of the timing of recombinant gene expression. PMID:20218961
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NASA Astrophysics Data System (ADS)
Zarifi, Keyvan; Gershman, Alex B.
2006-12-01
We analyze the performance of two popular blind subspace-based signature waveform estimation techniques proposed by Wang and Poor and Buzzi and Poor for direct-sequence code division multiple-access (DS-CDMA) systems with unknown correlated noise. Using the first-order perturbation theory, analytical expressions for the mean-square error (MSE) of these algorithms are derived. We also obtain simple high SNR approximations of the MSE expressions which explicitly clarify how the performance of these techniques depends on the environmental parameters and how it is related to that of the conventional techniques that are based on the standard white noise assumption. Numerical examples further verify the consistency of the obtained analytical results with simulation results.
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A framework for the recognition of 3D faces and expressions
NASA Astrophysics Data System (ADS)
Li, Chao; Barreto, Armando
2006-04-01
Face recognition technology has been a focus both in academia and industry for the last couple of years because of its wide potential applications and its importance to meet the security needs of today's world. Most of the systems developed are based on 2D face recognition technology, which uses pictures for data processing. With the development of 3D imaging technology, 3D face recognition emerges as an alternative to overcome the difficulties inherent with 2D face recognition, i.e. sensitivity to illumination conditions and orientation positioning of the subject. But 3D face recognition still needs to tackle the problem of deformation of facial geometry that results from the expression changes of a subject. To deal with this issue, a 3D face recognition framework is proposed in this paper. It is composed of three subsystems: an expression recognition system, a system for the identification of faces with expression, and neutral face recognition system. A system for the recognition of faces with one type of expression (happiness) and neutral faces was implemented and tested on a database of 30 subjects. The results proved the feasibility of this framework.
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Model Checking Artificial Intelligence Based Planners: Even the Best Laid Plans Must Be Verified
NASA Technical Reports Server (NTRS)
Smith, Margaret H.; Holzmann, Gerard J.; Cucullu, Gordon C., III; Smith, Benjamin D.
2005-01-01
Automated planning systems (APS) are gaining acceptance for use on NASA missions as evidenced by APS flown On missions such as Orbiter and Deep Space 1 both of which were commanded by onboard planning systems. The planning system takes high level goals and expands them onboard into a detailed of action fiat the spacecraft executes. The system must be verified to ensure that the automatically generated plans achieve the goals as expected and do not generate actions that would harm the spacecraft or mission. These systems are typically tested using empirical methods. Formal methods, such as model checking, offer exhaustive or measurable test coverage which leads to much greater confidence in correctness. This paper describes a formal method based on the SPIN model checker. This method guarantees that possible plans meet certain desirable properties. We express the input model in Promela, the language of SPIN and express the properties of desirable plans formally.
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Expression of the functional recombinant human glycosyltransferase GalNAcT2 in Escherichia coli.
Lauber, Jennifer; Handrick, René; Leptihn, Sebastian; Dürre, Peter; Gaisser, Sabine
2015-01-13
Recombinant protein-based therapeutics have become indispensable for the treatment of many diseases. They are produced using well-established expression systems based on bacteria, yeast, insect and mammalian cells. The majority of therapeutic proteins are glycoproteins and therefore the post-translational attachment of sugar residues is required. The development of an engineered Escherichia coli-based expression system for production of human glycoproteins could potentially lead to increased yields, as well as significant decreases in processing time and costs. This work describes the expression of functional human-derived glycosyltransferase UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 2 (GalNAcT2) in a recombinant E. coli strain. For expression, a codon-optimised gene encoding amino acids 52-571 of GalNAcT2 lacking the transmembrane N-terminal domain was inserted into a pET-23 derived vector encoding a polyhistidine-tag which was translationally fused to the N-terminus of the glycosyltransferase (HisDapGalNAcT2). The glycosyltransferase was produced in E. coli using a recently published expression system. Soluble HisDapGalNAcT2 produced in SHuffle® T7 host cells was purified using nickel affinity chromatography and was subsequently analysed by size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) and circular dichroism spectroscopy to determine molecular mass, folding state and thermal transitions of the protein. The activity of purified HisDapGalNAcT2 was monitored using a colorimetric assay based on the release of phosphate during transfer of glycosyl residues to a model acceptor peptide or, alternatively, to the granulocyte-colony stimulating growth factor (G-CSF). Modifications were assessed by Matrix Assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry analysis (MALDI-TOF-MS) and Electrospray Mass Spectrometry analysis (ESI-MS). The results clearly indicate the glycosylation of the acceptor peptide and of G-CSF. In the present work, we isolated a human-derived glycosyltransferase by expressing soluble HisDapGalNAcT2 in E. coli. The functional activity of the enzyme was shown in vitro. Further investigations are needed to assess the potential of in vivo glycosylation in E. coli.
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Xiao, Xiaodong; Chen, Yan; Mugabe, Sheila; Gao, Changshou; Tkaczyk, Christine; Mazor, Yariv; Pavlik, Peter; Wu, Herren; Dall'Acqua, William; Chowdhury, Partha Sarathi
2015-01-01
High throughput screenings of single chain Fv (scFv) antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays. The monovalent nature and lack of Fc in soluble scFvs prevent functional assays that are dependent on target cross linking and/or Fc functions. A convenient approach is to convert scFvs into scFv.Fc fusion proteins and express them in mammalian cell lines for screening. This approach is low throughput and is only taken after primary screening of monovalent scFvs that are expressed in bacteria. There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output. We have, therefore, developed a novel dual expression vector, called pSplice, which can be used to express scFv.Fc fusion proteins both in E.coli and mammalian cell lines. The hallmark of the vector is an engineered intron which houses the bacterial promoter and signal peptide for expression and secretion of scFv.Fc in E.coli. When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium. By applying basic knowledge of mammalian introns and splisosome, we designed this vector to enable screening of phage libraries in a product like format. Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions. Expression in E.coli maintains the speed of the bacterial expression platform and is used to triage clones based on binding and other assays that are not sensitive to endotoxin. Triaged clones are then expressed in a mammalian cell line without the need for any additional cloning steps. Conditioned media from the mammalian cell line containing the fusion proteins are then used for different types of cell based assays. Thus this system retains the speed of the current screening system for phage libraries and adds additional functionality to it.
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Watanabe, Colin; Cuellar, Trinna L.; Haley, Benjamin
2016-01-01
ABSTRACT Incorporating miRNA-like features into vector-based hairpin scaffolds has been shown to augment small RNA processing and RNAi efficiency. Therefore, defining an optimal, native hairpin context may obviate a need for hairpin-specific targeting design schemes, which confound the movement of functional siRNAs into shRNA/artificial miRNA backbones, or large-scale screens to identify efficacious sequences. Thus, we used quantitative cell-based assays to compare separate third generation artificial miRNA systems, miR-E (based on miR-30a) and miR-3G (based on miR-16-2 and first described in this study) to widely-adopted, first and second generation formats in both Pol-II and Pol-III expression vector contexts. Despite their unique structures and strandedness, and in contrast to first and second-generation RNAi triggers, the third generation formats operated with remarkable similarity to one another, and strong silencing was observed with a significant fraction of the evaluated target sequences within either promoter context. By pairing an established siRNA design algorithm with the third generation vectors we could readily identify targeting sequences that matched or exceeded the potency of those discovered through large-scale sensor-based assays. We find that third generation hairpin systems enable the maximal level of siRNA function, likely through enhanced processing and accumulation of precisely-defined guide RNAs. Therefore, we predict future gains in RNAi potency will come from improved hairpin expression and identification of optimal siRNA-intrinsic silencing properties rather than further modification of these scaffolds. Consequently, third generation systems should be the primary format for vector-based RNAi studies; miR-3G is advantageous due to its small expression cassette and simplified, cost-efficient cloning scheme. PMID:26786363
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Shamekova, Malika; Mendoza, Maria R; Hsieh, Yi-Cheng; Lindbo, John; Omarov, Rustem T; Scholthof, Herman B
2014-03-01
A next generation Tomato bushy stunt virus (TBSV) coat protein gene replacement vector system is described that can be applied by either RNA inoculation or through agroinfiltration. A vector expressing GFP rapidly yields high levels of transient gene expression in inoculated leaves of various plant species, as illustrated for Nicotiana benthamiana, cowpea, tomato, pepper, and lettuce. A start-codon mutation to down-regulate the dose of the P19 silencing suppressor reduces GFP accumulation, whereas mutations that result in undetectable levels of P19 trigger rapid silencing of GFP. Compared to existing virus vectors the TBSV system has a unique combination of a very broad host range, rapid and high levels of replication and gene expression, and the ability to regulate its suppressor. These features are attractive for quick transient assays in numerous plant species for over-expression of genes of interest, or as a sensor to monitor the efficacy of antiviral RNA silencing. Copyright © 2014. Published by Elsevier Inc.
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Khulape, S A; Maity, H K; Pathak, D C; Mohan, C Madhan; Dey, S
2015-09-01
The outer membrane glycoprotein, hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is important for virus infection and subsequent immune response by host, and offers target for development of recombinant antigen-based immunoassays and subunit vaccines. In this study, the expression of HN protein of NDV is attempted in yeast expression system. Yeast offers eukaryotic environment for protein processing and posttranslational modifications like glycosylation, in addition to higher growth rate and easy genetic manipulation. Saccharomyces cerevisiae was found to be better expression system for HN protein than Pichia pastoris as determined by codon usage analysis. The complete coding sequence of HN gene was amplified with the histidine tag, cloned in pESC-URA under GAL10 promotor and transformed in Saccharomyces cerevisiae. The recombinant HN (rHN) protein was characterized by western blot, showing glycosylation heterogeneity as observed with other eukaryotic expression systems. The recombinant protein was purified by affinity column purification. The protein could be further used as subunit vaccine.
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Zhang, Xiuren; Mason, Hugh
2006-02-05
A novel stable transgenic plant expression system was developed using elements of the replication machinery of Bean Yellow Dwarf Virus (BeYDV). The system contains two transgenes: 1) The BeYDV replicon vector with an expression cassette flanked by cis-acting DNA elements of BeYDV, and 2) The viral replication initiator protein (Rep) controlled by an alcohol-inducible promoter. When Rep expression was triggered by treatment with ethanol, it induced release of the BeYDV replicon from stably integrated T-DNA and episomal replication to high copy number. Replicon amplification resulted in substantially increased transgene mRNA levels (up to 80-fold) and translation products (up to 10-fold) after induction of Rep expression by ethanol treatment in tobacco NT1 cells and leaves of whole potato plants. Thus, the BeYDV stable transformant replicon system is a powerful tool for plant-based production of recombinant proteins. (c) 2005 Wiley Periodicals, Inc.
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Hutton, John J; Jegga, Anil G; Kong, Sue; Gupta, Ashima; Ebert, Catherine; Williams, Sarah; Katz, Jonathan D; Aronow, Bruce J
2004-01-01
Background In this study we have built and mined a gene expression database composed of 65 diverse mouse tissues for genes preferentially expressed in immune tissues and cell types. Using expression pattern criteria, we identified 360 genes with preferential expression in thymus, spleen, peripheral blood mononuclear cells, lymph nodes (unstimulated or stimulated), or in vitro activated T-cells. Results Gene clusters, formed based on similarity of expression-pattern across either all tissues or the immune tissues only, had highly significant associations both with immunological processes such as chemokine-mediated response, antigen processing, receptor-related signal transduction, and transcriptional regulation, and also with more general processes such as replication and cell cycle control. Within-cluster gene correlations implicated known associations of known genes, as well as immune process-related roles for poorly described genes. To characterize regulatory mechanisms and cis-elements of genes with similar patterns of expression, we used a new version of a comparative genomics-based cis-element analysis tool to identify clusters of cis-elements with compositional similarity among multiple genes. Several clusters contained genes that shared 5–6 cis-elements that included ETS and zinc-finger binding sites. cis-Elements AP2 EGRF ETSF MAZF SP1F ZF5F and AREB ETSF MZF1 PAX5 STAT were shared in a thymus-expressed set; AP4R E2FF EBOX ETSF MAZF SP1F ZF5F and CREB E2FF MAZF PCAT SP1F STAT cis-clusters occurred in activated T-cells; CEBP CREB NFKB SORY and GATA NKXH OCT1 RBIT occurred in stimulated lymph nodes. Conclusion This study demonstrates a series of analytic approaches that have allowed the implication of genes and regulatory elements that participate in the differentiation, maintenance, and function of the immune system. Polymorphism or mutation of these could adversely impact immune system functions. PMID:15504237
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Fan, Fangfang; Zhuo, Rui; Ma, Fuying; Gong, Yangmin; Wan, Xia; Jiang, Mulan
2012-01-01
Laccase is a copper-containing polyphenol oxidase that has great potential in industrial and biotechnological applications. Previous research has suggested that fungal laccase may be involved in the defense against oxidative stress, but there is little direct evidence supporting this hypothesis, and the mechanism by which laccase protects cells from oxidative stress also remains unclear. Here, we report that the expression of the laccase gene from white rot fungus in Pichia pastoris can significantly enhance the resistance of yeast to H2O2-mediated oxidative stress. The expression of laccase in yeast was found to confer a strong ability to scavenge intracellular H2O2 and to protect cells from lipid oxidative damage. The mechanism by which laccase gene expression increases resistance to oxidative stress was then investigated further. We found that laccase gene expression in Pichia pastoris could increase the level of glutathione-based antioxidative activity, including the intracellular glutathione levels and the enzymatic activity of glutathione peroxidase, glutathione reductase, and γ-glutamylcysteine synthetase. The transcription of the laccase gene in Pichia pastoris was found to be enhanced by the oxidative stress caused by exogenous H2O2. The stimulation of laccase gene expression in response to exogenous H2O2 stress further contributed to the transcriptional induction of the genes involved in the glutathione-dependent antioxidative system, including PpYAP1, PpGPX1, PpPMP20, PpGLR1, and PpGSH1. Taken together, these results suggest that the expression of the laccase gene in Pichia pastoris can enhance the resistance of yeast to H2O2-mediated oxidative stress by stimulating the glutathione-based antioxidative system to protect the cell from oxidative damage. PMID:22706050
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Chae, Heejoon; Lee, Sangseon; Seo, Seokjun; Jung, Daekyoung; Chang, Hyeonsook; Nephew, Kenneth P; Kim, Sun
2016-12-01
Measuring gene expression, DNA sequence variation, and DNA methylation status is routinely done using high throughput sequencing technologies. To analyze such multi-omics data and explore relationships, reliable bioinformatics systems are much needed. Existing systems are either for exploring curated data or for processing omics data in the form of a library such as R. Thus scientists have much difficulty in investigating relationships among gene expression, DNA sequence variation, and DNA methylation using multi-omics data. In this study, we report a system called BioVLAB-mCpG-SNP-EXPRESS for the integrated analysis of DNA methylation, sequence variation (SNPs), and gene expression for distinguishing cellular phenotypes at the pairwise and multiple phenotype levels. The system can be deployed on either the Amazon cloud or a publicly available high-performance computing node, and the data analysis and exploration of the analysis result can be conveniently done using a web-based interface. In order to alleviate analysis complexity, all the process are fully automated, and graphical workflow system is integrated to represent real-time analysis progression. The BioVLAB-mCpG-SNP-EXPRESS system works in three stages. First, it processes and analyzes multi-omics data as input in the form of the raw data, i.e., FastQ files. Second, various integrated analyses such as methylation vs. gene expression and mutation vs. methylation are performed. Finally, the analysis result can be explored in a number of ways through a web interface for the multi-level, multi-perspective exploration. Multi-level interpretation can be done by either gene, gene set, pathway or network level and multi-perspective exploration can be explored from either gene expression, DNA methylation, sequence variation, or their relationship perspective. The utility of the system is demonstrated by performing analysis of phenotypically distinct 30 breast cancer cell line data set. BioVLAB-mCpG-SNP-EXPRESS is available at http://biohealth.snu.ac.kr/software/biovlab_mcpg_snp_express/. Copyright © 2016 Elsevier Inc. All rights reserved.
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CRYPTOCHROME mediates behavioral executive choice in response to UV light
Baik, Lisa S.; Fogle, Keri J.; Roberts, Logan; Galschiodt, Alexis M.; Chevez, Joshua A.; Recinos, Yocelyn; Nguy, Vinh; Holmes, Todd C.
2017-01-01
Drosophila melanogaster CRYPTOCHROME (CRY) mediates behavioral and electrophysiological responses to blue light coded by circadian and arousal neurons. However, spectroscopic and biochemical assays of heterologously expressed CRY suggest that CRY may mediate functional responses to UV-A (ultraviolet A) light as well. To determine the relative contributions of distinct phototransduction systems, we tested mutants lacking CRY and mutants with disrupted opsin-based phototransduction for behavioral and electrophysiological responses to UV light. CRY and opsin-based external photoreceptor systems cooperate for UV light-evoked acute responses. CRY mediates behavioral avoidance responses related to executive choice, consistent with its expression in central brain neurons. PMID:28062690
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The CRISPR/Cas9 system is a powerful tool for studying gene function. Here, we describe a method that allows temporal control of CRISPR/Cas9 activity based on conditional Cas9 destabilization. We demonstrate that fusing an FKBP12-derived destabilizing domain to Cas9 (DD-Cas9) enables conditional Cas9 expression and temporal control of gene editing in the presence of an FKBP12 synthetic ligand. This system can be easily adapted to co-express, from the same promoter, DD-Cas9 with any other gene of interest without co-modulation of the latter.
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Tu, Samson W; Hrabak, Karen M; Campbell, James R; Glasgow, Julie; Nyman, Mark A; McClure, Robert; McClay, James; Abarbanel, Robert; Mansfield, James G; Martins, Susana M; Goldstein, Mary K; Musen, Mark A
2006-01-01
Developing computer-interpretable clinical practice guidelines (CPGs) to provide decision support for guideline-based care is an extremely labor-intensive task. In the EON/ATHENA and SAGE projects, we formulated substantial portions of CPGs as computable statements that express declarative relationships between patient conditions and possible interventions. We developed query and expression languages that allow a decision-support system (DSS) to evaluate these statements in specific patient situations. A DSS can use these guideline statements in multiple ways, including: (1) as inputs for determining preferred alternatives in decision-making, and (2) as a way to provide targeted commentaries in the clinical information system. The use of these declarative statements significantly reduces the modeling expertise and effort required to create and maintain computer-interpretable knowledge bases for decision-support purpose. We discuss possible implications for sharing of such knowledge bases.
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NASA Astrophysics Data System (ADS)
Okuno, Keisuke; Inamura, Tetsunari
A robotic coaching system can improve humans' learning performance of motions by intelligent usage of emphatic motions and adverbial expressions according to user reactions. In robotics, however, method to control both the motions and the expressions and how to bind them had not been adequately discussed from an engineering point of view. In this paper, we propose a method for controlling and binding emphatic motions and adverbial expressions by using two scalar parameters in a phase space. In the phase space, variety of motion patterns and verbal expressions are connected and can be expressed as static points. We show the feasibility of the proposing method through experiments of actual sport coaching tasks for beginners. From the results of participants' improvements in motion learning, we confirmed the feasibility of the methods to control and bind emphatic motions and adverbial expressions, as well as confirmed contribution of the emphatic motions and positive correlation of adverbial expressions for participants' improvements in motion learning. Based on the results, we introduce a hypothesis that individually optimized method for binding adverbial expression is required.
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Stevens, David Cole; Conway, Kyle R.; Pearce, Nelson; Villegas-Peñaranda, Luis Roberto; Garza, Anthony G.; Boddy, Christopher N.
2013-01-01
Background Heterologous expression of bacterial biosynthetic gene clusters is currently an indispensable tool for characterizing biosynthetic pathways. Development of an effective, general heterologous expression system that can be applied to bioprospecting from metagenomic DNA will enable the discovery of a wealth of new natural products. Methodology We have developed a new Escherichia coli-based heterologous expression system for polyketide biosynthetic gene clusters. We have demonstrated the over-expression of the alternative sigma factor σ54 directly and positively regulates heterologous expression of the oxytetracycline biosynthetic gene cluster in E. coli. Bioinformatics analysis indicates that σ54 promoters are present in nearly 70% of polyketide and non-ribosomal peptide biosynthetic pathways. Conclusions We have demonstrated a new mechanism for heterologous expression of the oxytetracycline polyketide biosynthetic pathway, where high-level pleiotropic sigma factors from the heterologous host directly and positively regulate transcription of the non-native biosynthetic gene cluster. Our bioinformatics analysis is consistent with the hypothesis that heterologous expression mediated by the alternative sigma factor σ54 may be a viable method for the production of additional polyketide products. PMID:23724102
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Vector modifications to eliminate transposase expression following piggyBac-mediated transgenesis
Chakraborty, Syandan; Ji, HaYeun; Chen, Jack; Gersbach, Charles A.; Leong, Kam W.
2014-01-01
Transgene insertion plays an important role in gene therapy and in biological studies. Transposon-based systems that integrate transgenes by transposase-catalyzed “cut-and-paste” mechanism have emerged as an attractive system for transgenesis. Hyperactive piggyBac transposon is particularly promising due to its ability to integrate large transgenes with high efficiency. However, prolonged expression of transposase can become a potential source of genotoxic effects due to uncontrolled transposition of the integrated transgene from one chromosomal locus to another. In this study we propose a vector design to decrease post-transposition expression of transposase and to eliminate the cells that have residual transposase expression. We design a single plasmid construct that combines the transposase and the transpositioning transgene element to share a single polyA sequence for termination. Consequently, the separation of the transposase element from the polyA sequence after transposition leads to its deactivation. We also co-express Herpes Simplex Virus thymidine kinase (HSV-tk) with the transposase. Therefore, cells having residual transposase expression can be eliminated by the administration of ganciclovir. We demonstrate the utility of this combination transposon system by integrating and expressing a model therapeutic gene, human coagulation Factor IX, in HEK293T cells. PMID:25492703
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Systemic bioinformatics analysis of skeletal muscle gene expression profiles of sepsis
Yang, Fang; Wang, Yumei
2018-01-01
Sepsis is a type of systemic inflammatory response syndrome with high morbidity and mortality. Skeletal muscle dysfunction is one of the major complications of sepsis that may also influence the outcome of sepsis. The aim of the present study was to explore and identify potential mechanisms and therapeutic targets of sepsis. Systemic bioinformatics analysis of skeletal muscle gene expression profiles from the Gene Expression Omnibus was performed. Differentially expressed genes (DEGs) in samples from patients with sepsis and control samples were screened out using the limma package. Differential co-expression and coregulation (DCE and DCR, respectively) analysis was performed based on the Differential Co-expression Analysis package to identify differences in gene co-expression and coregulation patterns between the control and sepsis groups. Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways of DEGs were identified using the Database for Annotation, Visualization and Integrated Discovery, and inflammatory, cancer and skeletal muscle development-associated biological processes and pathways were identified. DCE and DCR analysis revealed several potential therapeutic targets for sepsis, including genes and transcription factors. The results of the present study may provide a basis for the development of novel therapeutic targets and treatment methods for sepsis. PMID:29805480
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Baril, Patrick; Pichon, Chantal
2016-01-01
MicroRNAs (miRNAs) are a class of small, noncoding RNAs which regulate gene expression by directing their target mRNA for degradation or translational repression. Since their discovery in the early 1990s, miRNAs have emerged as key components in the posttranscriptional regulation of gene networks, shaping many biological processes from development, morphogenesis, differentiation, proliferation and apoptosis. Although understanding of the molecular basis of miRNA biology is improving, methods to monitor the dynamic and the spatiotemporal aspects of miRNA expression under physiopathological conditions are required. However, monitoring of miRNAs is difficult due to their small size, low abundance, high degree of sequence similarity, and their dynamic expression pattern which is subjected to tight transcriptional and post-transcriptional controls. Recently, we developed a miRNA monitoring system called RILES, standing for RNAi-inducible expression system, which relies on an engineered regulatable expression system, to switch on the expression of the luciferase gene when the targeted miRNA is expressed in cells. We demonstrated that RILES is a specific, sensitive, and robust method to determine the fine-tuning of miRNA expression during the development of an experimental pathological process in mice. Because RILES offers the possibility for longitudinal studies on individual subjects, sharper insights into miRNA regulation can be generated, with applications in physiology, pathophysiology and development of RNAi-based therapies. This chapter describes methods and protocols to monitor the expression of myomiR-206, -1, and -133 in the tibialis anterior muscle of mice. These protocols can be used and adapted to monitor the expression of other miRNAs in other biological processes.
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Mahjoub, Mohamed; Bouafia, Nabiha; Cheikh, Asma Ben; Ezzi, Olfa; Njah, Mansour
2016-11-25
This study provided an overview of healthcare professionals’ perception of patient safety based on analysis of the concept of freedom of expression and non-punitive response in order to identify and correct errors in our health system. This concept is a cornerstone of the patient safety culture among healthcare professionals and plays a central role in the quality improvement strategy..
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2014-11-03
Rob Mueller, NASA senior technologist in the Surface Systems Office in Kennedy Space Center's Engineering and Technology Directorate, demonstrates the Regolith Advanced Surface System Operations Robot, or RASSOR, during a media event at Kennedy's automated landing and hazard avoidance technology, or ALHAT, hazard field at the north end of the Shuttle Landing Facility. The event was held to announce Moon Express Inc., of Moffett Field, California is selected to utilize Kennedy facilities for NASA's Lunar Cargo Transportation and Landing by Soft Touchdown, or Lunar CATALYST, initiative. Moon Express is developing a lander with capabilities that will enable delivery of payloads to the surface of the moon, as well as new science and exploration missions of interest to NASA and scientific and academic communities. Moon Express will base its activities at Kennedy and utilize the Morpheus ALHAT field and a hangar nearby for CATALYST testing. The Advanced Exploration Systems Division of NASA's Human Exploration and Operations Mission Directorate manages Lunar CATALYST.
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Kinematics and constraints associated with swashplate blade pitch control
NASA Technical Reports Server (NTRS)
Leyland, Jane A.
1993-01-01
An important class of techniques to reduce helicopter vibration is based on using a Higher Harmonic controller to optimally define the Higher Harmonic blade pitch. These techniques typically require solution of a general optimization problem requiring the determination of a control vector which minimizes a performance index where functions of the control vector are subject to inequality constraints. Six possible constraint functions associated with swashplate blade pitch control were identified and defined. These functions constrain: (1) blade pitch Fourier Coefficients expressed in the Rotating System, (2) blade pitch Fourier Coefficients expressed in the Nonrotating System, (3) stroke of the individual actuators expressed in the Nonrotating System, (4) blade pitch expressed as a function of blade azimuth and actuator stroke, (5) time rate-of-change of the aforementioned parameters, and (6) required actuator power. The aforementioned constraints and the associated kinematics of swashplate blade pitch control by means of the strokes of the individual actuators are documented.
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Protocol for Uniformly Measuring and Expressing the Performance of Energy Storage Systems
DOE Office of Scientific and Technical Information (OSTI.GOV)
Conover, David R.; Crawford, Aladsair J.; Fuller, Jason C.
The Protocol for Uniformly Measuring and Expressing the Performance of Energy Storage Systems (PNNL-22010) was first issued in November 2012 as a first step toward providing a foundational basis for developing an initial standard for the uniform measurement and expression of energy storage system (ESS) performance. Based on experiences with the application and use of that document, and to include additional ESS applications and associated duty cycles, test procedures and performance metrics, a first revision of the November 2012 Protocol was issued in June 2014 (PNNL 22010 Rev. 1). As an update of the 2014 revision 1 to the Protocol,more » this document (the March 2016 revision 2 to the Protocol) is intended to supersede the June 2014 revision 1 to the Protocol and provide a more user-friendly yet more robust and comprehensive basis for measuring and expressing ESS performance.« less
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Case-based retrieval framework for gene expression data.
Anaissi, Ali; Goyal, Madhu; Catchpoole, Daniel R; Braytee, Ali; Kennedy, Paul J
2015-01-01
The process of retrieving similar cases in a case-based reasoning system is considered a big challenge for gene expression data sets. The huge number of gene expression values generated by microarray technology leads to complex data sets and similarity measures for high-dimensional data are problematic. Hence, gene expression similarity measurements require numerous machine-learning and data-mining techniques, such as feature selection and dimensionality reduction, to be incorporated into the retrieval process. This article proposes a case-based retrieval framework that uses a k-nearest-neighbor classifier with a weighted-feature-based similarity to retrieve previously treated patients based on their gene expression profiles. The herein-proposed methodology is validated on several data sets: a childhood leukemia data set collected from The Children's Hospital at Westmead, as well as the Colon cancer, the National Cancer Institute (NCI), and the Prostate cancer data sets. Results obtained by the proposed framework in retrieving patients of the data sets who are similar to new patients are as follows: 96% accuracy on the childhood leukemia data set, 95% on the NCI data set, 93% on the Colon cancer data set, and 98% on the Prostate cancer data set. The designed case-based retrieval framework is an appropriate choice for retrieving previous patients who are similar to a new patient, on the basis of their gene expression data, for better diagnosis and treatment of childhood leukemia. Moreover, this framework can be applied to other gene expression data sets using some or all of its steps.
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NASA Astrophysics Data System (ADS)
Chiu, Tzu-Keng; Chou, Wen-Pin; Huang, Song-Bin; Wang, Hung-Ming; Lin, Yung-Chang; Hsieh, Chia-Hsun; Wu, Min-Hsien
2016-09-01
Circulating tumour cells (CTCs) in a blood circulation system are associated with cancer metastasis. The analysis of the drug-resistance gene expression of cancer patients’ CTCs holds promise for selecting a more effective therapeutic regimen for an individual patient. However, the current CTC isolation schemes might not be able to harvest CTCs with sufficiently high purity for such applications. To address this issue, this study proposed to integrate the techniques of optically induced dielectrophoretic (ODEP) force-based cell manipulation and fluorescent microscopic imaging in a microfluidic system to further purify CTCs after the conventional CTC isolation methods. In this study, the microfluidic system was developed, and its optimal operating conditions and performance for CTC isolation were evaluated. The results revealed that the presented system was able to isolate CTCs with cell purity as high as 100%, beyond what is possible using the previously existing techniques. In the analysis of CTC gene expression, therefore, this method could exclude the interference of leukocytes in a cell sample and accordingly contribute to higher analytical sensitivity, as demonstrated in this study. Overall, this study has presented an ODEP-based microfluidic system capable of simply and effectively isolating a specific cell species from a cell mixture.
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Chiu, Tzu-Keng; Chou, Wen-Pin; Huang, Song-Bin; Wang, Hung-Ming; Lin, Yung-Chang; Hsieh, Chia-Hsun; Wu, Min-Hsien
2016-09-09
Circulating tumour cells (CTCs) in a blood circulation system are associated with cancer metastasis. The analysis of the drug-resistance gene expression of cancer patients' CTCs holds promise for selecting a more effective therapeutic regimen for an individual patient. However, the current CTC isolation schemes might not be able to harvest CTCs with sufficiently high purity for such applications. To address this issue, this study proposed to integrate the techniques of optically induced dielectrophoretic (ODEP) force-based cell manipulation and fluorescent microscopic imaging in a microfluidic system to further purify CTCs after the conventional CTC isolation methods. In this study, the microfluidic system was developed, and its optimal operating conditions and performance for CTC isolation were evaluated. The results revealed that the presented system was able to isolate CTCs with cell purity as high as 100%, beyond what is possible using the previously existing techniques. In the analysis of CTC gene expression, therefore, this method could exclude the interference of leukocytes in a cell sample and accordingly contribute to higher analytical sensitivity, as demonstrated in this study. Overall, this study has presented an ODEP-based microfluidic system capable of simply and effectively isolating a specific cell species from a cell mixture.
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Chiu, Tzu-Keng; Chou, Wen-Pin; Huang, Song-Bin; Wang, Hung-Ming; Lin, Yung-Chang; Hsieh, Chia-Hsun; Wu, Min-Hsien
2016-01-01
Circulating tumour cells (CTCs) in a blood circulation system are associated with cancer metastasis. The analysis of the drug-resistance gene expression of cancer patients’ CTCs holds promise for selecting a more effective therapeutic regimen for an individual patient. However, the current CTC isolation schemes might not be able to harvest CTCs with sufficiently high purity for such applications. To address this issue, this study proposed to integrate the techniques of optically induced dielectrophoretic (ODEP) force-based cell manipulation and fluorescent microscopic imaging in a microfluidic system to further purify CTCs after the conventional CTC isolation methods. In this study, the microfluidic system was developed, and its optimal operating conditions and performance for CTC isolation were evaluated. The results revealed that the presented system was able to isolate CTCs with cell purity as high as 100%, beyond what is possible using the previously existing techniques. In the analysis of CTC gene expression, therefore, this method could exclude the interference of leukocytes in a cell sample and accordingly contribute to higher analytical sensitivity, as demonstrated in this study. Overall, this study has presented an ODEP-based microfluidic system capable of simply and effectively isolating a specific cell species from a cell mixture. PMID:27609546
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2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori
Wang, Yuancheng; Wang, Feng; Wang, Riyuan; Zhao, Ping; Xia, Qingyou
2015-01-01
Fundamental and applied studies of silkworms have entered the functional genomics era. Here, we report a multi-gene expression system (MGES) based on 2A self-cleaving peptide (2A), which regulates the simultaneous expression and cleavage of multiple gene targets in the silk gland of transgenic silkworms. First, a glycine-serine-glycine spacer (GSG) was found to significantly improve the cleavage efficiency of 2A. Then, the cleavage efficiency of six types of 2As with GSG was analyzed. The shortest porcine teschovirus-1 2A (P2A-GSG) exhibited the highest cleavage efficiency in all insect cell lines that we tested. Next, P2A-GSG successfully cleaved the artificial human serum albumin (66 kDa) linked with human acidic fibroblast growth factor (20.2 kDa) fusion genes and vitellogenin receptor fragment (196 kD) of silkworm linked with EGFP fusion genes, importantly, vitellogenin receptor protein was secreted to the outside of cells. Furthermore, P2A-GSG successfully mediated the simultaneous expression and cleavage of a DsRed and EGFP fusion gene in silk glands and caused secretion into the cocoon of transgenic silkworms using our sericin1 expression system. We predicted that the MGES would be an efficient tool for gene function research and innovative research on various functional silk materials in medicine, cosmetics, and other biomedical areas. PMID:26537835
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Zhang, Bei; Rapolu, Madhusudhan; Liang, Zhibin; Han, Zhenlin; Williams, Philip G.; Su, Wei Wen
2015-01-01
Being able to coordinate co-expression of multiple proteins is necessary for a variety of important applications such as assembly of protein complexes, trait stacking, and metabolic engineering. Currently only few options are available for multiple recombinant protein co-expression, and most of them are not applicable to both prokaryotic and eukaryotic hosts. Here, we report a new polyprotein vector system that is based on a pair of self-excising mini-inteins fused in tandem, termed the dual-intein (DI) domain, to achieve synchronized co-expression of multiple proteins. The DI domain comprises an Ssp DnaE mini-intein N159A mutant and an Ssp DnaB mini-intein C1A mutant connected in tandem by a peptide linker to mediate efficient release of the flanking proteins via autocatalytic cleavage. Essentially complete release of constituent proteins, GFP and RFP (mCherry), from a polyprotein precursor, in bacterial, mammalian, and plant hosts was demonstrated. In addition, successful co-expression of GFP with chloramphenicol acetyltransferase, and thioredoxin with RFP, respectively, further substantiates the general applicability of the DI polyprotein system. Collectively, our results demonstrate the DI-based polyprotein technology as a highly valuable addition to the molecular toolbox for multi-protein co-expression which finds vast applications in biotechnology, biosciences, and biomedicine. PMID:25712612
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Neudert, Franziska; Nuernberger, Krishna-K Monique; Redies, Christoph
2008-12-20
The cerebellum shows remarkable variations in the relative size of its divisions among vertebrate species. In the present study, we compare the cerebella of two mammals (ferret and mouse) by mapping the expression of three cadherins (cadherin-8, protocadherin-7, and protocadherin-10) at similar postnatal stages. The three cadherins are expressed differentially in parasagittal stripes in the cerebellar cortex, in the portions of the deep cerebellar nuclei, in the divisions of the inferior olivary nucleus, and in the lateral vestibular nucleus. The expression profiles suggest that the cadherin-positive structures are interconnected. The expression patterns resemble each other in ferret and mouse, although some differences can be observed. The general resemblance indicates that cerebellar organization is based on a common set of embryonic divisions in the two species. Consequently, the large differences in cerebellar morphology between the two species are more likely caused by differential growth of these embryonic divisions than by differences in early embryonic patterning. Based on the cadherin expression patterns, a model of corticonuclear projection territories in ferret and mouse is proposed. In summary, our results indicate that the cerebellar systems of rodents and carnivores display a relatively large degree of similarity in their molecular and functional organization.
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Ma, W; Zhang, T-F; Lu, P; Lu, S H
2014-01-01
Breast cancer is categorized into two broad groups: estrogen receptor positive (ER+) and ER negative (ER-) groups. Previous study proposed that under trastuzumab-based neoadjuvant chemotherapy, tumor initiating cell (TIC) featured ER- tumors response better than ER+ tumors. Exploration of the molecular difference of these two groups may help developing new therapeutic strategies, especially for ER- patients. With gene expression profile from the Gene Expression Omnibus (GEO) database, we performed partial least squares (PLS) based analysis, which is more sensitive than common variance/regression analysis. We acquired 512 differentially expressed genes. Four pathways were found to be enriched with differentially expressed genes, involving immune system, metabolism and genetic information processing process. Network analysis identified five hub genes with degrees higher than 10, including APP, ESR1, SMAD3, HDAC2, and PRKAA1. Our findings provide new understanding for the molecular difference between TIC featured ER- and ER+ breast tumors with the hope offer supports for therapeutic studies.
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Hu, Chenggong; Zhou, Yongfang; Liu, Chang; Kang, Yan
2018-01-01
Gastric cancer (GC) is the fifth most common cancer and the third leading cause of cancer-associated mortality worldwide. In the current study, comprehensive bioinformatic analyses were performed to develop a novel scoring system for GC risk assessment based on CAP-Gly domain containing linker protein family member 4 (CLIP4) DNA methylation status. Two GC datasets with methylation sequencing information and mRNA expression profiling were downloaded from the The Cancer Genome Atlas and Gene Expression Omnibus databases. Differentially expressed genes (DEGs) between the CLIP4 hypermethylation and CLIP4 hypomethylation groups were screened using the limma package in R 3.3.1, and survival analysis of these DEGs was performed using the survival package. A risk scoring system was established via regression factor-weighted gene expression based on linear combination to screen the most important genes associated with CLIP4 methylation and prognosis. Genes associated with high/low-risk value were selected using the limma package. Functional enrichment analysis of the top 500 DEGs that positively and negatively associated with risk values was performed using DAVID 6.8 online and the gene set enrichment analysis (GSEA) software. In total, 35 genes were identified to be that significantly associated with prognosis and CLIP4 DNA methylation, and three prognostic signature genes, claudin-11 (CLDN11), apolipoprotein D (APOD), and chordin like 1 (CHRDL1), were used to establish a risk assessment system. The prognostic scoring system exhibited efficiency in classifying patients with different prognoses, where the low-risk groups had significantly longer overall survival times than those in the high-risk groups. CLDN11, APOD and CHRDL1 exhibited reduced expression in the hypermethylation and low-risk groups compare with the hypomethylation and high-risk groups, respectively. Multivariate Cox analysis indicated that risk value could be used as an independent prognostic factor. In functional analysis, six functional gene ontology terms and five GSEA pathways were associated with CLDN11, APOD and CHRDL1. The results established the credibility of the scoring system in this study. Additionally, these three genes, which were significantly associated with CLIP4 DNA methylation and GC risk assessment, were identified as potential prognostic biomarkers. PMID:29901187
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Cho, Hyunsoo; Kim, Se Hoon; Kim, Soo-Jeong; Chang, Jong Hee; Yang, Woo Ick; Suh, Chang-Ok; Cheong, June-Won; Kim, Yu Ri; Lee, Jung Yeon; Jang, Ji Eun; Kim, Yundeok; Min, Yoo Hong; Kim, Jin Seok
2017-07-01
The prognostic role of CD68 and FoxP3 in primary central nervous system lymphoma (PCNSL) has not been evaluated. Thus, we examined the prognostic significance of CD68 and FoxP3 expression in tumor samples of 76 newly diagnosed immunocompetent PCNSL patients. All patients were treated initially with high-dose methotrexate (HD-MTX)-based chemotherapy, and 16 (21.1%) patients received upfront autologous stem cell transplantation (ASCT) consolidation. High expression of CD68 (>55 cells/high-power field) or FoxP3 (>15 cells/high-power field) was observed in 10 patients, respectively. High CD68 expression was associated with inferior overall survival (OS) and progression-free survival (PFS) in multivariate analysis (P = 0.023 and P = 0.021, respectively). In addition, we performed subgroup analysis based on upfront ASCT. High CD68 expression was also associated with inferior OS and PFS in multivariate analysis (P = 0.013 and P < 0.001, respectively) among patients who did not receive upfront ASCT (n = 60), but not in patients who received upfront ASCT. The expression of FoxP3 was not significantly associated with survival. Therefore, we identified a prognostic significance of high CD68 expression in PCNSL, which suggests a need for further clinical trials and biological studies on the role of PCNSL tumor microenvironment.
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Regulated Expression of Adenoviral Vectors-Based Gene Therapies
Curtin, James F.; Candolfi, Marianela; Puntel, Mariana; Xiong, Weidong; Muhammad, A. K. M.; Kroeger, Kurt; Mondkar, Sonali; Liu, Chunyan; Bondale, Niyati; Lowenstein, Pedro R.; Castro, Maria G.
2008-01-01
Summary Regulatable promoter systems allow gene expression to be tightly controlled in vivo. This is highly desirable for the development of safe, efficacious adenoviral vectors that can be used to treat human diseases in the clinic. Ideally, regulatable cassettes should have minimal gene expression in the “OFF” state, and expression should quickly reach therapeutic levels in the “ON” state. In addition, the components of regulatable cassettes should be non-toxic at physiological concentrations and should not be immunogenic, especially when treating chronic illness that requires long-lasting gene expression. In this chapter, we will describe in detail protocols to develop and validate first generation (Ad) and high-capacity adenoviral (HC-Ad) vectors that express therapeutic genes under the control of the TetON regulatable system. Our laboratory has successfully used these protocols to regulate the expression of marker genes, immune stimulatory genes, and toxins for cancer gene therapeutics, i.e., glioma that is a deadly form of brain cancer. We have shown that this third generation TetON regulatable system, incorporating a doxycycline (DOX)-sensitive rtTA2S-M2 inducer and tTSKid silencer, is non-toxic, relatively non-immunogenic, and can tightly regulate reporter transgene expression downstream of a TRE promoter from adenoviral vectors in vitro and also in vivo. PMID:18470649
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Shim, Gayong; Han, Su-Eun; Yu, Yong-Hee; Lee, Sangbin; Lee, Han Young; Kim, Kwangmeyung; Kwon, Ick Chan; Park, Tae Gwan; Kim, Young Bong; Choi, Yong Seok; Kim, Chan-Wha; Oh, Yu-Kyoung
2011-10-10
Oligolysine-based cationic lipid derivatives were synthesized for delivery of siRNA, and formulated into cationic liposomes. Among various oligolysine-based lipid derivatives differing in lysine residue number and lipid moiety, trilysinoyl oleylamide (TLO)-based liposomes (TLOL) showed the highest delivery efficiency combined with minimal cytotoxicity. Delivery of siRNA using TLOL silenced target genes both in vitro and in vivo. In green fluorescent protein (GFP)-expressing tumor tissue, a significant reduction of fluorescence was observed after intratumoral administration of siGFP using TLOL compared with control siGL2. Intravenous administration of siMcl1 employing pegylated TLOL (pTLOL) reduced the expression of human Mcl1 protein in KB-xenografted tumor tissue. Despite the reduction in target protein Mcl1 expression following such systemic delivery, tumor growth was only slightly reduced compared to a siGL2-treated control group. To potentiate the anticancer activity of siMcl1, the anticancer drug suberoylanilide hydroxamic acid (SAHA) was additionally encapsulated in pTLOL. After intravenous administration of siMcl1 using SAHA-loaded pTLOL (pSTLOL), a significant reduction in tumor growth was observed compared to that seen in animals treated with free SAHA or siGL2 complexed with pSTLOL. The results indicate that pTLOL could be further developed as a systemic delivery system for synergistic anticancer siRNA and a drug. Copyright © 2010 Elsevier B.V. All rights reserved.
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Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells.
Benabdellah, Karim; Muñoz, Pilar; Cobo, Marién; Gutierrez-Guerrero, Alejandra; Sánchez-Hernández, Sabina; Garcia-Perez, Angélica; Anderson, Per; Carrillo-Gálvez, Ana Belén; Toscano, Miguel G; Martin, Francisco
2016-11-17
Conditional transgene expression in human stem cells has been difficult to achieve due to the low efficiency of existing delivery methods, the strong silencing of the transgenes and the toxicity of the regulators. Most of the existing technologies are based on stem cells clones expressing appropriate levels of tTA or rtTA transactivators (based on the TetR-VP16 chimeras). In the present study, we aim the generation of Tet-On all-in-one lentiviral vectors (LVs) that tightly regulate transgene expression in human stem cells using the original TetR repressor. By using appropriate promoter combinations and shielding the LVs with the Is2 insulator, we have constructed the Lent-On-Plus Tet-On system that achieved efficient transgene regulation in human multipotent and pluripotent stem cells. The generation of inducible stem cell lines with the Lent-ON-Plus LVs did not require selection or cloning, and transgene regulation was maintained after long-term cultured and upon differentiation toward different lineages. To our knowledge, Lent-On-Plus is the first all-in-one vector system that tightly regulates transgene expression in bulk populations of human pluripotent stem cells and its progeny.
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Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells
Benabdellah, Karim; Muñoz, Pilar; Cobo, Marién; Gutierrez-Guerrero, Alejandra; Sánchez-Hernández, Sabina; Garcia-Perez, Angélica; Anderson, Per; Carrillo-Gálvez, Ana Belén; Toscano, Miguel G.; Martin, Francisco
2016-01-01
Conditional transgene expression in human stem cells has been difficult to achieve due to the low efficiency of existing delivery methods, the strong silencing of the transgenes and the toxicity of the regulators. Most of the existing technologies are based on stem cells clones expressing appropriate levels of tTA or rtTA transactivators (based on the TetR-VP16 chimeras). In the present study, we aim the generation of Tet-On all-in-one lentiviral vectors (LVs) that tightly regulate transgene expression in human stem cells using the original TetR repressor. By using appropriate promoter combinations and shielding the LVs with the Is2 insulator, we have constructed the Lent-On-Plus Tet-On system that achieved efficient transgene regulation in human multipotent and pluripotent stem cells. The generation of inducible stem cell lines with the Lent-ON-Plus LVs did not require selection or cloning, and transgene regulation was maintained after long-term cultured and upon differentiation toward different lineages. To our knowledge, Lent-On-Plus is the first all-in-one vector system that tightly regulates transgene expression in bulk populations of human pluripotent stem cells and its progeny. PMID:27853296
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Vella, Danila; Zoppis, Italo; Mauri, Giancarlo; Mauri, Pierluigi; Di Silvestre, Dario
2017-12-01
The reductionist approach of dissecting biological systems into their constituents has been successful in the first stage of the molecular biology to elucidate the chemical basis of several biological processes. This knowledge helped biologists to understand the complexity of the biological systems evidencing that most biological functions do not arise from individual molecules; thus, realizing that the emergent properties of the biological systems cannot be explained or be predicted by investigating individual molecules without taking into consideration their relations. Thanks to the improvement of the current -omics technologies and the increasing understanding of the molecular relationships, even more studies are evaluating the biological systems through approaches based on graph theory. Genomic and proteomic data are often combined with protein-protein interaction (PPI) networks whose structure is routinely analyzed by algorithms and tools to characterize hubs/bottlenecks and topological, functional, and disease modules. On the other hand, co-expression networks represent a complementary procedure that give the opportunity to evaluate at system level including organisms that lack information on PPIs. Based on these premises, we introduce the reader to the PPI and to the co-expression networks, including aspects of reconstruction and analysis. In particular, the new idea to evaluate large-scale proteomic data by means of co-expression networks will be discussed presenting some examples of application. Their use to infer biological knowledge will be shown, and a special attention will be devoted to the topological and module analysis.
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NASA Technical Reports Server (NTRS)
Sitnikova, N.; Volkov, D.; Maximov, I.; Petrusevich, V.; Allen, D.
2003-01-01
This 12-part report documents the data obtained from various sensor measurements taken aboard the Russian Express-A2 and Express-A3 spacecraft in Geosynchronous Earth Orbit (GEO). These GEO communications satellites, which were designed and built by NPO Prikladnoy Mekhaniki (NPO PM) of Zheleznogorsk, Russia, utilize Hall thruster propulsion systems for north-south and east-west stationkeeping and as of June 2002, were still operating at 80 E. and 11 W., respectively. Express-A2 was launched on March 12, 2000, while Express-A3 was launched on June 24, 2000. The diagnostic equipment from which these data were taken includes electric field strength sensors, ion current and energy sensors, and pressure sensors. The diagnostics and the Hall thruster propulsion systems are described in detail along with lists of tabular data from those diagnostics and propulsion system and other satellite systems. Space Power, Inc., now part of Pratt & Whitney's Chemical Systems Division, under contract NAS3 99151 to the NASA Glenn Research Center, obtained these data over several periods from March 12, 2000, through September 30, 2001. Each of the 12 individual reports describe, in detail, the propulsion systems as well as the diagnostic sensors utilized. Finally, parts 11 and 12 include the requirements to which NPO PM prepared and delivered these data.
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Morgado, José Mário T; Sánchez-Muñoz, Laura; Teodósio, Cristina G; Jara-Acevedo, Maria; Alvarez-Twose, Iván; Matito, Almudena; Fernández-Nuñez, Elisa; García-Montero, Andrés; Orfao, Alberto; Escribano, Luís
2012-04-01
Aberrant expression of CD2 and/or CD25 by bone marrow, peripheral blood or other extracutaneous tissue mast cells is currently used as a minor World Health Organization diagnostic criterion for systemic mastocytosis. However, the diagnostic utility of CD2 versus CD25 expression by mast cells has not been prospectively evaluated in a large series of systemic mastocytosis. Here we evaluate the sensitivity and specificity of CD2 versus CD25 expression in the diagnosis of systemic mastocytosis. Mast cells from a total of 886 bone marrow and 153 other non-bone marrow extracutaneous tissue samples were analysed by multiparameter flow cytometry following the guidelines of the Spanish Network on Mastocytosis at two different laboratories. The 'CD25+ and/or CD2+ bone marrow mast cells' World Health Organization criterion showed an overall sensitivity of 100% with 99.0% specificity for the diagnosis of systemic mastocytosis whereas CD25 expression alone presented a similar sensitivity (100%) with a slightly higher specificity (99.2%). Inclusion of CD2 did not improve the sensitivity of the test and it decreased its specificity. In tissues other than bone marrow, the mast cell phenotypic criterion revealed to be less sensitive. In summary, CD2 expression does not contribute to improve the diagnosis of systemic mastocytosis when compared with aberrant CD25 expression alone, which supports the need to update and replace the minor World Health Organization 'CD25+ and/or CD2+' mast cell phenotypic diagnostic criterion by a major criterion based exclusively on CD25 expression.
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Viral delivery of genome-modifying proteins for cellular reprogramming.
Mikkelsen, Jacob Giehm
2018-06-18
Following the successful development of virus-based gene vehicles for genetic therapies, exploitation of viruses as carriers of genetic tools for cellular reprogramming and genome editing should be right up the street. However, whereas persistent, potentially life-long gene expression is the main goal of conventional genetic therapies, tools and bits for genome engineering should ideally be short-lived and active only for a limited time. Although viral vector systems have already been adapted for potent genome editing both in vitro and in vivo, regulatable gene expression systems or self-limiting expression circuits need to be implemented limiting exposure of chromatin to genome-modifying enzymes. As an alternative approach, emerging virus-based protein delivery technologies support direct protein delivery, providing a short, robust boost of enzymatic activity in transduced cells. Is this potentially the perfect way of shipping loads of cargo to many recipients and still maintain short-term activity? Copyright © 2018 Elsevier Ltd. All rights reserved.
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Windass, J D; Newton, C R; De Maeyer-Guignard, J; Moore, V E; Markham, A F; Edge, M D
1982-01-01
An 82 base pair DNA fragment has been synthesised which contains the E. coli trp promoter and operator sequences and also encodes the first Shine Dalgarno sequence of the trp operon. This DNA fragment is flanked by EcoRI and ClaI/TaqI cohesive ends and is thus easy to clone, transfer between vector systems and couple to genes to drive their expression. It has been cloned into plasmid pAT153, producing a convenient trp promoter vector. We have also joined the fragment to a synthetic IFN-alpha 1 gene, using synthetic oligonucleotides to generate a completely natural, highly efficient bacterial translation initiation signal on the promoter proximal side of the IFN gene. Plasmids carrying this construction enable E. coli cells to express IFN-alpha 1 almost constitutively and with significantly higher efficiency than from a lacUV5 promoter based system. Images PMID:6184675
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Extreme Facial Expressions Classification Based on Reality Parameters
NASA Astrophysics Data System (ADS)
Rahim, Mohd Shafry Mohd; Rad, Abdolvahab Ehsani; Rehman, Amjad; Altameem, Ayman
2014-09-01
Extreme expressions are really type of emotional expressions that are basically stimulated through the strong emotion. An example of those extreme expression is satisfied through tears. So to be able to provide these types of features; additional elements like fluid mechanism (particle system) plus some of physics techniques like (SPH) are introduced. The fusion of facile animation with SPH exhibits promising results. Accordingly, proposed fluid technique using facial animation is the real tenor for this research to get the complex expression, like laugh, smile, cry (tears emergence) or the sadness until cry strongly, as an extreme expression classification that's happens on the human face in some cases.
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Expression of short hairpin RNAs using the compact architecture of retroviral microRNA genes.
Burke, James M; Kincaid, Rodney P; Aloisio, Francesca; Welch, Nicole; Sullivan, Christopher S
2017-09-29
Short hairpin RNAs (shRNAs) are effective in generating stable repression of gene expression. RNA polymerase III (RNAP III) type III promoters (U6 or H1) are typically used to drive shRNA expression. While useful for some knockdown applications, the robust expression of U6/H1-driven shRNAs can induce toxicity and generate heterogeneous small RNAs with undesirable off-target effects. Additionally, typical U6/H1 promoters encompass the majority of the ∼270 base pairs (bp) of vector space required for shRNA expression. This can limit the efficacy and/or number of delivery vector options, particularly when delivery of multiple gene/shRNA combinations is required. Here, we develop a compact shRNA (cshRNA) expression system based on retroviral microRNA (miRNA) gene architecture that uses RNAP III type II promoters. We demonstrate that cshRNAs coded from as little as 100 bps of total coding space can precisely generate small interfering RNAs (siRNAs) that are active in the RNA-induced silencing complex (RISC). We provide an algorithm with a user-friendly interface to design cshRNAs for desired target genes. This cshRNA expression system reduces the coding space required for shRNA expression by >2-fold as compared to the typical U6/H1 promoters, which may facilitate therapeutic RNAi applications where delivery vector space is limiting. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
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Lancioni, Giulio E; Bellini, Domenico; Oliva, Doretta; Singh, Nirbhay N; O'reilly, Mark F; Lang, Russell; Didden, Robert; Bosco, Andrea
2011-01-01
To assess whether two persons with multiple disabilities could use smile expressions and new camera-based microswitch technology to select environmental stimuli. Within each session, a computer system provided samples/reminders of preferred and non-preferred stimuli. The camera-based microswitch determined whether the participants had smile expressions in relation to those samples. If they did, stimuli matching the specific samples to which they responded were presented for 20 seconds. The smile expression could be profitably used by the participants who managed to select means of ∼70% or 75% of the preferred stimulus opportunities made available by the environment while avoiding almost all the non-preferred stimulus opportunities. Smile expressions (a) might be an effective and rapid means for selecting preferred stimulation and (b) might develop into cognitively more elaborate forms of responding through the learning experience (i.e. their consistent association with positive/reinforcing consequences).
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Zhang, Liangyu; Ward, Jordan D.; Cheng, Ze; Dernburg, Abby F.
2015-01-01
Experimental manipulation of protein abundance in living cells or organisms is an essential strategy for investigation of biological regulatory mechanisms. Whereas powerful techniques for protein expression have been developed in Caenorhabditis elegans, existing tools for conditional disruption of protein function are far more limited. To address this, we have adapted the auxin-inducible degradation (AID) system discovered in plants to enable conditional protein depletion in C. elegans. We report that expression of a modified Arabidopsis TIR1 F-box protein mediates robust auxin-dependent depletion of degron-tagged targets. We document the effectiveness of this system for depletion of nuclear and cytoplasmic proteins in diverse somatic and germline tissues throughout development. Target proteins were depleted in as little as 20-30 min, and their expression could be re-established upon auxin removal. We have engineered strains expressing TIR1 under the control of various promoter and 3′ UTR sequences to drive tissue-specific or temporally regulated expression. The degron tag can be efficiently introduced by CRISPR/Cas9-based genome editing. We have harnessed this system to explore the roles of dynamically expressed nuclear hormone receptors in molting, and to analyze meiosis-specific roles for proteins required for germ line proliferation. Together, our results demonstrate that the AID system provides a powerful new tool for spatiotemporal regulation and analysis of protein function in a metazoan model organism. PMID:26552885
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Dynamic facial expression recognition based on geometric and texture features
NASA Astrophysics Data System (ADS)
Li, Ming; Wang, Zengfu
2018-04-01
Recently, dynamic facial expression recognition in videos has attracted growing attention. In this paper, we propose a novel dynamic facial expression recognition method by using geometric and texture features. In our system, the facial landmark movements and texture variations upon pairwise images are used to perform the dynamic facial expression recognition tasks. For one facial expression sequence, pairwise images are created between the first frame and each of its subsequent frames. Integration of both geometric and texture features further enhances the representation of the facial expressions. Finally, Support Vector Machine is used for facial expression recognition. Experiments conducted on the extended Cohn-Kanade database show that our proposed method can achieve a competitive performance with other methods.
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Liu, Zengjian; Tang, Buzhou; Wang, Xiaolong; Chen, Qingcai; Li, Haodi; Bu, Junzhao; Jiang, Jingzhi; Deng, Qiwen; Zhu, Suisong
2016-01-01
Time is an important aspect of information and is very useful for information utilization. The goal of this study was to analyze the challenges of temporal expression (TE) extraction and normalization in Chinese clinical notes by assessing the performance of a rule-based system developed by us on a manually annotated corpus (including 1,778 clinical notes of 281 hospitalized patients). In order to develop system conveniently, we divided TEs into three categories: direct, indirect and uncertain TEs, and designed different rules for each category of them. Evaluation on the independent test set shows that our system achieves an F-score of93.40% on TE extraction, and an accuracy of 92.58% on TE normalization under "exact-match" criterion. Compared with HeidelTime for Chinese newswire text, our system is much better, indicating that it is necessary to develop a specific TE extraction and normalization system for Chinese clinical notes because of domain difference.
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Artificial intelligence in hematology.
Zini, Gina
2005-10-01
Artificial intelligence (AI) is a computer based science which aims to simulate human brain faculties using a computational system. A brief history of this new science goes from the creation of the first artificial neuron in 1943 to the first artificial neural network application to genetic algorithms. The potential for a similar technology in medicine has immediately been identified by scientists and researchers. The possibility to store and process all medical knowledge has made this technology very attractive to assist or even surpass clinicians in reaching a diagnosis. Applications of AI in medicine include devices applied to clinical diagnosis in neurology and cardiopulmonary diseases, as well as the use of expert or knowledge-based systems in routine clinical use for diagnosis, therapeutic management and for prognostic evaluation. Biological applications include genome sequencing or DNA gene expression microarrays, modeling gene networks, analysis and clustering of gene expression data, pattern recognition in DNA and proteins, protein structure prediction. In the field of hematology the first devices based on AI have been applied to the routine laboratory data management. New tools concern the differential diagnosis in specific diseases such as anemias, thalassemias and leukemias, based on neural networks trained with data from peripheral blood analysis. A revolution in cancer diagnosis, including the diagnosis of hematological malignancies, has been the introduction of the first microarray based and bioinformatic approach for molecular diagnosis: a systematic approach based on the monitoring of simultaneous expression of thousands of genes using DNA microarray, independently of previous biological knowledge, analysed using AI devices. Using gene profiling, the traditional diagnostic pathways move from clinical to molecular based diagnostic systems.
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Taroni, Jaclyn N; Martyanov, Viktor; Mahoney, J Matthew; Whitfield, Michael L
2017-05-01
Systemic sclerosis is an orphan, systemic autoimmune disease with no FDA-approved treatments. Its heterogeneity and rarity often result in underpowered clinical trials making the analysis and interpretation of associated molecular data challenging. We performed a meta-analysis of gene expression data from skin biopsies of patients with systemic sclerosis treated with five therapies: mycophenolate mofetil, rituximab, abatacept, nilotinib, and fresolimumab. A common clinical improvement criterion of -20% or -5 modified Rodnan skin score was applied to each study. We applied a machine learning approach that captured features beyond differential expression and was better at identifying targets of therapies than the differential expression alone. Regardless of treatment mechanism, abrogation of inflammatory pathways accompanied clinical improvement in multiple studies suggesting that high expression of immune-related genes indicates active and targetable disease. Our framework allowed us to compare different trials and ask if patients who failed one therapy would likely improve on a different therapy, based on changes in gene expression. Genes with high expression at baseline in fresolimumab nonimprovers were downregulated in mycophenolate mofetil improvers, suggesting that immunomodulatory or combination therapy may have benefitted these patients. This approach can be broadly applied to increase tissue specificity and sensitivity of differential expression results. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.
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Art critic: Multisignal vision and speech interaction system in a gaming context.
Reale, Michael J; Liu, Peng; Yin, Lijun; Canavan, Shaun
2013-12-01
True immersion of a player within a game can only occur when the world simulated looks and behaves as close to reality as possible. This implies that the game must correctly read and understand, among other things, the player's focus, attitude toward the objects/persons in focus, gestures, and speech. In this paper, we proposed a novel system that integrates eye gaze estimation, head pose estimation, facial expression recognition, speech recognition, and text-to-speech components for use in real-time games. Both the eye gaze and head pose components utilize underlying 3-D models, and our novel head pose estimation algorithm uniquely combines scene flow with a generic head model. The facial expression recognition module uses the local binary patterns with three orthogonal planes approach on the 2-D shape index domain rather than the pixel domain, resulting in improved classification. Our system has also been extended to use a pan-tilt-zoom camera driven by the Kinect, allowing us to track a moving player. A test game, Art Critic, is also presented, which not only demonstrates the utility of our system but also provides a template for player/non-player character (NPC) interaction in a gaming context. The player alters his/her view of the 3-D world using head pose, looks at paintings/NPCs using eye gaze, and makes an evaluation based on the player's expression and speech. The NPC artist will respond with facial expression and synthetic speech based on its personality. Both qualitative and quantitative evaluations of the system are performed to illustrate the system's effectiveness.
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Mathematical Modeling of RNA-Based Architectures for Closed Loop Control of Gene Expression.
Agrawal, Deepak K; Tang, Xun; Westbrook, Alexandra; Marshall, Ryan; Maxwell, Colin S; Lucks, Julius; Noireaux, Vincent; Beisel, Chase L; Dunlop, Mary J; Franco, Elisa
2018-05-08
Feedback allows biological systems to control gene expression precisely and reliably, even in the presence of uncertainty, by sensing and processing environmental changes. Taking inspiration from natural architectures, synthetic biologists have engineered feedback loops to tune the dynamics and improve the robustness and predictability of gene expression. However, experimental implementations of biomolecular control systems are still far from satisfying performance specifications typically achieved by electrical or mechanical control systems. To address this gap, we present mathematical models of biomolecular controllers that enable reference tracking, disturbance rejection, and tuning of the temporal response of gene expression. These controllers employ RNA transcriptional regulators to achieve closed loop control where feedback is introduced via molecular sequestration. Sensitivity analysis of the models allows us to identify which parameters influence the transient and steady state response of a target gene expression process, as well as which biologically plausible parameter values enable perfect reference tracking. We quantify performance using typical control theory metrics to characterize response properties and provide clear selection guidelines for practical applications. Our results indicate that RNA regulators are well-suited for building robust and precise feedback controllers for gene expression. Additionally, our approach illustrates several quantitative methods useful for assessing the performance of biomolecular feedback control systems.
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Okada, D; Endo, S; Matsuda, H; Ogawa, S; Taniguchi, Y; Katsuta, T; Watanabe, T; Iwaisaki, H
2018-05-12
Genome-wide association studies (GWAS) of quantitative traits have detected numerous genetic associations, but they encounter difficulties in pinpointing prominent candidate genes and inferring gene networks. The present study used a systems genetics approach integrating GWAS results with external RNA-expression data to detect candidate gene networks in feed utilization and growth traits of Japanese Black cattle, which are matters of concern. A SNP co-association network was derived from significant correlations between SNPs with effects estimated by GWAS across seven phenotypic traits. The resulting network genes contained significant numbers of annotations related to the traits. Using bovine transcriptome data from a public database, an RNA co-expression network was inferred based on the similarity of expression patterns across different tissues. An intersection network was then generated by superimposing the SNP and RNA networks and extracting shared interactions. This intersection network contained four tissue-specific modules: nervous system, reproductive system, muscular system, and glands. To characterize the structure (topographical properties) of the three networks, their scale-free properties were evaluated, which revealed that the intersection network was the most scale-free. In the sub-network containing the most connected transcription factors (URI1, ROCK2 and ETV6), most genes were widely expressed across tissues, and genes previously shown to be involved in the traits were found. Results indicated that the current approach might be used to construct a gene network that better reflects biological information, providing encouragement for the genetic dissection of economically important quantitative traits.
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Architecture studies and system demonstrations for optical parallel processor for AI and NI
NASA Astrophysics Data System (ADS)
Lee, Sing H.
1988-03-01
In solving deterministic AI problems the data search for matching the arguments of a PROLOG expression causes serious bottleneck when implemented sequentially by electronic systems. To overcome this bottleneck we have developed the concepts for an optical expert system based on matrix-algebraic formulation, which will be suitable for parallel optical implementation. The optical AI system based on matrix-algebraic formation will offer distinct advantages for parallel search, adult learning, etc.
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Hablützel, Pascal I; Brown, Martha; Friberg, Ida M; Jackson, Joseph A
2016-09-01
The effect of anthropogenic environments on the function of the vertebrate immune system is a problem of general importance. For example, it relates to the increasing rates of immunologically-based disease in modern human populations and to the desirability of identifying optimal immune function in domesticated animals. Despite this importance, our present understanding is compromised by a deficit of experimental studies that make adequately matched comparisons between wild and captive vertebrates. We transferred post-larval fishes (three-spined sticklebacks), collected in the wild, to an anthropogenic (captive) environment. We then monitored, over 11 months, how the systemic expression of immunity genes changed in comparison to cohort-matched wild individuals in the originator population (total n = 299). We found that a range of innate (lyz, defbl2, il1r-like, tbk1) and adaptive (cd8a, igmh) immunity genes were up-regulated in captivity, accompanied by an increase in expression of the antioxidant enzyme, gpx4a. For some genes previously known to show seasonality in the wild, this appeared to be reduced in captive fishes. Captive fishes tended to express immunity genes, including igzh, foxp3b, lyz, defbl2, and il1r-like, more variably. Furthermore, although gene co-expression patterns (analyzed through gene-by-gene correlations and mutual information theory based networks) shared common structure in wild and captive fishes, there was also significant divergence. For one gene in particular, defbl2, high expression was associated with adverse health outcomes in captive fishes. Taken together, these results demonstrate widespread regulatory changes in the immune system in captive populations, and that the expression of immunity genes is more constrained in the wild. An increase in constitutive systemic immune activity, such as we observed here, may alter the risk of immunopathology and contribute to variance in health in vertebrate populations exposed to anthropogenic environments.
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Development of a Radar-Frequency Metamaterial Measurement and Characterization Apparatus
2012-03-01
Patterson Air Force Base, Ohio APPROVED FOR PUBLIC RELEASE; DISTRIBUTION IS UNLIMITED. The views expressed...17 GTRI Focused Beam System ................................................................................... 22 Shelby Parallel-Plate Waveguide...System ......................................................... 23 Figure 4: CST Model of Shelby PPWG
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Using rabies virus vaccine strain SRV9 as viral vector to express exogenous gene.
Wang, Hualei; Jin, Hongli; Feng, Na; Zheng, Xuexing; Li, Ling; Qi, Yinglin; Liang, Meng; Zhao, Yongkun; Wang, Tiecheng; Gao, Yuwei; Tu, Changchun; Jin, Ningyi; Yang, Songtao; Xia, Xianzhu
2015-04-01
Rabies virus (RABV) can cause a fatal neurological disease in human and animals, and vaccines were generally applied for the immunoprophylaxis of rabies. Here, a recombinant viral vector carrying the exogenous gene expression component between phosphoprotein (P) and matrix protein (M) genes of RABV was constructed based on the vaccine strain SRV9 used in China. To develop a reverse genetic system, the full-length cDNA plasmids of SRV9 were constructed using the eukaryotic expression vector pCI or pcDNA3.1(+). However, recovery efficiency based on the pcDNA3.1 vector was significantly higher than that of the pCI vector. The exogenous gene expression component PE-PS-BsiWI-PmeI or PS-BsiWI-PmeI-PE was introduced in different locations between the P and M genes of SRV9. When the enhanced green fluorescent protein (eGFP) was used as a reporter gene, both locations could rescue recombinant RABV (rRABV) expressing eGFP with high efficiency. Characterization of rRABV expressing eGFP in vitro revealed that its growth was similar to that of the parental virus. Animal experiments showed that rRABV expressing eGFP could replicate and express eGFP in the brains of suckling mice. Furthermore, rRABV of SRV9 was nonpathogenic for 3-week-old mice and could be cleared from the central nervous system at 5 days post-inoculation. Our results showed that the recombinant SRV9 virus could be used as a useful viral vector for exogenous gene expression.
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Data from clinical notes: a perspective on the tension between structure and flexible documentation
Denny, Joshua C; Xu, Hua; Lorenzi, Nancy; Stead, William W; Johnson, Kevin B
2011-01-01
Clinical documentation is central to patient care. The success of electronic health record system adoption may depend on how well such systems support clinical documentation. A major goal of integrating clinical documentation into electronic heath record systems is to generate reusable data. As a result, there has been an emphasis on deploying computer-based documentation systems that prioritize direct structured documentation. Research has demonstrated that healthcare providers value different factors when writing clinical notes, such as narrative expressivity, amenability to the existing workflow, and usability. The authors explore the tension between expressivity and structured clinical documentation, review methods for obtaining reusable data from clinical notes, and recommend that healthcare providers be able to choose how to document patient care based on workflow and note content needs. When reusable data are needed from notes, providers can use structured documentation or rely on post-hoc text processing to produce structured data, as appropriate. PMID:21233086
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FaceTOON: a unified platform for feature-based cartoon expression generation
NASA Astrophysics Data System (ADS)
Zaharia, Titus; Marre, Olivier; Prêteux, Françoise; Monjaux, Perrine
2008-02-01
This paper presents the FaceTOON system, a semi-automatic platform dedicated to the creation of verbal and emotional facial expressions, within the applicative framework of 2D cartoon production. The proposed FaceTOON platform makes it possible to rapidly create 3D facial animations with a minimum amount of user interaction. In contrast with existing commercial 3D modeling softwares, which usually require from the users advanced 3D graphics skills and competences, the FaceTOON system is based exclusively on 2D interaction mechanisms, the 3D modeling stage being completely transparent for the user. The system takes as input a neutral 3D face model, free of any facial feature, and a set of 2D drawings, representing the desired facial features. A 2D/3D virtual mapping procedure makes it possible to obtain a ready-for-animation model which can be directly manipulated and deformed for generating expressions. The platform includes a complete set of dedicated tools for 2D/3D interactive deformation, pose management, key-frame interpolation and MPEG-4 compliant animation and rendering. The proposed FaceTOON system is currently considered for industrial evaluation and commercialization by the Quadraxis company.
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Resilience of bacterial quorum sensing against fluid flow
NASA Astrophysics Data System (ADS)
Emge, Philippe; Moeller, Jens; Jang, Hongchul; Rusconi, Roberto; Yawata, Yutaka; Stocker, Roman; Vogel, Viola
2016-09-01
Quorum sensing (QS) is a population-density dependent chemical process that enables bacteria to communicate based on the production, secretion and sensing of small inducer molecules. While recombinant constructs have been widely used to decipher the molecular details of QS, how those findings translate to natural QS systems has remained an open question. Here, we compare the activation of natural and synthetic Pseudomonas aeruginosa LasI/R QS systems in bacteria exposed to quiescent conditions and controlled flows. Quantification of QS-dependent GFP expression in suspended cultures and in surface-attached microcolonies revealed that QS onset in both systems was similar under quiescent conditions but markedly differed under flow. Moderate flow (Pe > 25) was sufficient to suppress LasI/R QS recombinantly expressed in Escherichia coli, whereas only high flow (Pe > 102) suppressed QS in wild-type P. aeruginosa. We suggest that this difference stems from the differential production of extracellular matrix and that the matrix confers resilience against moderate flow to QS in wild-type organisms. These results suggest that the expression of a biofilm matrix extends the environmental conditions under which QS-based cell-cell communication is effective and that findings from synthetic QS circuits cannot be directly translated to natural systems.
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Compositional and enumerative designs for medical language representation.
Rassinoux, A. M.; Miller, R. A.; Baud, R. H.; Scherrer, J. R.
1997-01-01
Medical language is in essence highly compositional, allowing complex information to be expressed from more elementary pieces. Embedding the expressive power of medical language into formal systems of representation is recognized in the medical informatics community as a key step towards sharing such information among medical record, decision support, and information retrieval systems. Accordingly, such representation requires managing both the expressiveness of the formalism and its computational tractability, while coping with the level of detail expected by clinical applications. These desiderata can be supported by enumerative as well as compositional approaches, as argued in this paper. These principles have been applied in recasting a frame-based system for general medical findings developed during the 1980s. The new system captures the precise meaning of a subset of over 1500 medical terms for general internal medicine identified from the Quick Medical Reference (QMR) lexicon. In order to evaluate the adequacy of this formal structure in reflecting the deep meaning of the QMR findings, a validation process was implemented. It consists of automatically rebuilding the semantic representation of the QMR findings by analyzing them through the RECIT natural language analyzer, whose semantic components have been adjusted to this frame-based model for the understanding task. PMID:9357700
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Compositional and enumerative designs for medical language representation.
Rassinoux, A M; Miller, R A; Baud, R H; Scherrer, J R
1997-01-01
Medical language is in essence highly compositional, allowing complex information to be expressed from more elementary pieces. Embedding the expressive power of medical language into formal systems of representation is recognized in the medical informatics community as a key step towards sharing such information among medical record, decision support, and information retrieval systems. Accordingly, such representation requires managing both the expressiveness of the formalism and its computational tractability, while coping with the level of detail expected by clinical applications. These desiderata can be supported by enumerative as well as compositional approaches, as argued in this paper. These principles have been applied in recasting a frame-based system for general medical findings developed during the 1980s. The new system captures the precise meaning of a subset of over 1500 medical terms for general internal medicine identified from the Quick Medical Reference (QMR) lexicon. In order to evaluate the adequacy of this formal structure in reflecting the deep meaning of the QMR findings, a validation process was implemented. It consists of automatically rebuilding the semantic representation of the QMR findings by analyzing them through the RECIT natural language analyzer, whose semantic components have been adjusted to this frame-based model for the understanding task.
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Lai, Huafang; He, Junyun; Engle, Michael; Diamond, Michael S.; Chen, Qiang
2011-01-01
Summary Pharmaceutical protein production in plants has been greatly promoted by the development of viral-based vectors and transient expression systems. Tobacco and related Nicotiana species are currently the most common host plants for generation of plant-made pharmaceutical proteins (PMPs). Downstream processing of target PMPs from these plants, however, is hindered by potential technical and regulatory difficulties due to the presence of high levels of phenolics and toxic alkaloids. Here, we explored the use of lettuce, which grows quickly yet produces low levels of secondary metabolites, and viral vector-based transient expression systems to develop a robust PMP production platform. Our results showed that a geminiviral replicon system based on the bean yellow dwarf virus permits high-level expression in lettuce of virus-like particles (VLP) derived from the Norwalk virus capsid protein and therapeutic monoclonal antibodies (mAbs) against Ebola and West Nile viruses. These vaccine and therapeutic candidates can be readily purified from lettuce leaves with scalable processing methods while fully retaining functional activity. Furthermore, this study also demonstrated the feasibility of using commercially produced lettuce for high-level PMP production. This allows our production system to have access to unlimited quantities of inexpensive plant material for large-scale production. These results establish a new production platform for biological pharmaceutical agents that is effective, safe, low-cost, and amenable to large-scale manufacturing. PMID:21883868
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USDA-ARS?s Scientific Manuscript database
Expression of dsRNA fragments of rust pathogen genes in wheat seedlings through the barley stripe mosaic virus (BSMV) based host-induced gene silencing (HIGS) system can reduce the expression of the corresponding genes in the rust fungus. The highest levels of suppression have generally been observe...
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A flow cytometry-based strategy to identify and express IgM from VH1-69+ clonal peripheral B cells.
Charles, Edgar D; Orloff, Michael I M; Dustin, Lynn B
2011-01-05
Pathologic rheumatoid factor (RF) levels are hallmarks of several human diseases. Production of monoclonal RF in vitro is essential for studies of the antigenic specificities of RF, as well as for a dissection of the mechanisms of aberrant RF+ B cell activation. We have expanded upon previous methods to develop a flow cytometry-based method to efficiently clone monoclonal antibodies (mAbs) from humans with expansions of RF-like, immunoglobulin heavy chain variable region (IgVH) 1-69 gene segment-containing B cells. The cloned variable regions are expressed as IgM and produced during culture at concentrations between 5 and 20 μg/ml. Using this system, we show that clonal Igs from patients with HCV-related mixed cryoglobulinemia, when expressed as IgM, have RF activity. We anticipate that this system will be useful for the cloning and expression of mAbs partially encoded by VH1-69 and for determination of the reactivity patterns of polyspecific, low-affinity IgMs of human pathogenic importance. Copyright © 2010 Elsevier B.V. All rights reserved.
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A Systems' Biology Approach to Study MicroRNA-Mediated Gene Regulatory Networks
Kunz, Manfred; Vera, Julio; Wolkenhauer, Olaf
2013-01-01
MicroRNAs (miRNAs) are potent effectors in gene regulatory networks where aberrant miRNA expression can contribute to human diseases such as cancer. For a better understanding of the regulatory role of miRNAs in coordinating gene expression, we here present a systems biology approach combining data-driven modeling and model-driven experiments. Such an approach is characterized by an iterative process, including biological data acquisition and integration, network construction, mathematical modeling and experimental validation. To demonstrate the application of this approach, we adopt it to investigate mechanisms of collective repression on p21 by multiple miRNAs. We first construct a p21 regulatory network based on data from the literature and further expand it using algorithms that predict molecular interactions. Based on the network structure, a detailed mechanistic model is established and its parameter values are determined using data. Finally, the calibrated model is used to study the effect of different miRNA expression profiles and cooperative target regulation on p21 expression levels in different biological contexts. PMID:24350286
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Rational design of aptazyme riboswitches for efficient control of gene expression in mammalian cells
Zhong, Guocai; Wang, Haimin; Bailey, Charles C; Gao, Guangping; Farzan, Michael
2016-01-01
Efforts to control mammalian gene expression with ligand-responsive riboswitches have been hindered by lack of a general method for generating efficient switches in mammalian systems. Here we describe a rational-design approach that enables rapid development of efficient cis-acting aptazyme riboswitches. We identified communication-module characteristics associated with aptazyme functionality through analysis of a 32-aptazyme test panel. We then developed a scoring system that predicts an aptazymes’s activity by integrating three characteristics of communication-module bases: hydrogen bonding, base stacking, and distance to the enzymatic core. We validated the power and generality of this approach by designing aptazymes responsive to three distinct ligands, each with markedly wider dynamic ranges than any previously reported. These aptayzmes efficiently regulated adeno-associated virus (AAV)-vectored transgene expression in cultured mammalian cells and mice, highlighting one application of these broadly usable regulatory switches. Our approach enables efficient, protein-independent control of gene expression by a range of small molecules. DOI: http://dx.doi.org/10.7554/eLife.18858.001 PMID:27805569
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Hepatic differentiation of human iPSCs in different 3D models: A comparative study.
Meier, Florian; Freyer, Nora; Brzeszczynska, Joanna; Knöspel, Fanny; Armstrong, Lyle; Lako, Majlinda; Greuel, Selina; Damm, Georg; Ludwig-Schwellinger, Eva; Deschl, Ulrich; Ross, James A; Beilmann, Mario; Zeilinger, Katrin
2017-12-01
Human induced pluripotent stem cells (hiPSCs) are a promising source from which to derive distinct somatic cell types for in vitro or clinical use. Existent protocols for hepatic differentiation of hiPSCs are primarily based on 2D cultivation of the cells. In the present study, the authors investigated the generation of hiPSC-derived hepatocyte-like cells using two different 3D culture systems: A 3D scaffold-free microspheroid culture system and a 3D hollow-fiber perfusion bioreactor. The differentiation outcome in these 3D systems was compared with that in conventional 2D cultures, using primary human hepatocytes as a control. The evaluation was made based on specific mRNA expression, protein secretion, antigen expression and metabolic activity. The expression of α-fetoprotein was lower, while cytochrome P450 1A2 or 3A4 activities were higher in the 3D culture systems as compared with the 2D differentiation system. Cells differentiated in the 3D bioreactor showed an increased expression of albumin and hepatocyte nuclear factor 4α, as well as secretion of α-1-antitrypsin as compared with the 2D differentiation system, suggesting a higher degree of maturation. In contrast, the 3D scaffold-free microspheroid culture provides an easy and robust method to generate spheroids of a defined size for screening applications, while the bioreactor culture model provides an instrument for complex investigations under physiological-like conditions. In conclusion, the present study introduces two 3D culture systems for stem cell derived hepatic differentiation each demonstrating advantages for individual applications as well as benefits in comparison with 2D cultures.
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Hepatic differentiation of human iPSCs in different 3D models: A comparative study
Brzeszczynska, Joanna; Knöspel, Fanny; Armstrong, Lyle; Lako, Majlinda; Greuel, Selina; Damm, Georg; Ludwig-Schwellinger, Eva; Deschl, Ulrich; Ross, James A.
2017-01-01
Human induced pluripotent stem cells (hiPSCs) are a promising source from which to derive distinct somatic cell types for in vitro or clinical use. Existent protocols for hepatic differentiation of hiPSCs are primarily based on 2D cultivation of the cells. In the present study, the authors investigated the generation of hiPSC-derived hepatocyte-like cells using two different 3D culture systems: A 3D scaffold-free microspheroid culture system and a 3D hollow-fiber perfusion bioreactor. The differentiation outcome in these 3D systems was compared with that in conventional 2D cultures, using primary human hepatocytes as a control. The evaluation was made based on specific mRNA expression, protein secretion, antigen expression and metabolic activity. The expression of α-fetoprotein was lower, while cytochrome P450 1A2 or 3A4 activities were higher in the 3D culture systems as compared with the 2D differentiation system. Cells differentiated in the 3D bioreactor showed an increased expression of albumin and hepatocyte nuclear factor 4α, as well as secretion of α-1-antitrypsin as compared with the 2D differentiation system, suggesting a higher degree of maturation. In contrast, the 3D scaffold-free microspheroid culture provides an easy and robust method to generate spheroids of a defined size for screening applications, while the bioreactor culture model provides an instrument for complex investigations under physiological-like conditions. In conclusion, the present study introduces two 3D culture systems for stem cell derived hepatic differentiation each demonstrating advantages for individual applications as well as benefits in comparison with 2D cultures. PMID:29039463
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Maruthamuthu, Murali Kannan; Ganesh, Irisappan; Ravikumar, Sambandam; Hong, Soon Ho
2015-03-01
A ZraP-based lead sensing and removal system was constructed in E. coli. It was regulated by the ZraS/ZraR two-component system. The expression profile of the zraP gene towards extracellular lead was studied via real-time PCR. A dual-function bacterial system was also designed to express GFP and OmpC-lead binding peptide under the control of zraP for the simultaneous sensing and adsorption of environmental lead without additional manipulation. The constructed bacterial system can emit fluorescence and it adsorbed a maximum of 487 µmol lead/g cell DCW. From a study of artificial wastewater, the constructed bacteria adsorbed lead highly selectively (427 µmol lead/g cell DCW) among other metal ions. The newly-constructed dual function bacterial system can be applied for the development of an efficient process for the removal of lead from polluted wastes.
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REGIME CHANGES IN ECOLOGICAL SYSTEMS: AN INFORMATION THEORY APPROACH
We present our efforts at developing an ecological system using Information Theory. We derive an expression for Fisher Information based on sampling of the system trajectory as it evolves in the state space. The Fisher Information index as we have derived it captures the characte...
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Subgenomic Reporter RNA System for Detection of Alphavirus Infection in Mosquitoes
Steel, J. Jordan; Franz, Alexander W. E.; Sanchez-Vargas, Irma; Olson, Ken E.; Geiss, Brian J.
2013-01-01
Current methods for detecting real-time alphavirus (Family Togaviridae) infection in mosquitoes require the use of recombinant viruses engineered to express a visibly detectable reporter protein. These altered viruses expressing fluorescent proteins, usually from a duplicated viral subgenomic reporter, are effective at marking infection but tend to be attenuated due to the modification of the genome. Additionally, field strains of viruses cannot be visualized using this approach unless infectious clones can be developed to insert a reporter protein. To circumvent these issues, we have developed an insect cell-based system for detecting wild-type sindbis virus infection that uses a virus inducible promoter to express a fluorescent reporter gene only upon active virus infection. We have developed an insect expression system that produces sindbis virus minigenomes containing a subgenomic promoter sequence, which produces a translatable RNA species only when infectious virus is present and providing viral replication proteins. This subgenomic reporter RNA system is able to detect wild-type Sindbis infection in cultured mosquito cells. The detection system is relatively species specific and only detects closely related viruses, but can detect low levels of alphavirus specific replication early during infection. A chikungunya virus detection system was also developed that specifically detects chikungunya virus infection. Transgenic Aedes aegypti mosquito families were established that constitutively express the sindbis virus reporter RNA and were found to only express fluorescent proteins during virus infection. This virus inducible reporter system demonstrates a novel approach for detecting non-recombinant virus infection in mosquito cell culture and in live transgenic mosquitoes. PMID:24367703
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NASA Astrophysics Data System (ADS)
Sereda, T. G.; Kostarev, S. N.
2018-03-01
Theoretical bases of linkage of material streams of the machine-building enterprise and the automated system of decision-making are developed. The process of machine-building manufacture is submitted by the existential system. The equation of preservation of movement is based on calculation of volume of manufacture. The basis of resource variables includes capacities and operators of the equipment. Indignations such as a defect and failure are investigated in the existential basis. The equation of a stream of details on a manufacturing route is made. The received analytical expression expresses a condition of a stream of movement of details in view of influence of work of the equipment and traumatism of the personnel.
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The symbolic computation and automatic analysis of trajectories
NASA Technical Reports Server (NTRS)
Grossman, Robert
1991-01-01
Research was generally done on computation of trajectories of dynamical systems, especially control systems. Algorithms were further developed for rewriting expressions involving differential operators. The differential operators involved arise in the local analysis of nonlinear control systems. An initial design was completed of the system architecture for software to analyze nonlinear control systems using data base computing.
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Reynier, Frédéric; Petit, Fabien; Paye, Malick; Turrel-Davin, Fanny; Imbert, Pierre-Emmanuel; Hot, Arnaud; Mougin, Bruno; Miossec, Pierre
2011-01-01
The analysis of gene expression data shows that many genes display similarity in their expression profiles suggesting some co-regulation. Here, we investigated the co-expression patterns in gene expression data and proposed a correlation-based research method to stratify individuals. Using blood from rheumatoid arthritis (RA) patients, we investigated the gene expression profiles from whole blood using Affymetrix microarray technology. Co-expressed genes were analyzed by a biclustering method, followed by gene ontology analysis of the relevant biclusters. Taking the type I interferon (IFN) pathway as an example, a classification algorithm was developed from the 102 RA patients and extended to 10 systemic lupus erythematosus (SLE) patients and 100 healthy volunteers to further characterize individuals. We developed a correlation-based algorithm referred to as Classification Algorithm Based on a Biological Signature (CABS), an alternative to other approaches focused specifically on the expression levels. This algorithm applied to the expression of 35 IFN-related genes showed that the IFN signature presented a heterogeneous expression between RA, SLE and healthy controls which could reflect the level of global IFN signature activation. Moreover, the monitoring of the IFN-related genes during the anti-TNF treatment identified changes in type I IFN gene activity induced in RA patients. In conclusion, we have proposed an original method to analyze genes sharing an expression pattern and a biological function showing that the activation levels of a biological signature could be characterized by its overall state of correlation.
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Quinn, Michael C J; Wilson, Daniel J; Young, Fiona; Dempsey, Adam A; Arcand, Suzanna L; Birch, Ashley H; Wojnarowicz, Paulina M; Provencher, Diane; Mes-Masson, Anne-Marie; Englert, David; Tonin, Patricia N
2009-07-06
As gene expression signatures may serve as biomarkers, there is a need to develop technologies based on mRNA expression patterns that are adaptable for translational research. Xceed Molecular has recently developed a Ziplex technology, that can assay for gene expression of a discrete number of genes as a focused array. The present study has evaluated the reproducibility of the Ziplex system as applied to ovarian cancer research of genes shown to exhibit distinct expression profiles initially assessed by Affymetrix GeneChip analyses. The new chemiluminescence-based Ziplex gene expression array technology was evaluated for the expression of 93 genes selected based on their Affymetrix GeneChip profiles as applied to ovarian cancer research. Probe design was based on the Affymetrix target sequence that favors the 3' UTR of transcripts in order to maximize reproducibility across platforms. Gene expression analysis was performed using the Ziplex Automated Workstation. Statistical analyses were performed to evaluate reproducibility of both the magnitude of expression and differences between normal and tumor samples by correlation analyses, fold change differences and statistical significance testing. Expressions of 82 of 93 (88.2%) genes were highly correlated (p < 0.01) in a comparison of the two platforms. Overall, 75 of 93 (80.6%) genes exhibited consistent results in normal versus tumor tissue comparisons for both platforms (p < 0.001). The fold change differences were concordant for 87 of 93 (94%) genes, where there was agreement between the platforms regarding statistical significance for 71 (76%) of 87 genes. There was a strong agreement between the two platforms as shown by comparisons of log2 fold differences of gene expression between tumor versus normal samples (R = 0.93) and by Bland-Altman analysis, where greater than 90% of expression values fell within the 95% limits of agreement. Overall concordance of gene expression patterns based on correlations, statistical significance between tumor and normal ovary data, and fold changes was consistent between the Ziplex and Affymetrix platforms. The reproducibility and ease-of-use of the technology suggests that the Ziplex array is a suitable platform for translational research.
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Hennessy, Rosanna C; Stougaard, Peter; Olsson, Stefan
2017-03-21
Here, we report the development of a microplate reader-based system for visualizing gene expression dynamics in living bacterial cells in response to a fungus in space and real-time. A bacterium expressing the red fluorescent protein mCherry fused to the promoter region of a regulator gene nunF indicating activation of an antifungal secondary metabolite gene cluster was used as a reporter system. Time-lapse image recordings of the reporter red signal and a green signal from fluorescent metabolites combined with microbial growth measurements showed that nunF-regulated gene transcription is switched on when the bacterium enters the deceleration growth phase and upon physical encounter with fungal hyphae. This novel technique enables real-time live imaging of samples by time-series multi-channel automatic recordings using a microplate reader as both an incubator and image recorder of general use to researchers. The technique can aid in deciding when to destructively sample for other methods e.g. transcriptomics and mass spectrometry imaging to study gene expression and metabolites exchanged during the interaction.
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Multi-level Expression Design Language: Requirement level (MEDL-R) system evaluation
NASA Technical Reports Server (NTRS)
1980-01-01
An evaluation of the Multi-Level Expression Design Language Requirements Level (MEDL-R) system was conducted to determine whether it would be of use in the Goddard Space Flight Center Code 580 software development environment. The evaluation is based upon a study of the MEDL-R concept of requirement languages, the functions performed by MEDL-R, and the MEDL-R language syntax. Recommendations are made for changes to MEDL-R that would make it useful in the Code 580 environment.
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Balow, James E; Ryan, John G; Chae, Jae Jin; Booty, Matthew G; Bulua, Ariel; Stone, Deborah; Sun, Hong-Wei; Greene, James; Barham, Beverly; Goldbach-Mansky, Raphaela; Kastner, Daniel L; Aksentijevich, Ivona
2013-06-01
To analyse gene expression patterns and to define a specific gene expression signature in patients with the severe end of the spectrum of cryopyrin-associated periodic syndromes (CAPS). The molecular consequences of interleukin 1 inhibition were examined by comparing gene expression patterns in 16 CAPS patients before and after treatment with anakinra. We collected peripheral blood mononuclear cells from 22 CAPS patients with active disease and from 14 healthy children. Transcripts that passed stringent filtering criteria (p values≤false discovery rate 1%) were considered as differentially expressed genes (DEG). A set of DEG was validated by quantitative reverse transcription PCR and functional studies with primary cells from CAPS patients and healthy controls. We used 17 CAPS and 66 non-CAPS patient samples to create a set of gene expression models that differentiates CAPS patients from controls and from patients with other autoinflammatory conditions. Many DEG include transcripts related to the regulation of innate and adaptive immune responses, oxidative stress, cell death, cell adhesion and motility. A set of gene expression-based models comprising the CAPS-specific gene expression signature correctly classified all 17 samples from an independent dataset. This classifier also correctly identified 15 of 16 post-anakinra CAPS samples despite the fact that these CAPS patients were in clinical remission. We identified a gene expression signature that clearly distinguished CAPS patients from controls. A number of DEG were in common with other systemic inflammatory diseases such as systemic onset juvenile idiopathic arthritis. The CAPS-specific gene expression classifiers also suggest incomplete suppression of inflammation at low doses of anakinra.
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Balow, James E; Ryan, John G; Chae, Jae Jin; Booty, Matthew G; Bulua, Ariel; Stone, Deborah; Sun, Hong-Wei; Greene, James; Barham, Beverly; Goldbach-Mansky, Raphaela; Kastner, Daniel L; Aksentijevich, Ivona
2014-01-01
Objective To analyse gene expression patterns and to define a specific gene expression signature in patients with the severe end of the spectrum of cryopyrin-associated periodic syndromes (CAPS). The molecular consequences of interleukin 1 inhibition were examined by comparing gene expression patterns in 16 CAPS patients before and after treatment with anakinra. Methods We collected peripheral blood mononuclear cells from 22 CAPS patients with active disease and from 14 healthy children. Transcripts that passed stringent filtering criteria (p values ≤ false discovery rate 1%) were considered as differentially expressed genes (DEG). A set of DEG was validated by quantitative reverse transcription PCR and functional studies with primary cells from CAPS patients and healthy controls. We used 17 CAPS and 66 non-CAPS patient samples to create a set of gene expression models that differentiates CAPS patients from controls and from patients with other autoinflammatory conditions. Results Many DEG include transcripts related to the regulation of innate and adaptive immune responses, oxidative stress, cell death, cell adhesion and motility. A set of gene expression-based models comprising the CAPS-specific gene expression signature correctly classified all 17 samples from an independent dataset. This classifier also correctly identified 15 of 16 postanakinra CAPS samples despite the fact that these CAPS patients were in clinical remission. Conclusions We identified a gene expression signature that clearly distinguished CAPS patients from controls. A number of DEG were in common with other systemic inflammatory diseases such as systemic onset juvenile idiopathic arthritis. The CAPS-specific gene expression classifiers also suggest incomplete suppression of inflammation at low doses of anakinra. PMID:23223423
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Expression of non-toxic mutant of Escherichia coli heat-labile enterotoxin in tobacco chloroplasts.
Kang, Tae-Jin; Han, So-Chon; Kim, Mi-Young; Kim, Young-Sook; Yang, Moon-Sik
2004-11-01
Chloroplast transformation systems offer unique advantages in biotechnology, including high level of foreign gene expression, maternal inheritance, and polycistronic expression. We studied chloroplast expression of LTK63 (change Ser-->Lys at position 63 in the A subunit) which is the mutant of Escherichia coli heat-labile toxin. LTK63 is devoid of any toxic activity, but still retains its mucosal adjuvanticity. The LTK63 was cloned into chloroplast targeting vector and transformed to tobacco chloroplasts by particle bombardment. PCR and Southern blot analyses confirmed stable homologous recombination of the LTK63 gene into the chloroplast genome. The amount of LTK63 protein detected in tobacco chloroplasts was approximately 3.7% of the total soluble protein. The GM1-ganglioside binding assay confirmed that chloroplast-synthesized LTB of LTK63 binds to the intestinal membrane GM1-ganglioside receptor. Thus, the expression of LTK63 in chloroplasts provides a potential route toward the development of a plant-based edible vaccine for high expression system and environmentally friendly approach.
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Analysis of facial expressions in parkinson's disease through video-based automatic methods.
Bandini, Andrea; Orlandi, Silvia; Escalante, Hugo Jair; Giovannelli, Fabio; Cincotta, Massimo; Reyes-Garcia, Carlos A; Vanni, Paola; Zaccara, Gaetano; Manfredi, Claudia
2017-04-01
The automatic analysis of facial expressions is an evolving field that finds several clinical applications. One of these applications is the study of facial bradykinesia in Parkinson's disease (PD), which is a major motor sign of this neurodegenerative illness. Facial bradykinesia consists in the reduction/loss of facial movements and emotional facial expressions called hypomimia. In this work we propose an automatic method for studying facial expressions in PD patients relying on video-based METHODS: 17 Parkinsonian patients and 17 healthy control subjects were asked to show basic facial expressions, upon request of the clinician and after the imitation of a visual cue on a screen. Through an existing face tracker, the Euclidean distance of the facial model from a neutral baseline was computed in order to quantify the changes in facial expressivity during the tasks. Moreover, an automatic facial expressions recognition algorithm was trained in order to study how PD expressions differed from the standard expressions. Results show that control subjects reported on average higher distances than PD patients along the tasks. This confirms that control subjects show larger movements during both posed and imitated facial expressions. Moreover, our results demonstrate that anger and disgust are the two most impaired expressions in PD patients. Contactless video-based systems can be important techniques for analyzing facial expressions also in rehabilitation, in particular speech therapy, where patients could get a definite advantage from a real-time feedback about the proper facial expressions/movements to perform. Copyright © 2017 Elsevier B.V. All rights reserved.
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Realistic facial expression of virtual human based on color, sweat, and tears effects.
Alkawaz, Mohammed Hazim; Basori, Ahmad Hoirul; Mohamad, Dzulkifli; Mohamed, Farhan
2014-01-01
Generating extreme appearances such as scared awaiting sweating while happy fit for tears (cry) and blushing (anger and happiness) is the key issue in achieving the high quality facial animation. The effects of sweat, tears, and colors are integrated into a single animation model to create realistic facial expressions of 3D avatar. The physical properties of muscles, emotions, or the fluid properties with sweating and tears initiators are incorporated. The action units (AUs) of facial action coding system are merged with autonomous AUs to create expressions including sadness, anger with blushing, happiness with blushing, and fear. Fluid effects such as sweat and tears are simulated using the particle system and smoothed-particle hydrodynamics (SPH) methods which are combined with facial animation technique to produce complex facial expressions. The effects of oxygenation of the facial skin color appearance are measured using the pulse oximeter system and the 3D skin analyzer. The result shows that virtual human facial expression is enhanced by mimicking actual sweating and tears simulations for all extreme expressions. The proposed method has contribution towards the development of facial animation industry and game as well as computer graphics.
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Realistic Facial Expression of Virtual Human Based on Color, Sweat, and Tears Effects
Alkawaz, Mohammed Hazim; Basori, Ahmad Hoirul; Mohamad, Dzulkifli; Mohamed, Farhan
2014-01-01
Generating extreme appearances such as scared awaiting sweating while happy fit for tears (cry) and blushing (anger and happiness) is the key issue in achieving the high quality facial animation. The effects of sweat, tears, and colors are integrated into a single animation model to create realistic facial expressions of 3D avatar. The physical properties of muscles, emotions, or the fluid properties with sweating and tears initiators are incorporated. The action units (AUs) of facial action coding system are merged with autonomous AUs to create expressions including sadness, anger with blushing, happiness with blushing, and fear. Fluid effects such as sweat and tears are simulated using the particle system and smoothed-particle hydrodynamics (SPH) methods which are combined with facial animation technique to produce complex facial expressions. The effects of oxygenation of the facial skin color appearance are measured using the pulse oximeter system and the 3D skin analyzer. The result shows that virtual human facial expression is enhanced by mimicking actual sweating and tears simulations for all extreme expressions. The proposed method has contribution towards the development of facial animation industry and game as well as computer graphics. PMID:25136663
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Shrestha, Roshan P.; Hildebrand, Mark
2017-08-17
An inducible promoter for recombinant protein expression provides substantial benefits because under induction conditions cellular energy and metabolic capability can be directed into protein synthesis. The most widely used inducible promoter for diatoms is for nitrate reductase, however, nitrogen metabolism is tied into diverse aspects of cellular function, and the induction response is not necessarily robust. Silicon limitation offers a means to eliminate energy and metabolic flux into cell division processes, with little other detrimental effect on cellular function, and a protein expression system that works under those conditions could be advantageous. In this study, we evaluate a number ofmore » promoters for recombinant protein expression induced by silicon limitation and repressed by the presence of silicon in the diatoms Thalassiosira pseudonana and Cyclotella cryptica. In addition to silicon limitation, we describe additional strategies to elevate recombinant protein expression level, including inclusion of the 5' fragment of the coding region of the native gene and reducing carbon flow into ancillary processes of pigment synthesis and formation of photosynthetic storage products. We achieved yields of eGFP to 1.8% of total soluble protein in C. cryptica, which is about 3.6-fold higher than that obtained with chloroplast expression and ninefold higher than nuclear expression in another well-established algal system. Our studies demonstrate that the combination of inducible promoter and other strategies can result in robust expression of recombinant protein in a nuclear-based expression system in diatoms under silicon limited conditions, separating the protein expression regime from growth processes and improving overall recombinant protein yields.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Shrestha, Roshan P.; Hildebrand, Mark
An inducible promoter for recombinant protein expression provides substantial benefits because under induction conditions cellular energy and metabolic capability can be directed into protein synthesis. The most widely used inducible promoter for diatoms is for nitrate reductase, however, nitrogen metabolism is tied into diverse aspects of cellular function, and the induction response is not necessarily robust. Silicon limitation offers a means to eliminate energy and metabolic flux into cell division processes, with little other detrimental effect on cellular function, and a protein expression system that works under those conditions could be advantageous. In this study, we evaluate a number ofmore » promoters for recombinant protein expression induced by silicon limitation and repressed by the presence of silicon in the diatoms Thalassiosira pseudonana and Cyclotella cryptica. In addition to silicon limitation, we describe additional strategies to elevate recombinant protein expression level, including inclusion of the 5' fragment of the coding region of the native gene and reducing carbon flow into ancillary processes of pigment synthesis and formation of photosynthetic storage products. We achieved yields of eGFP to 1.8% of total soluble protein in C. cryptica, which is about 3.6-fold higher than that obtained with chloroplast expression and ninefold higher than nuclear expression in another well-established algal system. Our studies demonstrate that the combination of inducible promoter and other strategies can result in robust expression of recombinant protein in a nuclear-based expression system in diatoms under silicon limited conditions, separating the protein expression regime from growth processes and improving overall recombinant protein yields.« less
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Thovhogi, Ntevheleni; Sibuyi, Nicole Remaliah Samantha; Onani, Martin Opiyo; Meyer, Mervin; Madiehe, Abram Madimabe
2018-01-01
Obesity is a worldwide epidemic affecting millions of people. The current pharmacological treatment of obesity remains limited and ineffective due to drugs' undesirable side effects. Hence, there is a need for novel or improved strategies for long-term therapies that will help prevent the disease progression into other chronic diseases. Nanotechnology holds the future for the treatment of obesity because of its versatility, as shown by improved drug efficiency and safety in cancer clinical trials. Nano-based drug delivery systems could potentially do the same for obesity through targeted drug delivery. This study investigated the use of peptide-functionalized quantum dots (QDs) for the imaging of prohibitin (PHB)-expressing cells in vitro and in diet-induced obese rats, which could potentially be used as nanocarriers of antiobesity drugs. Cadmium (Cd)-based QDs were functionalized with an adipose homing peptide (AHP) and injected intravenously into lean and obese Wistar rats. Biodistribution of the QDs was analyzed by an IVIS ® Lumina XR imaging system and inductively coupled plasma optical emission spectroscopy (ICP-OES). For in vitro studies, PHB-expressing (Caco-2 and MCF-7) and non-PHB-expressing (KMST-6 and CHO) cells were exposed to either unfunctionalized QDs (QD625) or AHP-functionalized QDs (AHP-QD625) and analyzed by fluorescence microscopy. AHP-QD625 accumulated significantly in PHB-expressing cells in vitro when compared with non-PHB-expressing cells. In vivo data indicated that QD625 accumulated mainly in the reticuloendothelial system (RES) organs, while the AHP-QD625 accumulated mostly in the white adipose tissues (WATs). AHP-functionalized QDs were successfully and selectively delivered to the PHB-expressing cells in vitro (Caco-2 and MCF-7 cells) and in the WAT vasculature in vivo. This nanotechnology-based approach could potentially be used for dual targeted drug delivery and molecular imaging of adipose tissues in obese patients in real time.
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Parameterized Facial Expression Synthesis Based on MPEG-4
NASA Astrophysics Data System (ADS)
Raouzaiou, Amaryllis; Tsapatsoulis, Nicolas; Karpouzis, Kostas; Kollias, Stefanos
2002-12-01
In the framework of MPEG-4, one can include applications where virtual agents, utilizing both textual and multisensory data, including facial expressions and nonverbal speech help systems become accustomed to the actual feelings of the user. Applications of this technology are expected in educational environments, virtual collaborative workplaces, communities, and interactive entertainment. Facial animation has gained much interest within the MPEG-4 framework; with implementation details being an open research area (Tekalp, 1999). In this paper, we describe a method for enriching human computer interaction, focusing on analysis and synthesis of primary and intermediate facial expressions (Ekman and Friesen (1978)). To achieve this goal, we utilize facial animation parameters (FAPs) to model primary expressions and describe a rule-based technique for handling intermediate ones. A relation between FAPs and the activation parameter proposed in classical psychological studies is established, leading to parameterized facial expression analysis and synthesis notions, compatible with the MPEG-4 standard.
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Identification of FGF-dependent genes in the Drosophila tracheal system.
Stahl, Markus; Schuh, Reinhard; Adryan, Boris
2007-01-01
The embryonic development of the tracheal system of the fruit fly Drosophila provides a paradigm for genetic studies of branching morphogenesis. Efforts of many laboratories have identified Branchless (Bnl, a fibroblast growth factor homologue) and Breathless (Btl, the receptor homologue) as crucial factors at many stages of tracheal system development. The downstream targets of the Bnl/Btl signalling cascade, however, remain mostly unknown. Misexpression of the bnl gene results in specific tracheal phenotypes that lead to larval death. We characterised the transcriptional profiles of targeted over-expression of bnl in the embryonic trachea and of loss-of-function bnl(P1) mutant embryos. Gene expression data was mapped to high-throughput in situ hybridisation based ImaGO-annotation. Thus, we identified and confirmed by quantitative PCR 13 Bnl-dependent genes that are expressed in cells within and outside of the tracheal system.
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Cell-Free Optogenetic Gene Expression System.
Jayaraman, Premkumar; Yeoh, Jing Wui; Jayaraman, Sudhaghar; Teh, Ai Ying; Zhang, Jingyun; Poh, Chueh Loo
2018-04-20
Optogenetic tools provide a new and efficient way to dynamically program gene expression with unmatched spatiotemporal precision. To date, their vast potential remains untapped in the field of cell-free synthetic biology, largely due to the lack of simple and efficient light-switchable systems. Here, to bridge the gap between cell-free systems and optogenetics, we studied our previously engineered one component-based blue light-inducible Escherichia coli promoter in a cell-free environment through experimental characterization and mathematical modeling. We achieved >10-fold dynamic expression and demonstrated rapid and reversible activation of the target gene to generate oscillatory response. The deterministic model developed was able to recapitulate the system behavior and helped to provide quantitative insights to optimize dynamic response. This in vitro optogenetic approach could be a powerful new high-throughput screening technology for rapid prototyping of complex biological networks in both space and time without the need for chemical induction.
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Lin, Zhe; Lin, Yongsheng
2017-09-05
The aim of this study was to explore potential crucial genes associated with the steroid-induced necrosis of femoral head (SINFH) and to provide valid biological information for further investigation of SINFH. Gene expression profile of GSE26316, generated from 3 SINFH rat samples and 3 normal rat samples were downloaded from Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) were identified using LIMMA package. After functional enrichment analyses of DEGs, protein-protein interaction (PPI) network and sub-PPI network analyses were conducted based on the STRING database and cytoscape. In total, 59 up-regulated DEGs and 156 downregulated DEGs were identified. The up-regulated DEGs were mainly involved in functions about immunity (e.g. Fcer1A and Il7R), and the downregulated DEGs were mainly enriched in muscle system process (e.g. Tnni2, Mylpf and Myl1). The PPI network of DEGs consisted of 123 nodes and 300 interactions. Tnni2, Mylpf, and Myl1 were the top 3 outstanding genes based on both subgraph centrality and degree centrality evaluation. These three genes interacted with each other in the network. Furthermore, the significant network module was composed of 22 downregulated genes (e.g. Tnni2, Mylpf and Myl1). These genes were mainly enriched in functions like muscle system process. The DEGs related to the regulation of immune system process (e.g. Fcer1A and Il7R), and DEGs correlated with muscle system process (e.g. Tnni2, Mylpf and Myl1) may be closely associated with the progress of SINFH, which is still needed to be confirmed by experiments. Copyright © 2017 Elsevier B.V. All rights reserved.
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S locus-linked F-box genes expressed in anthers of Hordeum bulbosum.
Kakeda, Katsuyuki
2009-09-01
Diploid Hordeum bulbosum (a wild relative of cultivated barley) exhibits a two-locus self-incompatibility (SI) system gametophytically controlled by the unlinked multiallelic loci S and Z. This unique SI system is observed in the grasses (Poaceae) including the tribe Triticeae. This paper describes the identification and characterization of two F-box genes cosegregating with the S locus in H. bulbosum, named Hordeum S locus-linked F-box 1 (HSLF1) and HSLF2, which were derived from an S (3) haplotype-specific clone (HAS175) obtained by previous AMF (AFLP-based mRNA fingerprinting) analysis. Sequence analysis showed that both genes encode similar F-box proteins with a C-terminal leucine-rich repeat (LRR) domain, which are distinct from S locus (or S haplotype-specific) F-box protein (SLF/SFB), a class of F-box proteins identified as the pollen S determinant in S-RNase-based gametophytic SI systems. A number of homologous F-box genes with an LRR domain were found in the rice genome, although the functions of the gene family are unknown. One allele of the HSLF1 gene (HSLF1-S (3)) was expressed specifically in mature anthers, whereas no expression was detected from the other two alleles examined. Although the degree of sequence polymorphism among the three HSLF1 alleles was low, a frameshift mutation was found in one of the unexpressed alleles. The HSLF2 gene showed a low level of expression with no tissue specificity as well as little sequence polymorphism among the three alleles. The multiplicity of S locus-linked F-box genes is discussed in comparison with those found in the S-RNase-based SI system.
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Clémençon, Benjamin; Lüscher, Benjamin P.; Fine, Michael; Baumann, Marc U.; Surbek, Daniel V.; Bonny, Olivier; Hediger, Matthias A.
2014-01-01
The urate transporter, GLUT9, is responsible for the basolateral transport of urate in the proximal tubule of human kidneys and in the placenta, playing a central role in uric acid homeostasis. GLUT9 shares the least homology with other members of the glucose transporter family, especially with the glucose transporting members GLUT1-4 and is the only member of the GLUT family to transport urate. The recently published high-resolution structure of XylE, a bacterial D-xylose transporting homologue, yields new insights into the structural foundation of this GLUT family of proteins. While this represents a huge milestone, it is unclear if human GLUT9 can benefit from this advancement through subsequent structural based targeting and mutagenesis. Little progress has been made toward understanding the mechanism of GLUT9 since its discovery in 2000. Before work can begin on resolving the mechanisms of urate transport we must determine methods to express, purify and analyze hGLUT9 using a model system adept in expressing human membrane proteins. Here, we describe the surface expression, purification and isolation of monomeric protein, and functional analysis of recombinant hGLUT9 using the Xenopus laevis oocyte system. In addition, we generated a new homology-based high-resolution model of hGLUT9 from the XylE crystal structure and utilized our purified protein to generate a low-resolution single particle reconstruction. Interestingly, we demonstrate that the functional protein extracted from the Xenopus system fits well with the homology-based model allowing us to generate the predicted urate-binding pocket and pave a path for subsequent mutagenesis and structure-function studies. PMID:25286413
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System Biology Approach: Gene Network Analysis for Muscular Dystrophy.
Censi, Federica; Calcagnini, Giovanni; Mattei, Eugenio; Giuliani, Alessandro
2018-01-01
Phenotypic changes at different organization levels from cell to entire organism are associated to changes in the pattern of gene expression. These changes involve the entire genome expression pattern and heavily rely upon correlation patterns among genes. The classical approach used to analyze gene expression data builds upon the application of supervised statistical techniques to detect genes differentially expressed among two or more phenotypes (e.g., normal vs. disease). The use of an a posteriori, unsupervised approach based on principal component analysis (PCA) and the subsequent construction of gene correlation networks can shed a light on unexpected behaviour of gene regulation system while maintaining a more naturalistic view on the studied system.In this chapter we applied an unsupervised method to discriminate DMD patient and controls. The genes having the highest absolute scores in the discrimination between the groups were then analyzed in terms of gene expression networks, on the basis of their mutual correlation in the two groups. The correlation network structures suggest two different modes of gene regulation in the two groups, reminiscent of important aspects of DMD pathogenesis.
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[Cloning and characterization of a novel rat gene RSD-7 differentially expressed in testis].
Zhang, Xiao-dong; Gou, Da-wei; Miao, Shi-ying; Zhang, Jian-chao; Zong, Shu-dong; Wang, Lin-fang
2003-06-01
To isolate and identify the differentially expressed genes in spermatogenesis for the understanding molecular mechanism of spermatogenesis. Screening of the cDNA library, Northern blot, expression and purification in E. coli with GST expression system, immunocytochemical staining of testis sections were used. (1) A cDNA fragment designated as RSD-7 was isolated from rat testis cDNA library. It was 1,238 bp in length, coding a protein of 232 amino acids with the GenBank accession number AF315467. The encoding protein of RSD-7 cDNA had a Ubiquitin-like domain. (2) Northern blot indicated that RSD-7 was uniquely expressed in rat testis, and in the testis RSD-7 emerged on the 30th postnatal day and expressed until 120th postnatal day. (3) Expression and purification of RSD-7 protein in E. coli with GST expression system and were used to obtain anti-RSD-7 antibody. (4) Immunolocalization of RSD-7 in rat testis revealed that it is expressed only in Sertoli cells. Transcription pattern of RSD-7 and localization of RSD-7 protein in testis have been made, which established the base for the functional study of RSD-7.
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GECKO: a complete large-scale gene expression analysis platform.
Theilhaber, Joachim; Ulyanov, Anatoly; Malanthara, Anish; Cole, Jack; Xu, Dapeng; Nahf, Robert; Heuer, Michael; Brockel, Christoph; Bushnell, Steven
2004-12-10
Gecko (Gene Expression: Computation and Knowledge Organization) is a complete, high-capacity centralized gene expression analysis system, developed in response to the needs of a distributed user community. Based on a client-server architecture, with a centralized repository of typically many tens of thousands of Affymetrix scans, Gecko includes automatic processing pipelines for uploading data from remote sites, a data base, a computational engine implementing approximately 50 different analysis tools, and a client application. Among available analysis tools are clustering methods, principal component analysis, supervised classification including feature selection and cross-validation, multi-factorial ANOVA, statistical contrast calculations, and various post-processing tools for extracting data at given error rates or significance levels. On account of its open architecture, Gecko also allows for the integration of new algorithms. The Gecko framework is very general: non-Affymetrix and non-gene expression data can be analyzed as well. A unique feature of the Gecko architecture is the concept of the Analysis Tree (actually, a directed acyclic graph), in which all successive results in ongoing analyses are saved. This approach has proven invaluable in allowing a large (approximately 100 users) and distributed community to share results, and to repeatedly return over a span of years to older and potentially very complex analyses of gene expression data. The Gecko system is being made publicly available as free software http://sourceforge.net/projects/geckoe. In totality or in parts, the Gecko framework should prove useful to users and system developers with a broad range of analysis needs.
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Application of symbolic computations to the constitutive modeling of structural materials
NASA Technical Reports Server (NTRS)
Arnold, Steven M.; Tan, H. Q.; Dong, X.
1990-01-01
In applications involving elevated temperatures, the derivation of mathematical expressions (constitutive equations) describing the material behavior can be quite time consuming, involved and error-prone. Therefore intelligent application of symbolic systems to faciliate this tedious process can be of significant benefit. Presented here is a problem oriented, self contained symbolic expert system, named SDICE, which is capable of efficiently deriving potential based constitutive models in analytical form. This package, running under DOE MACSYMA, has the following features: (1) potential differentiation (chain rule), (2) tensor computations (utilizing index notation) including both algebraic and calculus; (3) efficient solution of sparse systems of equations; (4) automatic expression substitution and simplification; (5) back substitution of invariant and tensorial relations; (6) the ability to form the Jacobian and Hessian matrix; and (7) a relational data base. Limited aspects of invariant theory were also incorporated into SDICE due to the utilization of potentials as a starting point and the desire for these potentials to be frame invariant (objective). The uniqueness of SDICE resides in its ability to manipulate expressions in a general yet pre-defined order and simplify expressions so as to limit expression growth. Results are displayed, when applicable, utilizing index notation. SDICE was designed to aid and complement the human constitutive model developer. A number of examples are utilized to illustrate the various features contained within SDICE. It is expected that this symbolic package can and will provide a significant incentive to the development of new constitutive theories.
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Panthu, Baptiste; Ohlmann, Théophile; Perrier, Johan; Schlattner, Uwe; Jalinot, Pierre; Elena-Herrmann, Bénédicte; Rautureau, Gilles J P
2018-01-19
A counterintuitive cell-free protein synthesis (CFPS) strategy, based on reducing the ribosomal fraction in rabbit reticulocyte lysate (RRL), triggers the development of hybrid systems composed of RRL ribosome-free supernatant complemented with ribosomes from different mammalian cell-types. Hybrid RRL systems maintain translational properties of the original ribosome cell types, and deliver protein expression levels similar to RRL. Here, we show that persistent ribosome-associated metabolic activity consuming ATP is a major obstacle for maximal protein yield. We provide a detailed picture of hybrid CFPS systems energetic metabolism based on real-time nuclear magnetic resonance (NMR) investigation of metabolites kinetics. We demonstrate that protein synthesis capacity has an upper limit at native ribosome concentration and that lower amounts of the ribosomal fraction optimize energy fluxes toward protein translation, consequently increasing CFPS yield. These results provide a rationalized strategy for further mammalian CFPS developments and reveal the potential of real-time NMR metabolism phenotyping for optimization of cell-free protein expression systems.
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Higashiyama, Hiroyuki; Billin, Andrew N; Okamoto, Yuji; Kinoshita, Mine; Asano, Satoshi
2007-05-01
Peroxisome proliferator-activated receptor-delta (PPAR-delta) is known as a transcription factor involved in the regulation of fatty acid oxidation and mitochondrial biogenesis in several tissues, such as skeletal muscle, liver and adipose tissues. In this study, to elucidate systemic physiological functions of PPAR-delta, we examined the tissue distribution and localization of PPAR-delta in adult mouse tissues using tissue microarray (TMA)-based immunohistochemistry. PPAR-delta positive signals were observed on variety of tissues/cells in multiple systems including cardiovascular, urinary, respiratory, digestive, endocrine, nervous, hematopoietic, immune, musculoskeletal, sensory and reproductive organ systems. In these organs, PPAR-delta immunoreactivity was generally localized on the nucleus, although cytoplasmic localization was observed on several cell types including neurons in the nervous system and cells of the islet of Langerhans. These expression profiling data implicate various physiological roles of PPAR-delta in multiple organ systems. TMA-based immunohistochemistry enables to profile comprehensive protein localization and distribution in a high-throughput manner.
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Tsuchiya, Masa; Giuliani, Alessandro; Hashimoto, Midori; Erenpreisa, Jekaterina; Yoshikawa, Kenichi
2015-01-01
Background The underlying mechanism of dynamic control of the genome-wide expression is a fundamental issue in bioscience. We addressed it in terms of phase transition by a systemic approach based on both density analysis and characteristics of temporal fluctuation for the time-course mRNA expression in differentiating MCF-7 breast cancer cells. Methodology In a recent work, we suggested criticality as an essential aspect of dynamic control of genome-wide gene expression. Criticality was evident by a unimodal-bimodal transition through flattened unimodal expression profile. The flatness on the transition suggests the existence of a critical transition at which up- and down-regulated expression is balanced. Mean field (averaging) behavior of mRNAs based on the temporal expression changes reveals a sandpile type of transition in the flattened profile. Furthermore, around the transition, a self-similar unimodal-bimodal transition of the whole expression occurs in the density profile of an ensemble of mRNA expression. These singular and scaling behaviors identify the transition as the expression phase transition driven by self-organized criticality (SOC). Principal Findings Emergent properties of SOC through a mean field approach are revealed: i) SOC, as a form of genomic phase transition, consolidates distinct critical states of expression, ii) Coupling of coherent stochastic oscillations between critical states on different time-scales gives rise to SOC, and iii) Specific gene clusters (barcode genes) ranging in size from kbp to Mbp reveal similar SOC to genome-wide mRNA expression and ON-OFF synchronization to critical states. This suggests that the cooperative gene regulation of topological genome sub-units is mediated by the coherent phase transitions of megadomain-scaled conformations between compact and swollen chromatin states. Conclusion and Significance In summary, our study provides not only a systemic method to demonstrate SOC in whole-genome expression, but also introduces novel, physically grounded concepts for a breakthrough in the study of biological regulation. PMID:26067993
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NASA Astrophysics Data System (ADS)
Benitez-Garcia, Gibran; Nakamura, Tomoaki; Kaneko, Masahide
2017-01-01
Darwin was the first one to assert that facial expressions are innate and universal, which are recognized across all cultures. However, recent some cross-cultural studies have questioned this assumed universality. Therefore, this paper presents an analysis of the differences between Western and East-Asian faces of the six basic expressions (anger, disgust, fear, happiness, sadness and surprise) focused on three individual facial regions of eyes-eyebrows, nose and mouth. The analysis is conducted by applying PCA for two feature extraction methods: appearance-based by using the pixel intensities of facial parts, and geometric-based by handling 125 feature points from the face. Both methods are evaluated using 4 standard databases for both racial groups and the results are compared with a cross-cultural human study applied to 20 participants. Our analysis reveals that differences between Westerns and East-Asians exist mainly on the regions of eyes-eyebrows and mouth for expressions of fear and disgust respectively. This work presents important findings for a better design of automatic facial expression recognition systems based on the difference between two racial groups.
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RDFBuilder: a tool to automatically build RDF-based interfaces for MAGE-OM microarray data sources.
Anguita, Alberto; Martin, Luis; Garcia-Remesal, Miguel; Maojo, Victor
2013-07-01
This paper presents RDFBuilder, a tool that enables RDF-based access to MAGE-ML-compliant microarray databases. We have developed a system that automatically transforms the MAGE-OM model and microarray data stored in the ArrayExpress database into RDF format. Additionally, the system automatically enables a SPARQL endpoint. This allows users to execute SPARQL queries for retrieving microarray data, either from specific experiments or from more than one experiment at a time. Our system optimizes response times by caching and reusing information from previous queries. In this paper, we describe our methods for achieving this transformation. We show that our approach is complementary to other existing initiatives, such as Bio2RDF, for accessing and retrieving data from the ArrayExpress database. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.
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Buyel, Johannes Felix; Fischer, Rainer
2014-01-31
Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.
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Ternary particles for effective vaccine delivery to the pulmonary system
NASA Astrophysics Data System (ADS)
Terry, Treniece La'shay
Progress in the fields of molecular biology and genomics has provided great insight into the pathogenesis of disease and the defense mechanisms of the immune system. This knowledge has lead to the classification of an array of abnormal genes, for which, treatment relies on cellular expression of proteins. The utility of DNA-based vaccines hold great promise for the treatment of genetically based and infectious diseases, which ranges from hemophilia, cystic fibrosis, and HIV. Synthetic delivery systems consisting of cationic polymers, such as polyethylenimine (PEI), are capable of condensing DNA into compact structures, maximizing cellular uptake of DNA and yielding high levels of protein expression. To date, short term expression is a major obstacle in the development of gene therapies and has halted their expansion in clinical applications. This study intends to develop a sustained release vaccine delivery system using PLA-PEG block copolymers encapsulating PEI:DNA polyplexes. To enhance the effectiveness of such DNA-based vaccines, resident antigen presenting cells, macrophages and dendritic cells, will be targeted within the alveoli regions of the lungs. Porous microspheres will be engineered with aerodynamic properties capable of achieving deep lung deposition. A fabrication technique using concentric nozzles will be developed to produce porous microspheres. It was observed that modifications in the dispersed to continuous phase ratios have the largest influence on particle size distributions, release rates and encapsulation efficiency which ranged form 80--95% with fourteen days of release. Amphiphilic block copolymers were also used to fabricate porous microspheres. The confirmation of PEG within the biodegradable polymer backbone was found to have a tremendous impact on the microsphere morphology and encapsulation efficiency which varied from 50--90%. Porous microspheres were capable of providing sustained gene expression when tested in vitro using the luciferase reporter gene plasmid DNA. Prolonged expression was obtained for 9 days. PLGA and PLA-PEG microspheres were administered in vivo by intra-tracheal instillation and produced an acute inflammatory response, as observed from the large presence of neutrophils. The response using PLA-PEG microspheres yielded a lower total cell count signifying the incorporation of PEG into the copolymer backbone enhances the biocompatibility of the delivery system.
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Santi, Luca; Giritch, Anatoli; Roy, Chad J.; Marillonnet, Sylvestre; Klimyuk, Victor; Gleba, Yuri; Webb, Robert; Arntzen, Charles J.; Mason, Hugh S.
2006-01-01
Plague is still an endemic disease in different regions of the world. Increasing reports of incidence, the discovery of antibiotic resistance strains, and concern about a potential use of the causative bacteria Yersinia pestis as an agent of biological warfare have highlighted the need for a safe, efficacious, and rapidly producible vaccine. The use of F1 and V antigens and the derived protein fusion F1-V has shown great potential as a protective vaccine in animal studies. Plants have been extensively studied for the production of pharmaceutical proteins as an inexpensive and scalable alternative to common expression systems. In the current study the recombinant plague antigens F1, V, and fusion protein F1-V were produced by transient expression in Nicotiana benthamiana by using a deconstructed tobacco mosaic virus-based system that allowed very rapid and extremely high levels of expression. All of the plant-derived purified antigens, administered s.c. to guinea pigs, generated systemic immune responses and provided protection against an aerosol challenge of virulent Y. pestis. PMID:16410352
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Wang, Jing; Wang, Haoyuan; Yang, Le; Lv, Liping; Zhang, Zhe; Ren, Bin; Dong, Lichun; Li, Ning
2018-04-01
In this paper, a novel riboregulator Switch System of Gene Expression including an OFF-TO-ON switch and an ON-TO-OFF switch was designed to regulate the expression state of target genes between "ON" and "OFF" by switching the identifiability of ribosome recognition site (RBS) based on the thermodynamic stability of different RNA-RNA hybridizations between RBS and small noncoding RNAs. The proposed riboregulator switch system was employed for the fermentative production of succinic acid using an engineered strain of E. coli JW1021, during which the expression of mgtC gene was controlled at "ON" state and that of pepc and ecaA genes were controlled at the "OFF" state in the lag phase and switched to the "OFF" and "ON" state once the strain enters the logarithmic phase. The results showed that using the strain of JW1021, the yield and productivity of succinic acid can reach 0.91 g g -1 and 3.25 g L -1 h -1 , respectively, much higher than those using the strains without harboring the riboregulator switch system.
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Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System
Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; Escribano, José M.
2015-01-01
Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health. PMID:26458221
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Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System.
López-Vidal, Javier; Gómez-Sebastián, Silvia; Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; Escribano, José M
2015-01-01
Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health.
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Partitioning of functional gene expression data using principal points.
Kim, Jaehee; Kim, Haseong
2017-10-12
DNA microarrays offer motivation and hope for the simultaneous study of variations in multiple genes. Gene expression is a temporal process that allows variations in expression levels with a characterized gene function over a period of time. Temporal gene expression curves can be treated as functional data since they are considered as independent realizations of a stochastic process. This process requires appropriate models to identify patterns of gene functions. The partitioning of the functional data can find homogeneous subgroups of entities for the massive genes within the inherent biological networks. Therefor it can be a useful technique for the analysis of time-course gene expression data. We propose a new self-consistent partitioning method of functional coefficients for individual expression profiles based on the orthonormal basis system. A principal points based functional partitioning method is proposed for time-course gene expression data. The method explores the relationship between genes using Legendre coefficients as principal points to extract the features of gene functions. Our proposed method provides high connectivity in connectedness after clustering for simulated data and finds a significant subsets of genes with the increased connectivity. Our approach has comparative advantages that fewer coefficients are used from the functional data and self-consistency of principal points for partitioning. As real data applications, we are able to find partitioned genes through the gene expressions found in budding yeast data and Escherichia coli data. The proposed method benefitted from the use of principal points, dimension reduction, and choice of orthogonal basis system as well as provides appropriately connected genes in the resulting subsets. We illustrate our method by applying with each set of cell-cycle-regulated time-course yeast genes and E. coli genes. The proposed method is able to identify highly connected genes and to explore the complex dynamics of biological systems in functional genomics.
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Long, Dingpei; Lu, Weijian; Zhang, Yuli; Bi, Lihui; Xiang, Zhonghuai; Zhao, Aichun
2015-01-01
We developed an efficient strategy that combines a method for the post-integration elimination of all transposon sequences, a site-specific recombination system, and an optimized fibroin H-chain expression system to produce a stable, replaceable, highly efficient transgene expression system in the silkworm (Bombyx mori) that overcomes the disadvantages of random insertion and post-integration instability of transposons. Here, we generated four different transgenic silkworm strains, and of one the transgenic strains, designated TS1-RgG2, with up to 16% (w/w) of the target protein in the cocoons, was selected. The subsequent elimination of all the transposon sequences from TS1-RgG2 was completed by the heat-shock-induced expression of the transposase in vivo. The resulting transgenic silkworm strain was designated TS3-g2 and contained only the attP-flanked optimized fibroin H-chain expression cassette in its genome. A phiC31/att-system-based recombinase-mediated cassette exchange (RMCE) method could be used to integrate other genes of interest into the same genome locus between the attP sites in TS3-g2. Controlling for position effects with phiC31-mediated RMCE will also allow the optimization of exogenous protein expression and fine gene function analyses in the silkworm. The strategy developed here is also applicable to other lepidopteran insects, to improve the ecological safety of transgenic strains in biocontrol programs. PMID:25739894
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Yamada, Yuma; Ishikawa, Takuya; Harashima, Hideyoshi
2017-08-01
Mitochondria have their own gene expression system that is independent of the nuclear system, and control cellular functions in cooperation with the nucleus. While a number of useful technologies for achieving nuclear transgene expression have been reported, only a few have focused on mitochondria. In this study, we validated the utility of an artificial mitochondrial DNA vector with a virus promoter on mitochondrial transgene expression. We designed and constructed pCMV-mtLuc (CGG) that contains a CMV promotor derived from Cytomegalovirus and an artificial mitochondrial genome with a NanoLuc (Nluc) luciferase gene that records adjustments to the mitochondrial codon system. Nluc luciferase activity measurements showed that the pCMV-mtLuc (CGG) efficiently produced the Nluc luciferase protein in human HeLa cells. Moreover, we optimized the mitochondrial transfection of pCMV-mtLuc (CGG) using a MITO-Porter system, a liposome-based carrier for mitochondrial delivery via membrane fusion. As a result, we found that transfection of pCMV-mtLuc (CGG) by MITO-Porter modified with the KALA peptide (cationic amphipathic cell-penetrating peptide) showed a high mitochondrial transgene expression. The developed mitochondrial transgene expression system represents a potentially useful tool for the fields of nanoscience and nanotechnology for controlling the intracellular microenvironment via the regulation of mitochondrial function and promises to open additional innovative research fields of study. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Gene and enhancer trap tagging of vascular-expressed genes in poplar trees
Andrew Groover; Joseph R. Fontana; Gayle Dupper; Caiping Ma; Robert Martienssen; Steven Strauss; Richard Meilan
2004-01-01
We report a gene discovery system for poplar trees based on gene and enhancer traps. Gene and enhancer trap vectors carrying the β-glucuronidase (GUS) reporter gene were inserted into the poplar genome via Agrobacterium tumefaciens transformation, where they reveal the expression pattern of genes at or near the insertion sites. Because GUS...
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2014-11-03
Rob Mueller, left, NASA senior technologist in the Surface Systems Office in Kennedy Space Center's Engineering and Technology Directorate, talks with former NASA Apollo astronaut Buzz Aldrin during a demonstration of the Regolith Advanced Surface Systems Operations Robot, or RASSOR, at the automated landing and hazard avoidance technology, or ALHAT, hazard field at the north end of the Shuttle Landing Facility at NASA's Kennedy Space Center in Florida. The event was held to announce Moon Express Inc., of Moffett Field, California is selected to utilize Kennedy facilities for NASA's Lunar Cargo Transportation and Landing by Soft Touchdown, or Lunar CATALYST, initiative. Moon Express is developing a lander with capabilities that will enable delivery of payloads to the surface of the moon, as well as new science and exploration missions of interest to NASA and scientific and academic communities. Moon Express will base its activities at Kennedy and utilize the Morpheus ALHAT field and a hangar nearby for CATALYST testing. The Advanced Exploration Systems Division of NASA's Human Exploration and Operations Mission Directorate manages Lunar CATALYST.
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2014-11-03
Rob Mueller, left, NASA senior technologist in the Surface Systems Office in Kennedy Space Center's Engineering and Technology Directorate, talks with former NASA Apollo astronaut Buzz Aldrin during a demonstration of the Regolith Advanced Surface System Operations Robot, or RASSOR, at the automated landing and hazard avoidance technology, or ALHAT, hazard field at the north end of the Shuttle Landing Facility at NASA's Kennedy Space Center in Florida. The event was held to announce Moon Express Inc., of Moffett Field, California is selected to utilize Kennedy facilities for NASA's Lunar Cargo Transportation and Landing by Soft Touchdown, or Lunar CATALYST, initiative. Moon Express is developing a lander with capabilities that will enable delivery of payloads to the surface of the moon, as well as new science and exploration missions of interest to NASA and scientific and academic communities. Moon Express will base its activities at Kennedy and utilize the Morpheus ALHAT field and a hangar nearby for CATALYST testing. The Advanced Exploration Systems Division of NASA's Human Exploration and Operations Mission Directorate manages Lunar CATALYST.
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Del Piccolo, Lidia; de Haes, Hanneke; Heaven, Cathy; Jansen, Jesse; Verheul, William; Bensing, Jozien; Bergvik, Svein; Deveugele, Myriam; Eide, Hilde; Fletcher, Ian; Goss, Claudia; Humphris, Gerry; Kim, Young-Mi; Langewitz, Wolf; Mazzi, Maria Angela; Mjaaland, Trond; Moretti, Francesca; Nübling, Matthias; Rimondini, Michela; Salmon, Peter; Sibbern, Tonje; Skre, Ingunn; van Dulmen, Sandra; Wissow, Larry; Young, Bridget; Zandbelt, Linda; Zimmermann, Christa; Finset, Arnstein
2011-02-01
To present a method to classify health provider responses to patient cues and concerns according to the VR-CoDES-CC (Del Piccolo et al. (2009) [2] and Zimmermann et al. (submitted for publication) [3]). The system permits sequence analysis and a detailed description of how providers handle patient's expressions of emotion. The Verona-CoDES-P system has been developed based on consensus views within the "Verona Network of Sequence Analysis". The different phases of the creation process are described in detail. A reliability study has been conducted on 20 interviews from a convenience sample of 104 psychiatric consultations. The VR-CoDES-P has two main classes of provider responses, corresponding to the degree of explicitness (yes/no) and space (yes/no) that is given by the health provider to each cue/concern expressed by the patient. The system can be further subdivided into 17 individual categories. Statistical analyses showed that the VR-CoDES-P is reliable (agreement 92.86%, Cohen's kappa 0.90 (±0.04) p<0.0001). Once validity and reliability are tested in different settings, the system should be applied to investigate the relationship between provider responses to patients' expression of emotions and outcome variables. Research employing the VR-CoDES-P should be applied to develop research-based approaches to maximize appropriate responses to patients' indirect and overt expressions of emotional needs. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.
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Elementary School Students' Perceptions of Technology in their Pictorial Representations
ERIC Educational Resources Information Center
Eristi, Suzan Duygu; Kurt, Adile Askim
2011-01-01
The current study aimed to reveal elementary school students' perceptions of technology through their pictorial representations and their written expressions based on their pictorial representations. Content analysis based on the qualitative research method along with art-based inquiry was applied. The "coding system for the concepts revealed…
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Duan, Qiaonan; Flynn, Corey; Niepel, Mario; Hafner, Marc; Muhlich, Jeremy L; Fernandez, Nicolas F; Rouillard, Andrew D; Tan, Christopher M; Chen, Edward Y; Golub, Todd R; Sorger, Peter K; Subramanian, Aravind; Ma'ayan, Avi
2014-07-01
For the Library of Integrated Network-based Cellular Signatures (LINCS) project many gene expression signatures using the L1000 technology have been produced. The L1000 technology is a cost-effective method to profile gene expression in large scale. LINCS Canvas Browser (LCB) is an interactive HTML5 web-based software application that facilitates querying, browsing and interrogating many of the currently available LINCS L1000 data. LCB implements two compacted layered canvases, one to visualize clustered L1000 expression data, and the other to display enrichment analysis results using 30 different gene set libraries. Clicking on an experimental condition highlights gene-sets enriched for the differentially expressed genes from the selected experiment. A search interface allows users to input gene lists and query them against over 100 000 conditions to find the top matching experiments. The tool integrates many resources for an unprecedented potential for new discoveries in systems biology and systems pharmacology. The LCB application is available at http://www.maayanlab.net/LINCS/LCB. Customized versions will be made part of the http://lincscloud.org and http://lincs.hms.harvard.edu websites. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.
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Zhu, Yuerong; Zhu, Yuelin; Xu, Wei
2008-01-01
Background Though microarray experiments are very popular in life science research, managing and analyzing microarray data are still challenging tasks for many biologists. Most microarray programs require users to have sophisticated knowledge of mathematics, statistics and computer skills for usage. With accumulating microarray data deposited in public databases, easy-to-use programs to re-analyze previously published microarray data are in high demand. Results EzArray is a web-based Affymetrix expression array data management and analysis system for researchers who need to organize microarray data efficiently and get data analyzed instantly. EzArray organizes microarray data into projects that can be analyzed online with predefined or custom procedures. EzArray performs data preprocessing and detection of differentially expressed genes with statistical methods. All analysis procedures are optimized and highly automated so that even novice users with limited pre-knowledge of microarray data analysis can complete initial analysis quickly. Since all input files, analysis parameters, and executed scripts can be downloaded, EzArray provides maximum reproducibility for each analysis. In addition, EzArray integrates with Gene Expression Omnibus (GEO) and allows instantaneous re-analysis of published array data. Conclusion EzArray is a novel Affymetrix expression array data analysis and sharing system. EzArray provides easy-to-use tools for re-analyzing published microarray data and will help both novice and experienced users perform initial analysis of their microarray data from the location of data storage. We believe EzArray will be a useful system for facilities with microarray services and laboratories with multiple members involved in microarray data analysis. EzArray is freely available from . PMID:18218103
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Huang, Lihong; Liu, Shukai; Zang, Fuyu; Xing, Jinchao; Zhang, Youyue; Liang, Jiaqi; Zhang, Guihong
2017-01-01
In the swine industry, porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease which causes heavy economic losses worldwide. Effective prevention and disease control is an important issue. In this study, we described the construction of a Japanese encephalitis virus (JEV) DNA-based replicon with a cytomegalovirus (CMV) promoter based on the genome of Japanese encephalitis live vaccine virus SA14-14-2, which is capable of offering a potentially novel way to develop and produce vaccines against a major pathogen of global health. This JEV DNA-based replicon contains a large deletion in the structural genes (C-prM-E). A PRRSV GP5/M was inserted into the deletion position of JEV DNA-based replicons to develop a chimeric replicon vaccine candidate for PRRSV. The results showed that BALB/c mice models with the replicon vaccines pJEV-REP-G-2A-M-IRES and pJEV-REP-G-2A-M stimulated antibody responses and induced a cellular immune response. Analysis of ELSA data showed that vaccination with the replicon vaccine expressing GP5/M induced a better antibodies response than traditional DNA vaccines. Therefore, the results suggested that this ectopic expression system based on JEV DNA-based replicons may represent a useful molecular platform for various biological applications, and the JEV DNA-based replicons expressing GP5/M can be further developed into a novel, safe vaccine candidate for PRRS. PMID:28740748
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A stable RNA virus-based vector for citrus trees
DOE Office of Scientific and Technical Information (OSTI.GOV)
Folimonov, Alexey S.; Folimonova, Svetlana Y.; Bar-Joseph, Moshe
Virus-based vectors are important tools in plant molecular biology and plant genomics. A number of vectors based on viruses that infect herbaceous plants are in use for expression or silencing of genes in plants as well as screening unknown sequences for function. Yet there is a need for useful virus-based vectors for woody plants, which demand much greater stability because of the longer time required for systemic infection and analysis. We examined several strategies to develop a Citrus tristeza virus (CTV)-based vector for transient expression of foreign genes in citrus trees using a green fluorescent protein (GFP) as a reporter.more » These strategies included substitution of the p13 open reading frame (ORF) by the ORF of GFP, construction of a self-processing fusion of GFP in-frame with the major coat protein (CP), or expression of the GFP ORF as an extra gene from a subgenomic (sg) mRNA controlled either by a duplicated CTV CP sgRNA controller element (CE) or an introduced heterologous CE of Beet yellows virus. Engineered vector constructs were examined for replication, encapsidation, GFP expression during multiple passages in protoplasts, and for their ability to infect, move, express GFP, and be maintained in citrus plants. The most successful vectors based on the 'add-a-gene' strategy have been unusually stable, continuing to produce GFP fluorescence after more than 4 years in citrus trees.« less
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NASA Astrophysics Data System (ADS)
Toledo-Suárez, Carlos D.
It is proposed a way of increasing the cardinality of an alphabet used to write rules in a learning classifier system that extends the idea of relational schemata. Theoretical justifications regarding the possible reduction in the amount of rules for the solution of problems such extended alphabets (st-alphabets) imply are shown. It is shown that when expressed as bipolar neural networks, the matching process of rules over st-alphabets strongly resembles a gene expression mechanism applied to a system over {0,1,#}. In spite of the apparent drawbacks the explicit use of such relational alphabets would imply, their successful implementation in an information gain based classifier system (IGCS) is presented.
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González-Fuente, Santiago; Tubau, Susagna; Espinal, M. Teresa; Prieto, Pilar
2015-01-01
Previous research has proposed that languages diverge with respect to how their speakers confirm and contradict negative questions. Taking into account the classification between truth-based and polarity-based languages, this paper is mainly concerned with the expression of REJECT (a semantic operation that signals a contradiction move with respect to the common ground, along Krifka's lines) in two languages belonging to two typologically distinct answering systems, namely Catalan (polarity-based) and Russian (a mixed system using polarity-based, truth-based, and echoic strategies). This investigation has two goals. First, to assess empirically the relevance of prosodic and gestural patterns in the interpretation of confirming and rejecting responses to negative polar questions. Second, to test the claim that in fact speakers resort to strikingly similar universal strategies at the time of expressing rejecting answers to discourse accessible negative assertions and negative polar questions, namely the use of linguistic units that encode REJECT in combination with ASSERT. The results of our investigation support the existence of a universal answering system for rejecting negative polar questions that integrates lexical and syntactic strategies with prosodic and gestural patterns, and instantiate the REJECT and ASSERT operators. We will also discuss the implications these results have for the truth-based vs. polarity-based taxonomy. PMID:26217255
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Dammeyer, Thorben; Steinwand, Miriam; Krüger, Sarah-C; Dübel, Stefan; Hust, Michael; Timmis, Kenneth N
2011-02-21
Recombinant antibody fragments have a wide range of applications in research, diagnostics and therapy. For many of these, small fragments like single chain fragment variables (scFv) function well and can be produced inexpensively in bacterial expression systems. Although Escherichia coli K-12 production systems are convenient, yields of different fragments, even those produced from codon-optimized expression systems, vary significantly. Where yields are inadequate, alternative production systems are needed. Pseudomonas putida strain KT2440 is a versatile biosafety strain known for good expression of heterologous genes, so we have explored its utility as a cell factory for production of scFvs. We have generated new broad host range scFv expression constructs and assessed their production in the Pseudomonas putida KT2440 host. Two scFvs bind either to human C-reactive protein or to mucin1, proteins of significant medical diagnostic and therapeutic interest, whereas a third is a model anti-lysozyme scFv. The KT2440 antibody expression systems produce scFvs targeted to the periplasmic space that were processed precisely and were easily recovered and purified by single-step or tandem affinity chromatography. The influence of promoter system, codon optimization for P. putida, and medium on scFv yield was examined. Yields of up to 3.5 mg/l of pure, soluble, active scFv fragments were obtained from shake flask cultures of constructs based on the original codon usage and expressed from the Ptac expression system, yields that were 2.5-4 times higher than those from equivalent cultures of an E. coli K-12 expression host. Pseudomonas putida KT2440 is a good cell factory for the production of scFvs, and the broad host range constructs we have produced allow yield assessment in a number of different expression hosts when yields in one initially selected are insufficient. High cell density cultivation and further optimization and refinement of the KT2440 cell factory will achieve additional increases in the yields of scFvs.
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Knowledge-based requirements analysis for automating software development
NASA Technical Reports Server (NTRS)
Markosian, Lawrence Z.
1988-01-01
We present a new software development paradigm that automates the derivation of implementations from requirements. In this paradigm, informally-stated requirements are expressed in a domain-specific requirements specification language. This language is machine-understable and requirements expressed in it are captured in a knowledge base. Once the requirements are captured, more detailed specifications and eventually implementations are derived by the system using transformational synthesis. A key characteristic of the process is that the required human intervention is in the form of providing problem- and domain-specific engineering knowledge, not in writing detailed implementations. We describe a prototype system that applies the paradigm in the realm of communication engineering: the prototype automatically generates implementations of buffers following analysis of the requirements on each buffer.
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Construction of diagnosis system and gene regulatory networks based on microarray analysis.
Hong, Chun-Fu; Chen, Ying-Chen; Chen, Wei-Chun; Tu, Keng-Chang; Tsai, Meng-Hsiun; Chan, Yung-Kuan; Yu, Shyr Shen
2018-05-01
A microarray analysis generally contains expression data of thousands of genes, but most of them are irrelevant to the disease of interest, making analyzing the genes concerning specific diseases complicated. Therefore, filtering out a few essential genes as well as their regulatory networks is critical, and a disease can be easily diagnosed just depending on the expression profiles of a few critical genes. In this study, a target gene screening (TGS) system, which is a microarray-based information system that integrates F-statistics, pattern recognition matching, a two-layer K-means classifier, a Parameter Detection Genetic Algorithm (PDGA), a genetic-based gene selector (GBG selector) and the association rule, was developed to screen out a small subset of genes that can discriminate malignant stages of cancers. During the first stage, F-statistic, pattern recognition matching, and a two-layer K-means classifier were applied in the system to filter out the 20 critical genes most relevant to ovarian cancer from 9600 genes, and the PDGA was used to decide the fittest values of the parameters for these critical genes. Among the 20 critical genes, 15 are associated with cancer progression. In the second stage, we further employed a GBG selector and the association rule to screen out seven target gene sets, each with only four to six genes, and each of which can precisely identify the malignancy stage of ovarian cancer based on their expression profiles. We further deduced the gene regulatory networks of the 20 critical genes by applying the Pearson correlation coefficient to evaluate the correlationship between the expression of each gene at the same stages and at different stages. Correlationships between gene pairs were calculated, and then, three regulatory networks were deduced. Their correlationships were further confirmed by the Ingenuity pathway analysis. The prognostic significances of the genes identified via regulatory networks were examined using online tools, and most represented biomarker candidates. In summary, our proposed system provides a new strategy to identify critical genes or biomarkers, as well as their regulatory networks, from microarray data. Copyright © 2018. Published by Elsevier Inc.
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Analysis of a Plant Transcriptional Regulatory Network Using Transient Expression Systems.
Díaz-Triviño, Sara; Long, Yuchen; Scheres, Ben; Blilou, Ikram
2017-01-01
In plant biology, transient expression systems have become valuable approaches used routinely to rapidly study protein expression, subcellular localization, protein-protein interactions, and transcriptional activity prior to in vivo studies. When studying transcriptional regulation, luciferase reporter assays offer a sensitive readout for assaying promoter behavior in response to different regulators or environmental contexts and to confirm and assess the functional relevance of predicted binding sites in target promoters. This chapter aims to provide detailed methods for using luciferase reporter system as a rapid, efficient, and versatile assay to analyze transcriptional regulation of target genes by transcriptional regulators. We describe a series of optimized transient expression systems consisting of Arabidopsis thaliana protoplasts, infiltrated Nicotiana benthamiana leaves, and human HeLa cells to study the transcriptional regulations of two well-characterized transcriptional regulators SCARECROW (SCR) and SHORT-ROOT (SHR) on one of their targets, CYCLIN D6 (CYCD6).Here, we illustrate similarities and differences in outcomes when using different systems. The plant-based systems revealed that the SCR-SHR complex enhances CYCD6 transcription, while analysis in HeLa cells showed that the complex is not sufficient to strongly induce CYCD6 transcription, suggesting that additional, plant-specific regulators are required for full activation. These results highlight the importance of the system and suggest that including heterologous systems, such as HeLa cells, can provide a more comprehensive analysis of a complex gene regulatory network.
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Beneyton, Thomas; Thomas, Stéphane; Griffiths, Andrew D; Nicaud, Jean-Marc; Drevelle, Antoine; Rossignol, Tristan
2017-01-31
Droplet-based microfluidics is becoming an increasingly attractive alternative to microtiter plate techniques for enzymatic high-throughput screening (HTS), especially for exploring large diversities with lower time and cost footprint. In this case, the assayed enzyme has to be accessible to the substrate within the water-in-oil droplet by being ideally extracellular or displayed at the cell surface. However, most of the enzymes screened to date are expressed within the cytoplasm of Escherichia coli cells, which means that a lysis step must take place inside the droplets for enzyme activity to be assayed. Here, we take advantage of the excellent secretion abilities of the yeast Yarrowia lipolytica to describe a highly efficient expression system particularly suitable for the droplet-based microfluidic HTS. Five hydrolytic genes from Aspergillus niger genome were chosen and the corresponding five Yarrowia lipolytica producing strains were constructed. Each enzyme (endo-β-1,4-xylanase B and C; 1,4-β-cellobiohydrolase A; endoglucanase A; aspartic protease) was successfully overexpressed and secreted in an active form in the crude supernatant. A droplet-based microfluidic HTS system was developed to (a) encapsulate single yeast cells; (b) grow yeast in droplets; (c) inject the relevant enzymatic substrate; (d) incubate droplets on chip; (e) detect enzymatic activity; and (f) sort droplets based on enzymatic activity. Combining this integrated microfluidic platform with gene expression in Y. lipolytica results in remarkably low variability in the enzymatic activity at the single cell level within a given monoclonal population (<5%). Xylanase, cellobiohydrolase and protease activities were successfully assayed using this system. We then used the system to screen for thermostable variants of endo-β-1,4-xylanase C in error-prone PCR libraries. Variants displaying higher thermostable xylanase activities compared to the wild-type were isolated (up to 4.7-fold improvement). Yarrowia lipolytica was used to express fungal genes encoding hydrolytic enzymes of interest. We developed a successful droplet-based microfluidic platform for the high-throughput screening (10 5 strains/h) of Y. lipolytica based on enzyme secretion and activity. This approach provides highly efficient tools for the HTS of recombinant enzymatic activities. This should be extremely useful for discovering new biocatalysts via directed evolution or protein engineering approaches and should lead to major advances in microbial cell factory development.
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A statistical method for measuring activation of gene regulatory networks.
Esteves, Gustavo H; Reis, Luiz F L
2018-06-13
Gene expression data analysis is of great importance for modern molecular biology, given our ability to measure the expression profiles of thousands of genes and enabling studies rooted in systems biology. In this work, we propose a simple statistical model for the activation measuring of gene regulatory networks, instead of the traditional gene co-expression networks. We present the mathematical construction of a statistical procedure for testing hypothesis regarding gene regulatory network activation. The real probability distribution for the test statistic is evaluated by a permutation based study. To illustrate the functionality of the proposed methodology, we also present a simple example based on a small hypothetical network and the activation measuring of two KEGG networks, both based on gene expression data collected from gastric and esophageal samples. The two KEGG networks were also analyzed for a public database, available through NCBI-GEO, presented as Supplementary Material. This method was implemented in an R package that is available at the BioConductor project website under the name maigesPack.
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Zhang, Jing; Zhang, Lu; Zhang, Yan; Yang, Jing; Guo, Mengbiao; Sun, Liangdan; Pan, Hai-Feng; Hirankarn, Nattiya; Ying, Dingge; Zeng, Shuai; Lee, Tsz Leung; Lau, Chak Sing; Chan, Tak Mao; Leung, Alexander Moon Ho; Mok, Chi Chiu; Wong, Sik Nin; Lee, Ka Wing; Ho, Marco Hok Kung; Lee, Pamela Pui Wah; Chung, Brian Hon-Yin; Chong, Chun Yin; Wong, Raymond Woon Sing; Mok, Mo Yin; Wong, Wilfred Hing Sang; Tong, Kwok Lung; Tse, Niko Kei Chiu; Li, Xiang-Pei; Avihingsanon, Yingyos; Rianthavorn, Pornpimol; Deekajorndej, Thavatchai; Suphapeetiporn, Kanya; Shotelersuk, Vorasuk; Ying, Shirley King Yee; Fung, Samuel Ka Shun; Lai, Wai Ming; Garcia-Barceló, Maria-Mercè; Cherny, Stacey S; Sham, Pak Chung; Cui, Yong; Yang, Sen; Ye, Dong Qing; Zhang, Xue-Jun; Lau, Yu Lung; Yang, Wanling
2015-11-01
Previous genome-wide association studies (GWAS), which were mainly based on single-variant analysis, have identified many systemic lupus erythematosus (SLE) susceptibility loci. However, the genetic architecture of this complex disease is far from being understood. The aim of this study was to investigate whether using a gene-based analysis may help to identify novel loci, by considering global evidence of association from a gene or a genomic region rather than focusing on evidence for individual variants. Based on the results of a meta-analysis of 2 GWAS of SLE conducted in 2 Asian cohorts, we performed an in-depth gene-based analysis followed by replication in a total of 4,626 patients and 7,466 control subjects of Asian ancestry. Differential allelic expression was measured by pyrosequencing. More than one-half of the reported SLE susceptibility loci showed evidence of independent effects, and this finding is important for understanding the mechanisms of association and explaining disease heritability. ANXA6 was detected as a novel SLE susceptibility gene, with several single-nucleotide polymorphisms (SNPs) contributing independently to the association with disease. The risk allele of rs11960458 correlated significantly with increased expression of ANXA6 in peripheral blood mononuclear cells from heterozygous healthy control subjects. Several other associated SNPs may also regulate ANXA6 expression, according to data obtained from public databases. Higher expression of ANXA6 in patients with SLE was also reported previously. Our study demonstrated the merit of using gene-based analysis to identify novel susceptibility loci, especially those with independent effects, and also demonstrated the widespread presence of loci with independent effects in SLE susceptibility genes. © 2015, American College of Rheumatology.
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Liu, Fang-Teng; Chen, Han-Min; Xiong, Ying; Zhu, Zheng-Ming
2017-07-26
Numerous studies have investigated the relationship between deregulated HOXB7 expression with the clinical outcome in patients with digestive stem cancers, HOXB7 has showed negative impacts but with varying levels. We aimed to comprehensively evaluate the prediction and prognostic value of HOXB7 in digestive stem cancers. Electronic databases updated to December 1, 2016 were retrieved to collect relevant eligible studies to quantitatively explore the potential roles of HOXB7 as a prognostic indicator in digestive system cancers. A total of 9 studies (n = 1298 patients) was included in this synthetical meta-analysis. The pooled hazard ratios suggested that high expression of HOXB7 protein was associated with poor prognosis of OS in patients with digestive system cancers (HR = 1.97, 95% CI: 1.65-2.28, p= 0.000), and HOXB7 protein could act as an independent prognostic factor for predicting OS of patients with digestive system cancers (HR: 2.02, 95% CI: 1.69-2.36, p = 0.000). Statistical significance was also observed in subgroup meta-analysis based on the cancer type, histology type, country, sample size and publication date. Furthermore, we examined the correlations between HOXB7 protein and clinicopathological features. It showed that altered expression of HOXB7 protein was correlated with tumor invasion (p = 0.000), lymph node status (p = 0.000), distant metastasis (p = 0.001) and TNM stage (p = 0.000). However, the expression of HOXB7 protein was not associated with age (p = 0.64), gender (p = 0.40) or levels of differentiation (p = 0.19). High expression of HOXB7 protein was associated with poor prognosis of patients with digestive system cancers, as well as clinicopathologic characteristics, including the tumor invasion, lymph node status, distant metastasis and TNM stage. The expression of HOXB7 protein was not associated with age, gender or levels of differentiation. HOXB7 protein expression level in tumor tissue might serve as a novel prognostic marker for digestive system cancers.
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Calculating Stress: From Entropy to a Thermodynamic Concept of Health and Disease
Nečesánek, Ivo; Konečný, David; Vasku, Anna
2016-01-01
To date, contemporary science has lacked a satisfactory tool for the objective expression of stress. This text thus introduces a new–thermodynamically derived–approach to stress measurement, based on entropy production in time and independent of the quality or modality of a given stressor or a combination thereof. Hereto, we propose a novel model of stress response based on thermodynamic modelling of entropy production, both in the tissues/organs and in regulatory feedbacks. Stress response is expressed in our model on the basis of stress entropic load (SEL), a variable we introduced previously; the mathematical expression of SEL, provided here for the first time, now allows us to describe the various states of a living system, including differentiating between states of health and disease. The resulting calculation of stress response regardless of the type of stressor(s) in question is thus poised to become an entirely new tool for predicting the development of a living system. PMID:26771542
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Armstrong, Brent D; Herfst, Christine A; Tonial, Nicholas C; Wakabayashi, Adrienne T; Zeppa, Joseph J; McCormick, John K
2016-11-03
Streptococcus pyogenes is a globally prominent bacterial pathogen that exhibits strict tropism for the human host, yet bacterial factors responsible for the ability of S. pyogenes to compete within this limited biological niche are not well understood. Using an engineered recombinase-based in vivo expression technology (RIVET) system, we identified an in vivo-induced promoter region upstream of a predicted Class IIb bacteriocin system in the M18 serotype S. pyogenes strain MGAS8232. This promoter element was not active under in vitro laboratory conditions, but was highly induced within the mouse nasopharynx. Recombinant expression of the predicted mature S. pyogenes bacteriocin peptides (designated SpbM and SpbN) revealed that both peptides were required for antimicrobial activity. Using a gain of function experiment in Lactococcus lactis, we further demonstrated S. pyogenes immunity function is encoded downstream of spbN. These data highlight the importance of bacterial gene regulation within appropriate environments to help understand mechanisms of niche adaptation by bacterial pathogens.
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Armstrong, Brent D.; Herfst, Christine A.; Tonial, Nicholas C.; Wakabayashi, Adrienne T.; Zeppa, Joseph J.; McCormick, John K.
2016-01-01
Streptococcus pyogenes is a globally prominent bacterial pathogen that exhibits strict tropism for the human host, yet bacterial factors responsible for the ability of S. pyogenes to compete within this limited biological niche are not well understood. Using an engineered recombinase-based in vivo expression technology (RIVET) system, we identified an in vivo-induced promoter region upstream of a predicted Class IIb bacteriocin system in the M18 serotype S. pyogenes strain MGAS8232. This promoter element was not active under in vitro laboratory conditions, but was highly induced within the mouse nasopharynx. Recombinant expression of the predicted mature S. pyogenes bacteriocin peptides (designated SpbM and SpbN) revealed that both peptides were required for antimicrobial activity. Using a gain of function experiment in Lactococcus lactis, we further demonstrated S. pyogenes immunity function is encoded downstream of spbN. These data highlight the importance of bacterial gene regulation within appropriate environments to help understand mechanisms of niche adaptation by bacterial pathogens. PMID:27808235
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Genetic network inference as a series of discrimination tasks.
Kimura, Shuhei; Nakayama, Satoshi; Hatakeyama, Mariko
2009-04-01
Genetic network inference methods based on sets of differential equations generally require a great deal of time, as the equations must be solved many times. To reduce the computational cost, researchers have proposed other methods for inferring genetic networks by solving sets of differential equations only a few times, or even without solving them at all. When we try to obtain reasonable network models using these methods, however, we must estimate the time derivatives of the gene expression levels with great precision. In this study, we propose a new method to overcome the drawbacks of inference methods based on sets of differential equations. Our method infers genetic networks by obtaining classifiers capable of predicting the signs of the derivatives of the gene expression levels. For this purpose, we defined a genetic network inference problem as a series of discrimination tasks, then solved the defined series of discrimination tasks with a linear programming machine. Our experimental results demonstrated that the proposed method is capable of correctly inferring genetic networks, and doing so more than 500 times faster than the other inference methods based on sets of differential equations. Next, we applied our method to actual expression data of the bacterial SOS DNA repair system. And finally, we demonstrated that our approach relates to the inference method based on the S-system model. Though our method provides no estimation of the kinetic parameters, it should be useful for researchers interested only in the network structure of a target system. Supplementary data are available at Bioinformatics online.
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Govender, Nisha; Senan, Siju; Mohamed-Hussein, Zeti-Azura; Wickneswari, Ratnam
2018-06-15
The plant shoot system consists of reproductive organs such as inflorescences, buds and fruits, and the vegetative leaves and stems. In this study, the reproductive part of the Jatropha curcas shoot system, which includes the aerial shoots, shoots bearing the inflorescence and inflorescence were investigated in regard to gene-to-gene interactions underpinning yield-related biological processes. An RNA-seq based sequencing of shoot tissues performed on an Illumina HiSeq. 2500 platform generated 18 transcriptomes. Using the reference genome-based mapping approach, a total of 64 361 genes was identified in all samples and the data was annotated against the non-redundant database by the BLAST2GO Pro. Suite. After removing the outlier genes and samples, a total of 12 734 genes across 17 samples were subjected to gene co-expression network construction using petal, an R library. A gene co-expression network model built with scale-free and small-world properties extracted four vicinity networks (VNs) with putative involvement in yield-related biological processes as follow; heat stress tolerance, floral and shoot meristem differentiation, biosynthesis of chlorophyll molecules and laticifers, cell wall metabolism and epigenetic regulations. Our VNs revealed putative key players that could be adapted in breeding strategies for J. curcas shoot system improvements.
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Development of a PCR-Based Reverse Genetics System for an Attenuated Duck Tembusu Virus Strain
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
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Development of a PCR-Based Reverse Genetics System for an Attenuated Duck Tembusu Virus Strain.
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.
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2013-01-01
Background Respiratory syncytial virus (RSV) is an important cause of lower respiratory tract infection in young children. The degree of disease severity is determined by the host response to infection. Lung macrophages play an important early role in the host response to infection and we have used a systems-based approach to examine the host response in RSV-infected lung-derived macrophage cells. Results Lung macrophage cells could be efficiently infected (>95%) with RSV in vitro, and the expression of several virus structural proteins could be detected. Although we failed to detect significant levels of virus particle production, virus antigen could be detected up until 96 hours post-infection (hpi). Microarray analysis indicated that 20,086 annotated genes were expressed in the macrophage cells, and RSV infection induced an 8.9% and 11.3% change in the global gene transcriptome at 4 hpi and 24 hpi respectively. Genes showing up-regulated expression were more numerous and exhibited higher changes in expression compared to genes showing down-regulated expression. Based on gene ontology, genes with cytokine, antiviral, cell death, and signal transduction functions showed the highest increases in expression, while signalling transduction, RNA binding and protein kinase genes showed the greatest reduction in expression levels. Analysis of the global gene expression profile using pathway enrichment analysis confirmed that up-regulated expression of pathways related to pathogen recognition, interferon signalling and antigen presentation occurred in the lung macrophage cells challenged with RSV. Conclusion Our data provided a comprehensive analysis of RSV-induced gene expression changes in lung macrophages. Although virus gene expression was detected, our data was consistent with an abortive infection and this correlated with the activation of several antivirus signalling pathways such as interferon type I signalling and cell death signalling. RSV infection induced a relatively large increase in pro-inflammatory cytokine expression, however the maintenance of this pro-inflammatory response was not dependent on the production of infectious virus particles. The sustained pro-inflammatory response even in the absence of a productive infection suggests that drugs that control the pro-inflammatory response may be useful in the treatment of patients with severe RSV infection. PMID:23506210
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Majer, Eszter; Navarro, José-Antonio; Daròs, José-Antonio
2015-09-01
Plant virus-based expression systems allow quick and efficient production of recombinant proteins in plant biofactories. Among them, a system derived from tobacco etch virus (TEV; genus potyvirus) permits coexpression of equimolar amounts of several recombinant proteins. This work analyzed how to target recombinant proteins to different subcellular localizations in the plant cell using this system. We constructed TEV clones in which green fluorescent protein (GFP), with a chloroplast transit peptide (cTP), a nuclear localization signal (NLS) or a mitochondrial targeting peptide (mTP) was expressed either as the most amino-terminal product or embedded in the viral polyprotein. Results showed that cTP and mTP mediated efficient translocation of GFP to the corresponding organelle only when present at the amino terminus of the viral polyprotein. In contrast, the NLS worked efficiently at both positions. Viruses expressing GFP in the amino terminus of the viral polyprotein produced milder symptoms. Untagged GFPs and cTP and NLS tagged amino-terminal GFPs accumulated to higher amounts in infected tissues. Finally, viral progeny from clones with internal GFPs maintained the extra gene better. These observations will help in the design of potyvirus-based vectors able to coexpress several proteins while targeting different subcellular localizations, as required in plant metabolic engineering. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Angus, Steve P.; Beauchamp, Roberta L.; Blakeley, Jaishri O.; Bott, Marga; Burns, Sarah S.; Carlstedt, Annemarie; Chang, Long-Sheng; Chen, Xin; Clapp, D. Wade; Desouza, Patrick A.; Erdin, Serkan; Fernandez-Valle, Cristina; Guinney, Justin; Gusella, James F.; Haggarty, Stephen J.; Johnson, Gary L.; Morrison, Helen; Petrilli, Alejandra M.; Plotkin, Scott R.; Pratap, Abhishek; Ramesh, Vijaya; Sciaky, Noah; Stemmer-Rachamimov, Anat; Stuhlmiller, Tim J.; Talkowski, Michael E.; Yates, Charles W.; Zawistowski, Jon S.; Zhao, Wen-Ning
2018-01-01
Neurofibromatosis 2 (NF2) is a rare tumor suppressor syndrome that manifests with multiple schwannomas and meningiomas. There are no effective drug therapies for these benign tumors and conventional therapies have limited efficacy. Various model systems have been created and several drug targets have been implicated in NF2-driven tumorigenesis based on known effects of the absence of merlin, the product of the NF2 gene. We tested priority compounds based on known biology with traditional dose-concentration studies in meningioma and schwann cell systems. Concurrently, we studied functional kinome and gene expression in these cells pre- and post-treatment to determine merlin deficient molecular phenotypes. Cell viability results showed that three agents (GSK2126458, Panobinostat, CUDC-907) had the greatest activity across schwannoma and meningioma cell systems, but merlin status did not significantly influence response. In vivo, drug effect was tumor specific with meningioma, but not schwannoma, showing response to GSK2126458 and Panobinostat. In culture, changes in both the transcriptome and kinome in response to treatment clustered predominantly based on tumor type. However, there were differences in both gene expression and functional kinome at baseline between meningioma and schwannoma cell systems that may form the basis for future selective therapies. This work has created an openly accessible resource (www.synapse.org/SynodosNF2) of fully characterized isogenic schwannoma and meningioma cell systems as well as a rich data source of kinome and transcriptome data from these assay systems before and after treatment that enables single and combination drug discovery based on molecular phenotype. PMID:29897904
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Allaway, Robert; Angus, Steve P; Beauchamp, Roberta L; Blakeley, Jaishri O; Bott, Marga; Burns, Sarah S; Carlstedt, Annemarie; Chang, Long-Sheng; Chen, Xin; Clapp, D Wade; Desouza, Patrick A; Erdin, Serkan; Fernandez-Valle, Cristina; Guinney, Justin; Gusella, James F; Haggarty, Stephen J; Johnson, Gary L; La Rosa, Salvatore; Morrison, Helen; Petrilli, Alejandra M; Plotkin, Scott R; Pratap, Abhishek; Ramesh, Vijaya; Sciaky, Noah; Stemmer-Rachamimov, Anat; Stuhlmiller, Tim J; Talkowski, Michael E; Welling, D Bradley; Yates, Charles W; Zawistowski, Jon S; Zhao, Wen-Ning
2018-01-01
Neurofibromatosis 2 (NF2) is a rare tumor suppressor syndrome that manifests with multiple schwannomas and meningiomas. There are no effective drug therapies for these benign tumors and conventional therapies have limited efficacy. Various model systems have been created and several drug targets have been implicated in NF2-driven tumorigenesis based on known effects of the absence of merlin, the product of the NF2 gene. We tested priority compounds based on known biology with traditional dose-concentration studies in meningioma and schwann cell systems. Concurrently, we studied functional kinome and gene expression in these cells pre- and post-treatment to determine merlin deficient molecular phenotypes. Cell viability results showed that three agents (GSK2126458, Panobinostat, CUDC-907) had the greatest activity across schwannoma and meningioma cell systems, but merlin status did not significantly influence response. In vivo, drug effect was tumor specific with meningioma, but not schwannoma, showing response to GSK2126458 and Panobinostat. In culture, changes in both the transcriptome and kinome in response to treatment clustered predominantly based on tumor type. However, there were differences in both gene expression and functional kinome at baseline between meningioma and schwannoma cell systems that may form the basis for future selective therapies. This work has created an openly accessible resource (www.synapse.org/SynodosNF2) of fully characterized isogenic schwannoma and meningioma cell systems as well as a rich data source of kinome and transcriptome data from these assay systems before and after treatment that enables single and combination drug discovery based on molecular phenotype.
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Plavicki, Jessica S.; Squirrell, Jayne M.; Eliceiri, Kevin W.; Boekhoff-Falk, Grace
2015-01-01
Background Distal-less (Dll) encodes a homeodomain transcription factor expressed in developing appendages of organisms throughout metazoan phylogeny. Based on earlier observations in the limbless nematode Caenorhabditis elegans and the primitive chordate amphioxus, it was proposed that Dll had an ancestral function in nervous system development. Consistent with this hypothesis, Dll is necessary for the development of both peripheral and central components of the Drosophila olfactory system. Furthermore, vertebrate homologs of Dll, the Dlx genes, play critical roles in mammalian brain development. Results Using fluorescent immunohistochemistry of fixed samples and multiphoton microscopy of living Drosophila embryos we show that Dll is expressed in the embryonic, larval and adult CNS and PNS in embryonic and larval neurons, brain and ventral nerve cord (VNC) glia, as well as in PNS structures associated with chemosensation. In adult flies, Dll expression is expressed in the optic lobes, central brain regions and the antennal lobes. Conclusions Characterization of Dll expression in the developing nervous system supports a role of Dll in neural development and function and establishes an important basis for determining the specific functional roles of Dll in Drosophila development and for comparative studies of Drosophila Dll functions with those of its vertebrate counterparts. PMID:26472170
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Gene expression platform for synthetic biology in the human pathogen Streptococcus pneumoniae.
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.
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NASA Technical Reports Server (NTRS)
Hague, D. S.; Woodbury, N. W.
1975-01-01
The Mars system is a tool for rapid prediction of aircraft or engine characteristics based on correlation-regression analysis of past designs stored in the data bases. An example of output obtained from the MARS system, which involves derivation of an expression for gross weight of subsonic transport aircraft in terms of nine independent variables is given. The need is illustrated for careful selection of correlation variables and for continual review of the resulting estimation equations. For Vol. 1, see N76-10089.
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Gene network analysis: from heart development to cardiac therapy.
Ferrazzi, Fulvia; Bellazzi, Riccardo; Engel, Felix B
2015-03-01
Networks offer a flexible framework to represent and analyse the complex interactions between components of cellular systems. In particular gene networks inferred from expression data can support the identification of novel hypotheses on regulatory processes. In this review we focus on the use of gene network analysis in the study of heart development. Understanding heart development will promote the elucidation of the aetiology of congenital heart disease and thus possibly improve diagnostics. Moreover, it will help to establish cardiac therapies. For example, understanding cardiac differentiation during development will help to guide stem cell differentiation required for cardiac tissue engineering or to enhance endogenous repair mechanisms. We introduce different methodological frameworks to infer networks from expression data such as Boolean and Bayesian networks. Then we present currently available temporal expression data in heart development and discuss the use of network-based approaches in published studies. Collectively, our literature-based analysis indicates that gene network analysis constitutes a promising opportunity to infer therapy-relevant regulatory processes in heart development. However, the use of network-based approaches has so far been limited by the small amount of samples in available datasets. Thus, we propose to acquire high-resolution temporal expression data to improve the mathematical descriptions of regulatory processes obtained with gene network inference methodologies. Especially probabilistic methods that accommodate the intrinsic variability of biological systems have the potential to contribute to a deeper understanding of heart development.
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Ito, Akira; Nagai, Momoko; Tajino, Junichi; Yamaguchi, Shoki; Iijima, Hirotaka; Zhang, Xiangkai; Aoyama, Tomoki; Kuroki, Hiroshi
2015-01-01
Cell-based therapy has been explored for articular cartilage regeneration. Autologous chondrocyte implantation is a promising cell-based technique for repairing articular cartilage defects. However, there are several issues such as chondrocyte de-differentiation. While numerous studies have been designed to overcome some of these issues, only a few have focused on the thermal environment that can affect chondrocyte metabolism and phenotype. In this study, the effects of different culture temperatures on human chondrocyte metabolism- and phenotype-related gene expression were investigated in 2D and 3D environments. Human chondrocytes were cultured in a monolayer or in a pellet culture system at three different culture temperatures (32°C, 37°C, and 41°C) for 3 days. The results showed that the total RNA level, normalized to the threshold cycle value of internal reference genes, was higher at lower temperatures in both culture systems. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and citrate synthase (CS), which are involved in glycolysis and the citric acid cycle, respectively, were expressed at similar levels at 32°C and 37°C in pellet cultures, but the levels were significantly lower at 41°C. Expression of the chondrogenic markers, collagen type IIA1 (COL2A1) and aggrecan (ACAN), was higher at 37°C than at 32°C and 41°C in both culture systems. However, this phenomenon did not coincide with SRY (sex-determining region Y)-box 9 (SOX9), which is a fundamental transcription factor for chondrogenesis, indicating that a SOX9-independent pathway might be involved in this phenomenon. In conclusion, the expression of chondrocyte metabolism-related genes at 32°C was maintained or enhanced compared to that at 37°C. However, chondrogenesis-related genes were further induced at 37°C in both culture systems. Therefore, manipulating the culture temperature may be an advantageous approach for regulating human chondrocyte metabolic activity and chondrogenesis.
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Automatic Generation of Caricatures with Multiple Expressions Using Transformative Approach
NASA Astrophysics Data System (ADS)
Liao, Wen-Hung; Lai, Chien-An
The proliferation of digital cameras has changed the way we create and share photos. Novel forms of photo composition and reproduction have surfaced in recent years. In this paper, we present an automatic caricature generation system using transformative approaches. By combing facial feature detection, image segmentation and image warping/morphing techniques, the system is able to generate stylized caricature using only one reference image. When more than one reference sample are available, the system can either choose the best fit based on shape matching, or synthesize a composite style using polymorph technique. The system can also produce multiple expressions by controlling a subset of MPEG-4 facial animation parameters (FAP). Finally, to enable flexible manipulation of the synthetic caricature, we also investigate issues such as color quantization and raster-to-vector conversion. A major strength of our method is that the synthesized caricature bears a higher degree of resemblance to the real person than traditional component-based approaches.
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Droplet-based microfluidic analysis and screening of single plant cells.
Yu, Ziyi; Boehm, Christian R; Hibberd, Julian M; Abell, Chris; Haseloff, Jim; Burgess, Steven J; Reyna-Llorens, Ivan
2018-01-01
Droplet-based microfluidics has been used to facilitate high-throughput analysis of individual prokaryote and mammalian cells. However, there is a scarcity of similar workflows applicable to rapid phenotyping of plant systems where phenotyping analyses typically are time-consuming and low-throughput. We report on-chip encapsulation and analysis of protoplasts isolated from the emergent plant model Marchantia polymorpha at processing rates of >100,000 cells per hour. We use our microfluidic system to quantify the stochastic properties of a heat-inducible promoter across a population of transgenic protoplasts to demonstrate its potential for assessing gene expression activity in response to environmental conditions. We further demonstrate on-chip sorting of droplets containing YFP-expressing protoplasts from wild type cells using dielectrophoresis force. This work opens the door to droplet-based microfluidic analysis of plant cells for applications ranging from high-throughput characterisation of DNA parts to single-cell genomics to selection of rare plant phenotypes.
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Tang, Chao; Yang, Chuanjun; Yu, Hui; Tian, Shen; Huang, Xiaomei; Wang, Weiyi; Cai, Peng
2018-01-11
Photosynthesis of Microcystis aeruginosa under Electromagnetic Radiation (1.8 GHz, 40 V/m) was studied by using the proteomics. A total of 30 differentially expressed proteins, including 15 up-regulated and 15 down-regulated proteins, were obtained in this study. The differentially expressed proteins were significantly enriched in the photosynthesis pathway, in which the protein expression levels of photosystems II cytochrome b559 α subunit, cytochrome C550, PsbY, and F-type ATP synthase (a, b) decreased. Our results indicated that electromagnetic radiation altered the photosynthesis-related protein expression levels, and aimed at the function of photosynthetic pigments, photosystems II potential activity, photosynthetic electron transport process, and photosynthetic phosphorylation process of M. aeruginosa. Based on the above evidence, that photoreaction system may be deduced as a target of electromagnetic radiation on the photosynthesis in cyanobacteria; the photoreaction system of cyanobacteria is a hypothetical "shared target effector" that responds to light and electromagnetic radiation; moreover, electromagnetic radiation does not act on the functional proteins themselves but their expression processes.
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Lu, Jiamiao; Williams, James A.; Luke, Jeremy; Zhang, Feijie; Chu, Kirk; Kay, Mark A.
2017-01-01
We previously developed a mini-intronic plasmid (MIP) expression system in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within a universal 5′ UTR noncoding exon. Like minicircle DNA plasmids (devoid of bacterial backbone sequences), MIP plasmids overcome transcriptional silencing of the transgene. However, in addition MIP plasmids increase transgene expression by 2 and often >10 times higher than minicircle vectors in vivo and in vitro. Based on these findings, we examined the effects of the MIP intronic sequences in a recombinant adeno-associated virus (AAV) vector system. Recombinant AAV vectors containing an intron with a bacterial replication origin and bacterial selectable marker increased transgene expression by 40 to 100 times in vivo when compared with conventional AAV vectors. Therefore, inclusion of this noncoding exon/intron sequence upstream of the coding region can substantially enhance AAV-mediated gene expression in vivo. PMID:27903072
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Wang Lei; Sasai, Ken; Akagi, Tsuyoshi
2008-08-29
The AKT pathway is frequently activated in glioblastoma, and as such, inhibitors of this pathway could prove very useful as anti-glioblastoma therapies. Here we established immortalized astrocytes expressing Renilla luciferase as well as those expressing both an active form of AKT and firefly luciferase. Since both luciferase activities represent the numbers of corresponding cell lines, novel inhibitors of the AKT pathway can be identified by treating co-cultures containing the two types of luciferase-expressing cells with individual compounds. Indeed, such a screening system succeeded in identifying fumitremorgin C as an efficient inhibitor of the AKT pathway, which was further confirmed bymore » the ability of fumitremorgin C to selectively inhibit the growth of immortalized astrocytes expressing an active form of AKT. The present study proposes a broadly applicable approach for identifying therapeutic agents that target the pathways and/or molecules responsible for cancer development.« less
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Sevillano, Laura; Díaz, Margarita; Santamaría, Ramón I
2017-09-26
The industrial use of enzymes produced by microorganisms is continuously growing due to the need for sustainable solutions. Nevertheless, many of the plasmids used for recombinant production of proteins in bacteria are based on the use of antibiotic resistance genes as selection markers. The safety concerns and legal requirements surrounding the increased use of antibiotic resistance genes have made the development of new antibiotic-free approaches essential. In this work, a system completely free of antibiotic resistance genes and useful for the production of high yields of proteins in Streptomyces is described. This system is based on the separation of the two components of the yefM/yoeBsl (antitoxin/toxin) operon; the toxin (yoeBsl) gene, responsible for host death, is integrated into the genome and the antitoxin gene (yefMsl), which inactivates the toxin, is located in the expression plasmid. To develop this system, the toxin gene was integrated into the genome of a strain lacking the complete operon, and the antibiotic resistance gene integrated along with the toxin was eliminated by Cre recombinase to generate a final host strain free of any antibiotic resistance marker. In the same way, the antibiotic resistance gene from the final expression plasmid was removed by Dre recombinase. The usefulness of this system was analysed by checking the production of two hydrolases from different Streptomyces. Production of both proteins, with potential industrial use, was high and stable over time after strain storage and after serial subcultures. These results support the robustness and stability of the positive selection system developed. The total absence of antibiotic resistance genes makes this system a powerful tool for using Streptomyces as a host to produce proteins at the industrial level. This work is the first Streptomyces antibiotic marker-free system to be described. Graphical abstract Antibiotic marker-free platform for protein expression in Streptomyces. The antitoxin gene present in the expression plasmid counteracts the effect of the toxin gene in the genome. In absence of the expression plasmid, the toxin causes cell death ensuring that only plasmid-containing cells persist.
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Display-based communications for advanced transport aircraft
NASA Technical Reports Server (NTRS)
Lee, Alfred T.
1989-01-01
The next generation of civil transport aircraft will depend increasingly upon ground-air-ground and satellite data link for information critical to safe and efficient air transportation. Previous studies which examined the concept of display-based communications in addition to, or in lieu of, conventional voice transmissions are reviewed. A full-mission flight simulation comparing voice and display-based communication modes in an advanced transport aircraft is also described. The results indicate that a display-based mode of information transfer does not result in significantly increased aircrew workload, but does result in substantially increased message acknowledgment times when compared to conventional voice transmissions. User acceptance of the display-based communication system was generally high, replicating the findings of previous studies. However, most pilots tested expressed concern over the potential loss of information available from frequency monitoring which might result from the introduction of discrete address communications. Concern was expressed by some pilots for the reduced time available to search for conflicting traffic when using the communications display system. The implications of the findings for the design of display-based communications are discussed.
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Modelling multimodal expression of emotion in a virtual agent.
Pelachaud, Catherine
2009-12-12
Over the past few years we have been developing an expressive embodied conversational agent system. In particular, we have developed a model of multimodal behaviours that includes dynamism and complex facial expressions. The first feature refers to the qualitative execution of behaviours. Our model is based on perceptual studies and encompasses several parameters that modulate multimodal behaviours. The second feature, the model of complex expressions, follows a componential approach where a new expression is obtained by combining facial areas of other expressions. Lately we have been working on adding temporal dynamism to expressions. So far they have been designed statically, typically at their apex. Only full-blown expressions could be modelled. To overcome this limitation, we have defined a representation scheme that describes the temporal evolution of the expression of an emotion. It is no longer represented by a static definition but by a temporally ordered sequence of multimodal signals.
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RIPS: a UNIX-based reference information program for scientists.
Klyce, S D; Rózsa, A J
1983-09-01
A set of programs is described which implement a personal reference management and information retrieval system on a UNIX-based minicomputer. The system operates in a multiuser configuration with a host of user-friendly utilities that assist entry of reference material, its retrieval, and formatted printing for associated tasks. A search command language was developed without restriction in keyword vocabulary, number of keywords, or level of parenthetical expression nesting. The system is readily transported, and by design is applicable to any academic specialty.
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Targeted and inducible regulation of mammalian gene expression is a broadly important research capability that may also enable development of novel therapeutics for treating human diseases. Here we demonstrate that a catalytically inactive RNA-guided CRISPR-Cpf1 nuclease fused to transcriptional activation domains can up-regulate endogenous human gene expression. We engineered drug-inducible Cpf1-based activators and show how this system can be used to tune the regulation of endogenous gene transcription in human cells.
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A High-Level Language for Modeling Algorithms and Their Properties
NASA Astrophysics Data System (ADS)
Akhtar, Sabina; Merz, Stephan; Quinson, Martin
Designers of concurrent and distributed algorithms usually express them using pseudo-code. In contrast, most verification techniques are based on more mathematically-oriented formalisms such as state transition systems. This conceptual gap contributes to hinder the use of formal verification techniques. Leslie Lamport introduced PlusCal, a high-level algorithmic language that has the "look and feel" of pseudo-code, but is equipped with a precise semantics and includes a high-level expression language based on set theory. PlusCal models can be compiled to TLA + and verified using the model checker tlc.
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A versatile and efficient high-throughput cloning tool for structural biology.
Geertsma, Eric R; Dutzler, Raimund
2011-04-19
Methods for the cloning of large numbers of open reading frames into expression vectors are of critical importance for challenging structural biology projects. Here we describe a system termed fragment exchange (FX) cloning that facilitates the high-throughput generation of expression constructs. The method is based on a class IIS restriction enzyme and negative selection markers. FX cloning combines attractive features of established recombination- and ligation-independent cloning methods: It allows the straightforward transfer of an open reading frame into a variety of expression vectors and is highly efficient and very economic in its use. In addition, FX cloning avoids the common but undesirable feature of significantly extending target open reading frames with cloning related sequences, as it leaves a minimal seam of only a single extra amino acid to either side of the protein. The method has proven to be very robust and suitable for all common pro- and eukaryotic expression systems. It considerably speeds up the generation of expression constructs compared to traditional methods and thus facilitates a broader expression screening.
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Guardado, Pedro; Olivera, Anlys; Rusch, Heather L; Roy, Michael; Martin, Christiana; Lejbman, Natasha; Lee, Hwyunhwa; Gill, Jessica M
2016-03-01
Whole transcriptome analysis provides an unbiased examination of biological activity, and likely, unique insight into the mechanisms underlying posttraumatic stress disorder (PTSD) and comorbid depression and traumatic brain injury. This study compared gene-expression profiles in military personnel with PTSD (n=28) and matched controls without PTSD (n=27) using HG-U133 Plus 2.0 microarrays (Affymetrix), which contain 54,675 probe sets representing more than 38,500 genes. Analysis of expression profiles revealed 203 differentially expressed genes in PTSD, of which 72% were upregulated. Using Partek Genomics Suite 6.6, differentially expressed transcription clusters were filtered based on a selection criterion of ≥1.5 relative fold change at a false discovery rate of ≤5%. Ingenuity Pathway Analysis (Qiagen) of the differentially expressed genes indicated a dysregulation of genes associated with the innate immune, neuroendocrine, and NF-κB systems. These findings provide novel insights that may lead to new pharmaceutical agents for PTSD treatments and help mitigate mental and physical comorbidity risk. Copyright © 2016. Published by Elsevier Ltd.
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Jailani, A Abdul Kader; Solanki, Vikas; Roy, Anirban; Sivasudha, T; Mandal, Bikash
2017-04-02
A highly infectious clone of Cucumber green mottle mosaic virus (CGMMV), a cucurbit-infecting tobamovirus was utilized for designing of gene expression vectors. Two versions of vector were examined for their efficacy in expressing the green fluorescent protein (GFP) in Nicotiana benthamiana. When the GFP gene was inserted at the stop codon of coat protein (CP) gene of the CGMMV genome without any read-through codon, systemic expression of GFP, as well as virion formation and systemic symptoms expression were obtained in N. benthamiana. The qRT-PCR analysis showed 23 fold increase of GFP over actin at 10days post inoculation (dpi), which increased to 45 fold at 14dpi and thereafter the GFP expression was significantly declined. Further, we show that when the most of the CP sequence is deleted retaining only the first 105 nucleotides, the shortened vector containing GFP in frame of original CP open reading frame (ORF) resulted in 234 fold increase of GFP expression over actin at 5dpi in N. benthamiana without the formation of virions and disease symptoms. Our study demonstrated that a simple manipulation of CP gene in the CGMMV genome while preserving the translational frame of CP resulted in developing a virus-free, rapid and efficient foreign protein expression system in the plant. The CGMMV based vectors developed in this study may be potentially useful for the production of edible vaccines in cucurbits. Copyright © 2017 Elsevier B.V. All rights reserved.
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Radiologists' preferences for just-in-time learning.
Kahn, Charles E; Ehlers, Kevin C; Wood, Beverly P
2006-09-01
Effective learning can occur at the point of care, when opportunities arise to acquire information and apply it to a clinical problem. To assess interest in point-of-care learning, we conducted a survey to explore radiologists' attitudes and preferences regarding the use of just-in-time learning (JITL) in radiology. Following Institutional Review Board approval, we invited 104 current radiology residents and 86 radiologists in practice to participate in a 12-item Internet-based survey to assess their attitudes toward just-in-time learning. Voluntary participation in the survey was solicited by e-mail; respondents completed the survey on a web-based form. Seventy-nine physicians completed the questionnaire, including 47 radiology residents and 32 radiologists in practice; the overall response rate was 42%. Respondents generally expressed a strong interest for JITL: 96% indicated a willingness to try such a system, and 38% indicated that they definitely would use a JITL system. They expressed a preference for learning interventions of 5-10 min in length. Current and recent radiology trainees have expressed a strong interest in just-in-time learning. The information from this survey should be useful in pursuing the design of learning interventions and systems for delivering just-in-time learning to radiologists.
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Yoshimoto, Nobuo; Kuroda, Shun'ichi
2014-04-01
For efficient biomolecule production (e.g., antibodies, recombinant proteins), mammalian cells with high expression rates should be selected from cell libraries, propagated while maintaining a homogenous expression rate, and subsequently stabilized at their high expression rate. Clusters of isogenic cells (i.e., colonies) have been used for these processes. However, cellular heterogeneity makes it difficult to obtain cell lines with the highest expression rates by using single-colony-based breeding. Furthermore, even among the single cells in an isogenic cell population, the desired cell properties fluctuate stochastically during long-term culture. Therefore, although the molecular mechanisms underlying stochastic fluctuation are poorly understood, it is necessary to establish excellent cell lines in order to breed single cells to have higher expression, higher stability, and higher homogeneity while suppressing stochastic fluctuation (i.e., single-cell-based breeding). In this review, we describe various methods for manipulating single cells and facilitating single-cell analysis in order to better understand stochastic fluctuation. We demonstrated that single-cell-based breeding is practical and promising by using a high-throughput automated system to analyze and manipulate single cells. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
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Chen, Hui; Huang, Rui; Zhang, Y-H Percival
2017-06-01
The precise control of multiple heterologous enzyme expression levels in one Escherichia coli strain is important for cascade biocatalysis, metabolic engineering, synthetic biology, natural product synthesis, and studies of complexed proteins. We systematically investigated the co-expression of up to four thermophilic enzymes (i.e., α-glucan phosphorylase (αGP), phosphoglucomutase (PGM), glucose 6-phosphate dehydrogenase (G6PDH), and 6-phosphogluconate dehydrogenase (6PGDH)) in E. coli BL21(DE3) by adding T7 promoter or T7 terminator of each gene for multiple genes in tandem, changing gene alignment, and comparing one or two plasmid systems. It was found that the addition of T7 terminator after each gene was useful to decrease the influence of the upstream gene. The co-expression of the four enzymes in E. coli BL21(DE3) was demonstrated to generate two NADPH molecules from one glucose unit of maltodextrin, where NADPH was oxidized to convert xylose to xylitol. The best four-gene co-expression system was based on two plasmids (pET and pACYC) which harbored two genes. As a result, apparent enzymatic activities of the four enzymes were regulated to be at similar levels and the overall four-enzyme activity was the highest based on the formation of xylitol. This study provides useful information for the precise control of multi-enzyme-coordinated expression in E. coli BL21(DE3).
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In Silico Prediction and Validation of Gfap as an miR-3099 Target in Mouse Brain.
Abidin, Shahidee Zainal; Leong, Jia-Wen; Mahmoudi, Marzieh; Nordin, Norshariza; Abdullah, Syahril; Cheah, Pike-See; Ling, King-Hwa
2017-08-01
MicroRNAs are small non-coding RNAs that play crucial roles in the regulation of gene expression and protein synthesis during brain development. MiR-3099 is highly expressed throughout embryogenesis, especially in the developing central nervous system. Moreover, miR-3099 is also expressed at a higher level in differentiating neurons in vitro, suggesting that it is a potential regulator during neuronal cell development. This study aimed to predict the target genes of miR-3099 via in-silico analysis using four independent prediction algorithms (miRDB, miRanda, TargetScan, and DIANA-micro-T-CDS) with emphasis on target genes related to brain development and function. Based on the analysis, a total of 3,174 miR-3099 target genes were predicted. Those predicted by at least three algorithms (324 genes) were subjected to DAVID bioinformatics analysis to understand their overall functional themes and representation. The analysis revealed that nearly 70% of the target genes were expressed in the nervous system and a significant proportion were associated with transcriptional regulation and protein ubiquitination mechanisms. Comparison of in situ hybridization (ISH) expression patterns of miR-3099 in both published and in-house-generated ISH sections with the ISH sections of target genes from the Allen Brain Atlas identified 7 target genes (Dnmt3a, Gabpa, Gfap, Itga4, Lxn, Smad7, and Tbx18) having expression patterns complementary to miR-3099 in the developing and adult mouse brain samples. Of these, we validated Gfap as a direct downstream target of miR-3099 using the luciferase reporter gene system. In conclusion, we report the successful prediction and validation of Gfap as an miR-3099 target gene using a combination of bioinformatics resources with enrichment of annotations based on functional ontologies and a spatio-temporal expression dataset.
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Jung, Seung-Hyun; Shin, Seung-Hun; Yim, Seon-Hee; Choi, Hye-Sun; Lee, Sug-Hyung; Chung, Yeun-Jun
2009-07-31
Recently, microarray-based comparative genomic hybridization (array-CGH) has emerged as a very efficient technology with higher resolution for the genome-wide identification of copy number alterations (CNA). Although CNAs are thought to affect gene expression, there is no platform currently available for the integrated CNA-expression analysis. To achieve high-resolution copy number analysis integrated with expression profiles, we established human 30k oligoarray-based genome-wide copy number analysis system and explored the applicability of this system for integrated genome and transcriptome analysis using MDA-MB-231 cell line. We compared the CNAs detected by the oligoarray with those detected by the 3k BAC array for validation. The oligoarray identified the single copy difference more accurately and sensitively than the BAC array. Seventeen CNAs detected by both platforms in MDA-MB-231 such as gains of 5p15.33-13.1, 8q11.22-8q21.13, 17p11.2, and losses of 1p32.3, 8p23.3-8p11.21, and 9p21 were consistently identified in previous studies on breast cancer. There were 122 other small CNAs (mean size 1.79 mb) that were detected by oligoarray only, not by BAC-array. We performed genomic qPCR targeting 7 CNA regions, detected by oligoarray only, and one non-CNA region to validate the oligoarray CNA detection. All qPCR results were consistent with the oligoarray-CGH results. When we explored the possibility of combined interpretation of both DNA copy number and RNA expression profiles, mean DNA copy number and RNA expression levels showed a significant correlation. In conclusion, this 30k oligoarray-CGH system can be a reasonable choice for analyzing whole genome CNAs and RNA expression profiles at a lower cost.
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Zhang, Xiaomeng; Shao, Bin; Wu, Yangle; Qi, Ouyang
2013-01-01
One of the major objectives in systems biology is to understand the relation between the topological structures and the dynamics of biological regulatory networks. In this context, various mathematical tools have been developed to deduct structures of regulatory networks from microarray expression data. In general, from a single data set, one cannot deduct the whole network structure; additional expression data are usually needed. Thus how to design a microarray expression experiment in order to get the most information is a practical problem in systems biology. Here we propose three methods, namely, maximum distance method, trajectory entropy method, and sampling method, to derive the optimal initial conditions for experiments. The performance of these methods is tested and evaluated in three well-known regulatory networks (budding yeast cell cycle, fission yeast cell cycle, and E. coli. SOS network). Based on the evaluation, we propose an efficient strategy for the design of microarray expression experiments.
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Michaud, Sébastien; Tanguay, Robert M
2003-01-01
Background In addition to their strong induction following stress, small heat shock proteins (Hsp) are also expressed during development in a wide variety of organisms. However, the precise identity of cell(s) expressing these proteins and the functional contribution of small heat shock proteins in such developmental context remain to be determined. The present study provides a detailed description of the Drosophila small heat shock protein Hsp23 expression pattern during embryogenesis and evaluates its functional contribution to central nervous system development. Results Throughout embryogenesis, Hsp23 is expressed in a stage-specific manner by a restricted number of neuronal and glial lineages of the central nervous system. Hsp23 is also detected in the amnioserosa and within a single lateral chordotonal organ. Its expression within the MP2 lineage does not require the presence of a functional midline nor the activity of the Notch signaling pathway. Transactivation assays demonstrate that transcription factors implicated in the differentiation of the midline also regulate hsp23 promoter activity. Phenotypic analysis of a transgenic line exhibiting loss of Hsp23 expression in the central nervous system suggests that Hsp23 is not required for development and function of this tissue. Likewise, its overexpression does not cause deleterious effects, as development remains unaffected. Conclusions Based on the presented data, we suggest that the tightly regulated developmental expression of Hsp23 is not actively involved in cell differentiation and central nervous system development per se but rather reflects a putative role in preventive "pre-stress" neuroprotection or in non-vital process(es) common to the identified cell lineages. PMID:14617383
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Nguyen, Tien-Thanh; Nguyen, Hoang-Minh; Geiger, Barbara; Mathiesen, Geir; Eijsink, Vincent G H; Peterbauer, Clemens K; Haltrich, Dietmar; Nguyen, Thu-Ha
2015-03-07
Two overlapping genes lacL and lacM (lacLM) encoding for heterodimeric β-galactosidase from Lactobacillus reuteri were previously cloned and over-expressed in the food-grade host strain Lactobacillus plantarum WCFS1, using the inducible lactobacillal pSIP expression system. In this study, we analyzed different factors that affect the production of recombinant L. reuteri β-galactosidase. Various factors related to the cultivation, i.e. culture pH, growth temperature, glucose concentration, as well as the induction conditions, including cell concentration at induction point and inducer concentration, were tested. Under optimal fermentation conditions, the maximum β-galactosidase levels obtained were 130 U/mg protein and 35-40 U/ml of fermentation broth corresponding to the formation of approximately 200 mg of recombinant protein per litre of fermentation medium. As calculated from the specific activity of the purified enzyme (190 U/mg), β-galactosidase yield amounted to roughly 70% of the total soluble intracellular protein of the host organism. It was observed that pH and substrate (glucose) concentration are the most prominent factors affecting the production of recombinant β-galactosidase. The over-expression of recombinant L. reuteri β-galactosidase in a food-grade host strain was optimized, which is of interest for applications of this enzyme in the food industry. The results provide more detailed insight into these lactobacillal expression systems and confirm the potential of the pSIP system for efficient, tightly controlled expression of enzymes and proteins in lactobacilli.
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Chang, Le; Wang, Guojing; Jia, Tingting; Zhang, Lei; Li, Yulong; Han, Yanxi; Zhang, Kuo; Lin, Guigao; Zhang, Rui; Li, Jinming; Wang, Lunan
2016-04-26
Hepatocellular carcinoma (HCC) is one of the most frequently diagnosed cancers worldwide. However, the treatment of patients with HCC is particularly challenging. Long non-coding RNA maternally expressed gene 3 (MEG3) has been identified as a potential suppressor of several types of tumors, but the delivery of long RNA remains problematic, limiting its applications. In the present study, we designed a novel delivery system based on MS2 virus-like particles (VLPs) crosslinked with GE11 polypeptide. This vector was found to be fast, effective and safe for the targeted delivery of lncRNA MEG3 RNA to the epidermal growth factor receptor (EGFR)-positive HCC cell lines without the activation of EGFR downstream pathways, and significantly attenuated both in vitro and in vivo tumor cell growth. Our study also revealed that the targeted delivery was mainly dependent on clathrin-mediated endocytosis and MEG3 RNA suppresses tumor growth mainly via increasing the expression of p53 and its downstream gene GDF15, but decreasing the expression of MDM2. Thus, this vector is promising as a novel delivery system and may facilitate a new approach to lncRNA based cancer therapy.
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Quality Assurance of Cancer Study Common Data Elements Using A Post-Coordination Approach
Jiang, Guoqian; Solbrig, Harold R.; Prud’hommeaux, Eric; Tao, Cui; Weng, Chunhua; Chute, Christopher G.
2015-01-01
Domain-specific common data elements (CDEs) are emerging as an effective approach to standards-based clinical research data storage and retrieval. A limiting factor, however, is the lack of robust automated quality assurance (QA) tools for the CDEs in clinical study domains. The objectives of the present study are to prototype and evaluate a QA tool for the study of cancer CDEs using a post-coordination approach. The study starts by integrating the NCI caDSR CDEs and The Cancer Genome Atlas (TCGA) data dictionaries in a single Resource Description Framework (RDF) data store. We designed a compositional expression pattern based on the Data Element Concept model structure informed by ISO/IEC 11179, and developed a transformation tool that converts the pattern-based compositional expressions into the Web Ontology Language (OWL) syntax. Invoking reasoning and explanation services, we tested the system utilizing the CDEs extracted from two TCGA clinical cancer study domains. The system could automatically identify duplicate CDEs, and detect CDE modeling errors. In conclusion, compositional expressions not only enable reuse of existing ontology codes to define new domain concepts, but also provide an automated mechanism for QA of terminological annotations for CDEs. PMID:26958201
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The Study on Network Examinational Database based on ASP Technology
NASA Astrophysics Data System (ADS)
Zhang, Yanfu; Han, Yuexiao; Zhou, Yanshuang
This article introduces the structure of the general test base system based on .NET technology, discussing the design of the function modules and its implementation methods. It focuses on key technology of the system, proposing utilizing the WEB online editor control to solve the input problem and regular expression to solve the problem HTML code, making use of genetic algorithm to optimize test paper and the automated tools of WORD to solve the problem of exporting papers and others. Practical effective design and implementation technology can be used as reference for the development of similar systems.
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Capaldi, Stefano
2014-01-01
In recent years, the production of recombinant pharmaceutical proteins in heterologous systems has increased significantly. Most applications involve complex proteins and glycoproteins that are difficult to produce, thus promoting the development and improvement of a wide range of production platforms. No individual system is optimal for the production of all recombinant proteins, so the diversity of platforms based on plants offers a significant advantage. Here, we discuss the production of four recombinant pharmaceutical proteins using different platforms, highlighting from these examples the unique advantages of plant-based systems over traditional fermenter-based expression platforms. PMID:24745008
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Lyles, Courtney Rees; Harris, Lynne T; Le, Tung; Flowers, Jan; Tufano, James; Britt, Diane; Hoath, James; Hirsch, Irl B; Goldberg, Harold I; Ralston, James D
2011-05-01
Drawing on previous web-based diabetes management programs based on the Chronic Care Model, we expanded an intervention to include care management through mobile phones and a game console web browser. The pilot intervention enrolled eight diabetes patients from the University of Washington in Seattle into a collaborative care program: connecting them to a care provider specializing in diabetes, providing access to their full electronic medical record, allowing wireless glucose uploads and e-mail with providers, and connecting them to the program's web services through a game system. To evaluate the study, we conducted qualitative thematic analysis of semistructured interviews. Participants expressed frustrations with using the cell phones and the game system in their everyday lives, but liked the wireless system for collaborating with a provider on uploaded glucoses and receiving automatic feedback on their blood sugar trends. A majority of participants also expressed that their participation in the trial increased their health awareness. Mobile communication technologies showed promise within a web-based collaborative care program for type 2 diabetes. Future intervention design should focus on integrating easy-to-use applications within mobile technologies already familiar to patients and ensure the system allows for sufficient collaboration with a care provider.
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An audiovisual emotion recognition system
NASA Astrophysics Data System (ADS)
Han, Yi; Wang, Guoyin; Yang, Yong; He, Kun
2007-12-01
Human emotions could be expressed by many bio-symbols. Speech and facial expression are two of them. They are both regarded as emotional information which is playing an important role in human-computer interaction. Based on our previous studies on emotion recognition, an audiovisual emotion recognition system is developed and represented in this paper. The system is designed for real-time practice, and is guaranteed by some integrated modules. These modules include speech enhancement for eliminating noises, rapid face detection for locating face from background image, example based shape learning for facial feature alignment, and optical flow based tracking algorithm for facial feature tracking. It is known that irrelevant features and high dimensionality of the data can hurt the performance of classifier. Rough set-based feature selection is a good method for dimension reduction. So 13 speech features out of 37 ones and 10 facial features out of 33 ones are selected to represent emotional information, and 52 audiovisual features are selected due to the synchronization when speech and video fused together. The experiment results have demonstrated that this system performs well in real-time practice and has high recognition rate. Our results also show that the work in multimodules fused recognition will become the trend of emotion recognition in the future.
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Automated Assessment of Child Vocalization Development Using LENA.
Richards, Jeffrey A; Xu, Dongxin; Gilkerson, Jill; Yapanel, Umit; Gray, Sharmistha; Paul, Terrance
2017-07-12
To produce a novel, efficient measure of children's expressive vocal development on the basis of automatic vocalization assessment (AVA), child vocalizations were automatically identified and extracted from audio recordings using Language Environment Analysis (LENA) System technology. Assessment was based on full-day audio recordings collected in a child's unrestricted, natural language environment. AVA estimates were derived using automatic speech recognition modeling techniques to categorize and quantify the sounds in child vocalizations (e.g., protophones and phonemes). These were expressed as phone and biphone frequencies, reduced to principal components, and inputted to age-based multiple linear regression models to predict independently collected criterion-expressive language scores. From these models, we generated vocal development AVA estimates as age-standardized scores and development age estimates. AVA estimates demonstrated strong statistical reliability and validity when compared with standard criterion expressive language assessments. Automated analysis of child vocalizations extracted from full-day recordings in natural settings offers a novel and efficient means to assess children's expressive vocal development. More research remains to identify specific mechanisms of operation.
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NASA Astrophysics Data System (ADS)
Levashov, Valentin A.; Egami, Takeshi; Morris, James R.
2009-03-01
We present a new approach to the issue of correlation range in supercooled liquids based on Green-Kubo expression for viscosity. The integrand of this expression is the average stress-stress autocorrelation function. This correlation function could be rewritten in terms of correlations among local atomic stresses at different times and distances. The features of the autocorrelation function decay with time depend on temperature and correlation range. Through this approach we can study the development of spatial correlation with time, thus directly addressing the question of dynamic heterogeneity. We performed MD simulations on a single component system of particles interacting through short range pair potential. Our results indicate that even above the crossover temperature correlations extend well beyond the nearest neighbors. Surprisingly we found that the system size effects exist even on relatively large systems. We also address the role of diffusion in decay of stress-stress correlation function.
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Kajino, T; Ohto, C; Muramatsu, M; Obata, S; Udaka, S; Yamada, Y; Takahashi, H
2000-02-01
We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system.
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Ho, Kwun Yin; Murray, Victoria L.; Liu, Allen P.
2015-01-01
Generation of artificial cells provides the bridge needed to cover the gap between studying the complexity of biological processes in whole cells and studying these same processes in an in vitro reconstituted system. Artificial cells are defined as the encapsulation of biologically active material in a biological or synthetic membrane. Here, we describe a robust and general method to produce artificial cells for the purpose of mimicking one or more behaviors of a cell. A microfluidic double emulsion system is used to encapsulate a mammalian cell free expression system that is able to express membrane proteins into the bilayer or soluble proteins inside the vesicles. The development of a robust platform that allows the assembly of artificial cells is valuable in understanding subcellular functions and emergent behaviors in a more cell-like environment as well as for creating novel signaling pathways to achieve specific cellular behaviors. PMID:25997354
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Kajino, Tsutomu; Ohto, Chikara; Muramatsu, Masayoshi; Obata, Shusei; Udaka, Shigezo; Yamada, Yukio; Takahashi, Haruo
2000-01-01
We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system. PMID:10653729
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Kuipers, Grietje; Karyolaimos, Alexandros; Zhang, Zhe; Ismail, Nurzian; Trinco, Gianluca; Vikström, David; Slotboom, Dirk Jan; de Gier, Jan-Willem
2017-12-16
To optimize the production of membrane and secretory proteins in Escherichia coli, it is critical to harmonize the expression rates of the genes encoding these proteins with the capacity of their biogenesis machineries. Therefore, we engineered the Lemo21(DE3) strain, which is derived from the T7 RNA polymerase-based BL21(DE3) protein production strain. In Lemo21(DE3), the T7 RNA polymerase activity can be modulated by the controlled co-production of its natural inhibitor T7 lysozyme. This setup enables to precisely tune target gene expression rates in Lemo21(DE3). The t7lys gene is expressed from the pLemo plasmid using the titratable rhamnose promoter. A disadvantage of the Lemo21(DE3) setup is that the system is based on two plasmids, a T7 expression vector and pLemo. The aim of this study was to simplify the Lemo21(DE3) setup by incorporating the key elements of pLemo in a standard T7-based expression vector. By incorporating the gene encoding the T7 lysozyme under control of the rhamnose promoter in a standard T7-based expression vector, pReX was created (ReX stands for Regulated gene eXpression). For two model membrane proteins and a model secretory protein we show that the optimized production yields obtained with the pReX expression vector in BL21(DE3) are similar to the ones obtained with Lemo21(DE3) using a standard T7 expression vector. For another secretory protein, a c-type cytochrome, we show that pReX, in contrast to Lemo21(DE3), enables the use of a helper plasmid that is required for the maturation and hence the production of this heme c protein. Here, we created pReX, a T7-based expression vector that contains the gene encoding the T7 lysozyme under control of the rhamnose promoter. pReX enables regulated T7-based target gene expression using only one plasmid. We show that with pReX the production of membrane and secretory proteins can be readily optimized. Importantly, pReX facilitates the use of helper plasmids. Furthermore, the use of pReX is not restricted to BL21(DE3), but it can in principle be used in any T7 RNAP-based strain. Thus, pReX is a versatile alternative to Lemo21(DE3).
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Aptamer Selection Express: A Novel Method for Rapid Single-Step Selection and Sensing of Aptamers
Fan, Maomian; McBurnett, Shelly Roper; Andrews, Carrie J.; Allman, Amity M.; Bruno, John G.; Kiel, Johnathan L.
2008-01-01
Here we describe a new DNA capture element (DCE) sensing system, based on the quenching and dequenching of a double-stranded aptamer. This system shows very good sensitivity and thermal stability. While quenching, dequenching, and separating the DCE systems made from different aptamers (all selected by SELEX), an alternative method to rapidly select aptamers was developed—the Aptamer Selection Express (ASExp). This process has been used to select aptamers against different types of targets (Bacillus anthracis spores, Bacillus thuringiensis spores, MS-2 bacteriophage, ovalbumin, and botulinum neurotoxin). The DCE systems made from botulinum neurotoxin aptamers selected by ASExp have been investigated. The results of this investigation indicate that ASExp can be used to rapidly select aptamers for the DCE sensing system. PMID:19183794
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Chen, Wei-Guan; Zheng, Jia-Xuan; Xu, Xi; Hu, Yu-Ming; Ma, Yu-Min
2018-06-01
As a well-known bile acid receptor, the role of Farnesoid X receptor (FXR) in the digestive system and cardiovascular system has been widely explored. However, there are very few studies involving FXR in the central nervous system. In this study, we explored the role of FXR in the pathogenesis of depression, a serious and worldwide neuropsychiatric disease. It was found that chronic unpredictable mild stress (CUMS) fully enhanced the protein and mRNA expressions of FXR in hippocampus, but not medial prefrontal cortex (mPFC). Overexpression of hippocampal FXR induced notable depressive-like behaviors and decreased expression of brain-derived neurotrophic factor (BDNF) in naïve rats, while knockdown of hippocampal FXR fully prevented the effects of CUMS on rat behaviors and hippocampal BDNF expression. Taken together, our research extends the knowledge of FXR's role in the central nervous system, and may provide a potential and novel therapeutic target for treating depression. Copyright © 2018 Elsevier B.V. All rights reserved.
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Expression of RYamide in the nervous and endocrine system of Bombyx mori.
Roller, Ladislav; Čižmár, Daniel; Bednár, Branislav; Žitňan, Dušan
2016-06-01
RYamides are neuropeptides encoded by a gene whose precise expression and function have not yet been determined. We identified the RYamide gene transcript (fmgV1g15f, SilkBase database) and predicted two candidates for G-protein coupled RYamide receptors (A19-BAG68418 and A22-BAG68421) in the silkworm Bombyx mori. We cloned the RYamide transcript and described its spatial expression using in situ hybridisation. In the larval central nervous system (CNS) expression of RYamide was restricted to 12-14 small neurons in the brain and two posterior neurons in the terminal abdominal ganglion. During metamorphosis their number decreased to eight protocerebral neurons in the adults. Multiple staining, using various insect neuropeptide antibodies, revealed that neurons expressing RYamide are different from other peptidergic cells in the CNS. We also found RYamide expression in the enteroendocrine cells (EC) of the anterior midgut of larvae, pupae and adults. Two minor subpopulations of these EC were also immunoreactive to antibodies against tachykinin and myosupressin. This expression pattern suggests RYamides may play a role in the regulation of feeding and digestion. Copyright © 2016 Elsevier Inc. All rights reserved.
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Andries, Erik; Hagstrom, Thomas; Atlas, Susan R; Willman, Cheryl
2007-02-01
Linear discrimination, from the point of view of numerical linear algebra, can be treated as solving an ill-posed system of linear equations. In order to generate a solution that is robust in the presence of noise, these problems require regularization. Here, we examine the ill-posedness involved in the linear discrimination of cancer gene expression data with respect to outcome and tumor subclasses. We show that a filter factor representation, based upon Singular Value Decomposition, yields insight into the numerical ill-posedness of the hyperplane-based separation when applied to gene expression data. We also show that this representation yields useful diagnostic tools for guiding the selection of classifier parameters, thus leading to improved performance.
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Information services and information processing
NASA Technical Reports Server (NTRS)
1975-01-01
Attempts made to design and extend space system capabilities are reported. Special attention was given to establishing user needs for information or services which might be provided by space systems. Data given do not attempt to detail scientific, technical, or economic bases for the needs expressed by the users.
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Decision-Guided Recommenders with Composite Alternatives
ERIC Educational Resources Information Center
Alodhaibi, Khalid
2011-01-01
Recommender systems aim to support users in their decision-making process while interacting with large information spaces and recommend items of interest to users based on preferences they have expressed, either explicitly or implicitly. Recommender systems are increasingly used with product and service selection over the Internet. Although…
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A Rapid and Quantitative Recombinase Activity Assay
USDA-ARS?s Scientific Manuscript database
We present here a comparison between the recombinase systems FLP-FRT and Cre-loxP. A transient excision based dual luciferase expression assay is used for its rapid and repeatable nature. The detection system was designed within an intron to remove the remaining recombinase recognition site and no...
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mirEX: a platform for comparative exploration of plant pri-miRNA expression data.
Bielewicz, Dawid; Dolata, Jakub; Zielezinski, Andrzej; Alaba, Sylwia; Szarzynska, Bogna; Szczesniak, Michal W; Jarmolowski, Artur; Szweykowska-Kulinska, Zofia; Karlowski, Wojciech M
2012-01-01
mirEX is a comprehensive platform for comparative analysis of primary microRNA expression data. RT-qPCR-based gene expression profiles are stored in a universal and expandable database scheme and wrapped by an intuitive user-friendly interface. A new way of accessing gene expression data in mirEX includes a simple mouse operated querying system and dynamic graphs for data mining analyses. In contrast to other publicly available databases, the mirEX interface allows a simultaneous comparison of expression levels between various microRNA genes in diverse organs and developmental stages. Currently, mirEX integrates information about the expression profile of 190 Arabidopsis thaliana pri-miRNAs in seven different developmental stages: seeds, seedlings and various organs of mature plants. Additionally, by providing RNA structural models, publicly available deep sequencing results, experimental procedure details and careful selection of auxiliary data in the form of web links, mirEX can function as a one-stop solution for Arabidopsis microRNA information. A web-based mirEX interface can be accessed at http://bioinfo.amu.edu.pl/mirex.
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Specifying the behavior of concurrent systems
NASA Technical Reports Server (NTRS)
Furtek, F. C.
1984-01-01
A framework for rigorously specifying the behavior of concurrent systems is proposed. It is based on the view of a concurrent system as a collection of interacting processes but no assumptions are made about the mechanisms for process synchronization and communication. A formal language is described that permits the expression of a broad range of logical and timing dependencies.
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Goos, Jeroen A C M; Coupé, Veerle M H; van de Wiel, Mark A; Diosdado, Begoña; Delis-Van Diemen, Pien M; Hiemstra, Annemieke C; de Cuba, Erienne M V; Beliën, Jeroen A M; Menke-van der Houven van Oordt, C Willemien; Geldof, Albert A; Meijer, Gerrit A; Hoekstra, Otto S; Fijneman, Remond J A
2016-01-12
Prognosis of patients with colorectal cancer liver metastasis (CRCLM) is estimated based on clinicopathological models. Stratifying patients based on tumor biology may have additional value. Tissue micro-arrays (TMAs), containing resected CRCLM and corresponding primary tumors from a multi-institutional cohort of 507 patients, were immunohistochemically stained for 18 candidate biomarkers. Cross-validated hazard rate ratios (HRRs) for overall survival (OS) and the proportion of HRRs with opposite effect (P(HRR < 1) or P(HRR > 1)) were calculated. A classifier was constructed by classification and regression tree (CART) analysis and its prognostic value determined by permutation analysis. Correlations between protein expression in primary tumor-CRCLM pairs were calculated. Based on their putative prognostic value, EGFR (P(HRR < 1) = .02), AURKA (P(HRR < 1) = .02), VEGFA (P(HRR < 1) = .02), PTGS2 (P(HRR < 1) = .01), SLC2A1 (P(HRR > 1) < 01), HIF1α (P(HRR > 1) = .06), KCNQ1 (P(HRR > 1) = .09), CEA (P (HRR > 1) = .05) and MMP9 (P(HRR < 1) = .07) were included in the CART analysis (n = 201). The resulting classifier was based on AURKA, PTGS2 and MMP9 expression and was associated with OS (HRR 2.79, p < .001), also after multivariate analysis (HRR 3.57, p < .001). The prognostic value of the biomarker-based classifier was superior to the clinicopathological model (p = .001). Prognostic value was highest for colon cancer patients (HRR 5.71, p < .001) and patients not treated with systemic therapy (HRR 3.48, p < .01). Classification based on protein expression in primary tumors could be based on AURKA expression only (HRR 2.59, p = .04). A classifier was generated for patients with CRCLM with improved prognostic value compared to the standard clinicopathological prognostic parameters, which may aid selection of patients who may benefit from adjuvant systemic therapy.
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Biomarkers of adult and developmental neurotoxicity
DOE Office of Scientific and Technical Information (OSTI.GOV)
Slikker, William; Bowyer, John F.
2005-08-07
Neurotoxicity may be defined as any adverse effect on the structure or function of the central and/or peripheral nervous system by a biological, chemical, or physical agent. A multidisciplinary approach is necessary to assess adult and developmental neurotoxicity due to the complex and diverse functions of the nervous system. The overall strategy for understanding developmental neurotoxicity is based on two assumptions: (1) significant differences in the adult versus the developing nervous system susceptibility to neurotoxicity exist and they are often developmental stage dependent; (2) a multidisciplinary approach using neurobiological, including gene expression assays, neurophysiological, neuropathological, and behavioral function is necessarymore » for a precise assessment of neurotoxicity. Application of genomic approaches to developmental studies must use the same criteria for evaluating microarray studies as those in adults including consideration of reproducibility, statistical analysis, homogenous cell populations, and confirmation with non-array methods. A study using amphetamine to induce neurotoxicity supports the following: (1) gene expression data can help define neurotoxic mechanism(s) (2) gene expression changes can be useful biomarkers of effect, and (3) the site-selective nature of gene expression in the nervous system may mandate assessment of selective cell populations.« less
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Personalization of Rule-based Web Services.
Choi, Okkyung; Han, Sang Yong
2008-04-04
Nowadays Web users have clearly expressed their wishes to receive personalized services directly. Personalization is the way to tailor services directly to the immediate requirements of the user. However, the current Web Services System does not provide any features supporting this such as consideration of personalization of services and intelligent matchmaking. In this research a flexible, personalized Rule-based Web Services System to address these problems and to enable efficient search, discovery and construction across general Web documents and Semantic Web documents in a Web Services System is proposed. This system utilizes matchmaking among service requesters', service providers' and users' preferences using a Rule-based Search Method, and subsequently ranks search results. A prototype of efficient Web Services search and construction for the suggested system is developed based on the current work.
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ISOL@: an Italian SOLAnaceae genomics resource.
Chiusano, Maria Luisa; D'Agostino, Nunzio; Traini, Alessandra; Licciardello, Concetta; Raimondo, Enrico; Aversano, Mario; Frusciante, Luigi; Monti, Luigi
2008-03-26
Present-day '-omics' technologies produce overwhelming amounts of data which include genome sequences, information on gene expression (transcripts and proteins) and on cell metabolic status. These data represent multiple aspects of a biological system and need to be investigated as a whole to shed light on the mechanisms which underpin the system functionality. The gathering and convergence of data generated by high-throughput technologies, the effective integration of different data-sources and the analysis of the information content based on comparative approaches are key methods for meaningful biological interpretations. In the frame of the International Solanaceae Genome Project, we propose here ISOLA, an Italian SOLAnaceae genomics resource. ISOLA (available at http://biosrv.cab.unina.it/isola) represents a trial platform and it is conceived as a multi-level computational environment.ISOLA currently consists of two main levels: the genome and the expression level. The cornerstone of the genome level is represented by the Solanum lycopersicum genome draft sequences generated by the International Tomato Genome Sequencing Consortium. Instead, the basic element of the expression level is the transcriptome information from different Solanaceae species, mainly in the form of species-specific comprehensive collections of Expressed Sequence Tags (ESTs). The cross-talk between the genome and the expression levels is based on data source sharing and on tools that enhance data quality, that extract information content from the levels' under parts and produce value-added biological knowledge. ISOLA is the result of a bioinformatics effort that addresses the challenges of the post-genomics era. It is designed to exploit '-omics' data based on effective integration to acquire biological knowledge and to approach a systems biology view. Beyond providing experimental biologists with a preliminary annotation of the tomato genome, this effort aims to produce a trial computational environment where different aspects and details are maintained as they are relevant for the analysis of the organization, the functionality and the evolution of the Solanaceae family.
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Gurunathan, Rajalakshmi; Van Emden, Bernard; Panchanathan, Sethuraman; Kumar, Sudhir
2004-01-01
Background Modern developmental biology relies heavily on the analysis of embryonic gene expression patterns. Investigators manually inspect hundreds or thousands of expression patterns to identify those that are spatially similar and to ultimately infer potential gene interactions. However, the rapid accumulation of gene expression pattern data over the last two decades, facilitated by high-throughput techniques, has produced a need for the development of efficient approaches for direct comparison of images, rather than their textual descriptions, to identify spatially similar expression patterns. Results The effectiveness of the Binary Feature Vector (BFV) and Invariant Moment Vector (IMV) based digital representations of the gene expression patterns in finding biologically meaningful patterns was compared for a small (226 images) and a large (1819 images) dataset. For each dataset, an ordered list of images, with respect to a query image, was generated to identify overlapping and similar gene expression patterns, in a manner comparable to what a developmental biologist might do. The results showed that the BFV representation consistently outperforms the IMV representation in finding biologically meaningful matches when spatial overlap of the gene expression pattern and the genes involved are considered. Furthermore, we explored the value of conducting image-content based searches in a dataset where individual expression components (or domains) of multi-domain expression patterns were also included separately. We found that this technique improves performance of both IMV and BFV based searches. Conclusions We conclude that the BFV representation consistently produces a more extensive and better list of biologically useful patterns than the IMV representation. The high quality of results obtained scales well as the search database becomes larger, which encourages efforts to build automated image query and retrieval systems for spatial gene expression patterns. PMID:15603586
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Effects of gravity changes on gene expression of BDNF and serotonin receptors in the mouse brain.
Ishikawa, Chihiro; Li, Haiyan; Ogura, Rin; Yoshimura, Yuko; Kudo, Takashi; Shirakawa, Masaki; Shiba, Dai; Takahashi, Satoru; Morita, Hironobu; Shiga, Takashi
2017-01-01
Spaceflight entails various stressful environmental factors including microgravity. The effects of gravity changes have been studied extensively on skeletal, muscular, cardiovascular, immune and vestibular systems, but those on the nervous system are not well studied. The alteration of gravity in ground-based animal experiments is one of the approaches taken to address this issue. Here we investigated the effects of centrifugation-induced gravity changes on gene expression of brain-derived neurotrophic factor (BDNF) and serotonin receptors (5-HTRs) in the mouse brain. Exposure to 2g hypergravity for 14 days showed differential modulation of gene expression depending on regions of the brain. BDNF expression was decreased in the ventral hippocampus and hypothalamus, whereas increased in the cerebellum. 5-HT1BR expression was decreased in the cerebellum, whereas increased in the ventral hippocampus and caudate putamen. In contrast, hypergravity did not affect gene expression of 5-HT1AR, 5-HT2AR, 5-HT2CR, 5-HT4R and 5-HT7R. In addition to hypergravity, decelerating gravity change from 2g hypergravity to 1g normal gravity affected gene expression of BDNF, 5-HT1AR, 5-HT1BR, and 5-HT2AR in various regions of the brain. We also examined involvement of the vestibular organ in the effects of hypergravity. Surgical lesions of the inner ear's vestibular organ removed the effects induced by hypergravity on gene expression, which suggests that the effects of hypergravity are mediated through the vestibular organ. In summary, we showed that gravity changes induced differential modulation of gene expression of BDNF and 5-HTRs (5-HT1AR, 5-HT1BR and 5-HT2AR) in some brain regions. The modulation of gene expression may constitute molecular bases that underlie behavioral alteration induced by gravity changes.
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Effects of gravity changes on gene expression of BDNF and serotonin receptors in the mouse brain
Yoshimura, Yuko; Kudo, Takashi; Shirakawa, Masaki; Shiba, Dai; Takahashi, Satoru; Morita, Hironobu
2017-01-01
Spaceflight entails various stressful environmental factors including microgravity. The effects of gravity changes have been studied extensively on skeletal, muscular, cardiovascular, immune and vestibular systems, but those on the nervous system are not well studied. The alteration of gravity in ground-based animal experiments is one of the approaches taken to address this issue. Here we investigated the effects of centrifugation-induced gravity changes on gene expression of brain-derived neurotrophic factor (BDNF) and serotonin receptors (5-HTRs) in the mouse brain. Exposure to 2g hypergravity for 14 days showed differential modulation of gene expression depending on regions of the brain. BDNF expression was decreased in the ventral hippocampus and hypothalamus, whereas increased in the cerebellum. 5-HT1BR expression was decreased in the cerebellum, whereas increased in the ventral hippocampus and caudate putamen. In contrast, hypergravity did not affect gene expression of 5-HT1AR, 5-HT2AR, 5-HT2CR, 5-HT4R and 5-HT7R. In addition to hypergravity, decelerating gravity change from 2g hypergravity to 1g normal gravity affected gene expression of BDNF, 5-HT1AR, 5-HT1BR, and 5-HT2AR in various regions of the brain. We also examined involvement of the vestibular organ in the effects of hypergravity. Surgical lesions of the inner ear’s vestibular organ removed the effects induced by hypergravity on gene expression, which suggests that the effects of hypergravity are mediated through the vestibular organ. In summary, we showed that gravity changes induced differential modulation of gene expression of BDNF and 5-HTRs (5-HT1AR, 5-HT1BR and 5-HT2AR) in some brain regions. The modulation of gene expression may constitute molecular bases that underlie behavioral alteration induced by gravity changes. PMID:28591153
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Autonomous system for Web-based microarray image analysis.
Bozinov, Daniel
2003-12-01
Software-based feature extraction from DNA microarray images still requires human intervention on various levels. Manual adjustment of grid and metagrid parameters, precise alignment of superimposed grid templates and gene spots, or simply identification of large-scale artifacts have to be performed beforehand to reliably analyze DNA signals and correctly quantify their expression values. Ideally, a Web-based system with input solely confined to a single microarray image and a data table as output containing measurements for all gene spots would directly transform raw image data into abstracted gene expression tables. Sophisticated algorithms with advanced procedures for iterative correction function can overcome imminent challenges in image processing. Herein is introduced an integrated software system with a Java-based interface on the client side that allows for decentralized access and furthermore enables the scientist to instantly employ the most updated software version at any given time. This software tool is extended from PixClust as used in Extractiff incorporated with Java Web Start deployment technology. Ultimately, this setup is destined for high-throughput pipelines in genome-wide medical diagnostics labs or microarray core facilities aimed at providing fully automated service to its users.
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Definition, Detection and Generation of Iyashi Expressions
NASA Astrophysics Data System (ADS)
Kitaoka, Tetsuko; Diago, Luis A.; Hagiwara, Ichiro; Kitazaki, Satoshi; Yamane, Shigeru
This paper concerns the engineering analysis of “Iyashi”, a peculiar concept to the Japanese, which affect person's heart and may change their expression and behavior. We have integrated the advocator's view of “Iyashi”, analyzed the social background of “Iyashi” and have defined Iyashi and also the Iyashi expression. As the facial expression is the special and important stimulus for both observers and people who show expressions, we want to prove the existence of expressions that change the observer's emotion with Iyashi. We have developed the system to clarify the combination of facial features important for Iyashi through the psychological experiments and the analysis by Holographic Neural Networks (HNN). HNN analysis gave the structure of the Iyashi expression, that is the important combination of the physical facial parameters contributing to the high degree of Iyashi. Based on the structure of Iyashi we are able to generate the Iyashi expression appropriate for each person.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Hummel, K.E.
1987-12-01
Expert systems are artificial intelligence programs that solve problems requiring large amounts of heuristic knowledge, based on years of experience and tradition. Production systems are domain-independent tools that support the development of rule-based expert systems. This document describes a general purpose production system known as HERB. This system was developed to support the programming of expert systems using hierarchically structured rule bases. HERB encourages the partitioning of rules into multiple rule bases and supports the use of multiple conflict resolution strategies. Multiple rule bases can also be placed on a system stack and simultaneously searched during each interpreter cycle. Bothmore » backward and forward chaining rules are supported by HERB. The condition portion of each rule can contain both patterns, which are matched with facts in a data base, and LISP expressions, which are explicitly evaluated in the LISP environment. Properties of objects can also be stored in the HERB data base and referenced within the scope of each rule. This document serves both as an introduction to the principles of LISP-based production systems and as a user's manual for the HERB system. 6 refs., 17 figs.« less
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Spatial pattern of receptor expression in the olfactory epithelium.
Nef, P; Hermans-Borgmeyer, I; Artières-Pin, H; Beasley, L; Dionne, V E; Heinemann, S F
1992-01-01
A PCR-based strategy for amplifying putative receptors involved in murine olfaction was employed to isolate a member (OR3) of the seven-transmembrane-domain receptor superfamily. During development, the first cells that express OR3 appear adjacent to the wall of the telencephalic vesicle at embryonic day 10. The OR3 receptor is uniquely expressed in a subset of olfactory cells that have a characteristic bilateral symmetry in the adult olfactory epithelium. This receptor and its specific pattern of expression may serve a functional role in odor coding or, alternatively, may play a role in the development of the olfactory system. Images PMID:1384038
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Kidd, M.E.C.
1997-02-01
The goal of our work is to provide a high level of confidence that critical software driven event sequences are maintained in the face of hardware failures, malevolent attacks and harsh or unstable operating environments. This will be accomplished by providing dynamic fault management measures directly to the software developer and to their varied development environments. The methodology employed here is inspired by previous work in path expressions. This paper discusses the perceived problems, a brief overview of path expressions, the proposed methods, and a discussion of the differences between the proposed methods and traditional path expression usage and implementation.
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Ratzka, Andreas; Baron, Olga; Grothe, Claudia
2011-01-01
Secreted proteins of the fibroblast growth factor (FGF) family play important roles during development of various organ systems. A detailed knowledge of their temporal and spatial expression profiles, especially of closely related FGF family members, are essential to further identification of specific functions in distinct tissues. In the central nervous system dopaminergic neurons of the substantia nigra and their axonal projections into the striatum progressively degenerate in Parkinson's disease. In contrast, FGF-2 deficient mice display increased numbers of dopaminergic neurons. In this study, we determined the expression profiles of all 22 FGF-ligands and 10 FGF-receptor isoforms, in order to clarify, if FGF-2 deficiency leads to compensatory up-regulation of other FGFs in the nigrostriatal system. Three tissues, ventral mesencephalon (VM), striatum (STR) and as reference tissue spinal cord (SC) of wild-type and FGF-2 deficient mice at four developmental stages E14.5, P0, P28, and adult were comparatively analyzed by quantitative RT-PCR. As no differences between the genotypes were observed, a compensatory up-regulation can be excluded. Moreover, this analysis revealed that the majority of FGF-ligands (18/22) and FGF-receptors (9/10) are expressed during normal development of the nigrostriatal system and identified dynamic changes for some family members. By comparing relative expression level changes to SC reference tissue, general alterations in all 3 tissues, such as increased expression of FGF-1, -2, -22, FgfR-2c, -3c and decreased expression of FGF-13 during postnatal development were identified. Further, specific changes affecting only one tissue, such as increased FGF-16 (STR) or decreased FGF-17 (VM) expression, or two tissues, such as decreased expression of FGF-8 (VM, STR) and FGF-15 (SC, VM) were found. Moreover, 3 developmentally down-regulated FGFs (FGF-8b, FGF-15, FGF-17a) were functionally characterized by plasmid-based over-expression in dissociated E11.5 VM cell cultures, however, such a continuous exposure had no influence on the yield of dopaminergic neurons in vitro.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Yu Xiaozhong; Hong, Sung Woo; Moreira, Estefania G.
Gonocytes exist in the neonatal testis and represent a transient population of male germ-line stem cells. It has been shown that stem cell self-renewal and progeny production is probably controlled by the neighboring differentiated cells and extracellular matrix (ECM) in vivo known as niches. Recently, we developed an in vitro three-dimensional (3D) Sertoli cell/gonocyte co-culture (SGC) model with ECM overlay, which creates an in vivo-like niche and supports germ-line stem cell functioning within a 3D environment. In this study, we applied morphological and cytotoxicity evaluations, as well as microarray-based gene expression to examine the effects of different phthalate esters (PE)more » on this model. Known in vivo male developmentally toxic PEs (DTPE) and developmentally non-toxic PEs (DNTPE) were evaluated. We observed that DTPE induced significantly greater dose-dependent morphological changes, a decrease in cell viability and an increase in cytotoxicity compared to those treated with DNTPE. Moreover, the gene expression was more greatly altered by DTPE than by DNTPE and non-supervised cluster analysis allowed the discrimination of DTPE from the DNTPE. Our systems-based GO-Quant analysis showed significant alterations in the gene pathways involved in cell cycle, phosphate transport and apoptosis regulation with DTPE but not with DNTPE treatment. Disruptions of steroidogenesis related-gene expression such as Star, Cyp19a1, Hsd17b8, and Nr4a3 were observed in the DTPE group, but not in the DNTPE group. In summary, our observation on cell viability, cytotoxicity, and microarray-based gene expression analysis induced by PEs demonstrate that our in vitro 3D-SGC system mimicked in vivo responses for PEs and suggests that the 3D-SGC system might be useful in identifying developmental reproductive toxicants.« less
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Hortsch, Ralf; Weuster-Botz, Dirk
2011-04-01
Parallel operated milliliter-scale stirred tank bioreactors were applied for recombinant protein expression studies in simple batch experiments without pH titration. An enzymatic glucose release system (EnBase), a complex medium, and the frequently used LB and TB media were compared with regard to growth of Escherichia coli and recombinant protein expression (alcohol dehydrogenase (ADH) from Lactobacillus brevis and formate dehydrogenase (FDH) from Candida boidinii). Dissolved oxygen and pH were recorded online, optical densities were measured at-line, and the activities of ADH and FDH were analyzed offline. Best growth was observed in a complex medium with maximum dry cell weight concentrations of 14 g L(-1). EnBase cultivations enabled final dry cell weight concentrations between 6 and 8 g L(-1). The pH remained nearly constant in EnBase cultivations due to the continuous glucose release, showing the usefulness of this glucose release system especially for pH-sensitive bioprocesses. Cell-specific enzyme activities varied considerably depending on the different media used. Maximum specific ADH activities were measured with the complex medium, 6 h after induction with IPTG, whereas the highest specific FDH activities were achieved with the EnBase medium at low glucose release profiles 24 h after induction. Hence, depending on the recombinant protein, different medium compositions, times for induction, and times for cell harvest have to be evaluated to achieve efficient expression of recombinant proteins in E. coli. A rapid experimental evaluation can easily be performed with parallel batch operated small-scale stirred tank bioreactors.
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Yigang, X U; Yijing, L I
2008-05-01
Lactobacillus casei ATCC 393 was selected as a bacterial carrier for the development of mucosal vaccine against porcine parvovirus (PPV) infection. The PPV major structural polypeptide VP2 was used as the model parvovirus antigen. Two inducible expression systems, namely pPG611.1 of the cell-surface expression system and pPG612.1 of the secretion expression system based on the xylose operon promoter were used to express the VP2 protein. The immunogenicity of recombinant strains producing VP2 protein in two cellular locations, cell-surface exposed and secreted, was compared to each other by immunizing mice through the intragastric administration. The two types of constructs were able to induce strong specific immune responses against VP2 via intragastric administration and maximum titres of IgA and IgG were attained on days 46 post oral immunization, while the highest antibody levels were obtained with the strain producing the VP2 protein in extracellular milieu. The induced antibodies demonstrated neutralizing effects on PPV infection.
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Miyazaki, Maiko; Yuba, Eiji; Hayashi, Hiroshi; Harada, Atsushi; Kono, Kenji
2018-01-17
For the enhancement of therapeutic effects and reduction of side effects derived from anticancer drugs in cancer chemotherapy, it is imperative to develop drug delivery systems with cancer-specificity and controlled release function inside cancer cells. pH-sensitive liposomes are useful as an intracellular drug delivery system because of their abilities to transfer their contents into the cell interior through fusion or destabilization of endosome, which has weakly acidic environment. We earlier reported liposomes modified with various types of pH-sensitive polymers based on synthetic polymers and biopolymers as vehicles for intracellular drug delivery systems. In this study, hyaluronic acid (HA)-based pH-sensitive polymers were designed as multifunctional polymers having not only pH-sensitivity but also targeting properties to cells expressing CD44, which is known as a cancer cell surface marker. Carboxyl group-introduced HA derivatives of two types, MGlu-HA and CHex-HA, which have a more hydrophobic side chain structure than that of MGlu-HA, were synthesized by reaction with various dicarboxylic anhydrides. These polymer-modified liposomes were stable at neutral pH, but showed content release under weakly acidic conditions. CHex-HA-modified liposomes delivered their contents into CD44-expressing cells more efficiently than HA-modified or MGlu-HA-modified liposomes or unmodified liposomes, whereas the same liposomes were taken up only slightly by cells expressing CD44 proteins less. Competition assay using free HA or other polymers revealed that HA derivative-modified liposomes might be recognized by CD44. Therefore, HA-derivative-modified liposomes are useful as cell-specific intracellular drug delivery systems.
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An Event-Based Approach to Distributed Diagnosis of Continuous Systems
NASA Technical Reports Server (NTRS)
Daigle, Matthew; Roychoudhurry, Indranil; Biswas, Gautam; Koutsoukos, Xenofon
2010-01-01
Distributed fault diagnosis solutions are becoming necessary due to the complexity of modern engineering systems, and the advent of smart sensors and computing elements. This paper presents a novel event-based approach for distributed diagnosis of abrupt parametric faults in continuous systems, based on a qualitative abstraction of measurement deviations from the nominal behavior. We systematically derive dynamic fault signatures expressed as event-based fault models. We develop a distributed diagnoser design algorithm that uses these models for designing local event-based diagnosers based on global diagnosability analysis. The local diagnosers each generate globally correct diagnosis results locally, without a centralized coordinator, and by communicating a minimal number of measurements between themselves. The proposed approach is applied to a multi-tank system, and results demonstrate a marked improvement in scalability compared to a centralized approach.
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2014-01-01
Oxytocin is thought to play a central role in promoting close social bonds via influence on social interactions. The current investigation targeted interactions involving expressed gratitude between members of romantic relationships because recent evidence suggests gratitude and its expression provides behavioral and psychological ‘glue’ to bind individuals closer together. Specifically, we took a genetic approach to test the hypothesis that social interactions involving expressed gratitude would be associated with variation in a gene, CD38, which has been shown to affect oxytocin secretion. A polymorphism (rs6449182) that affects CD38 expression was significantly associated with global relationship satisfaction, perceived partner responsiveness and positive emotions (particularly love) after lab-based interactions, observed behavioral expression of gratitude toward a romantic partner in the lab, and frequency of expressed gratitude in daily life. A separate polymorphism in CD38 (rs3796863) previously associated with plasma oxytocin levels and social engagement was also associated with perceived responsiveness in the benefactor after an expression of gratitude. The combined influence of the two polymorphisms was associated with a broad range of gratitude-related behaviors and feelings. The consistent pattern of findings suggests that the oxytocin system is associated with solidifying the glue that binds adults into meaningful and important relationships. PMID:24396004
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Prevention of bubonic and pneumonic plague using plant-derived vaccines.
Alvarez, M Lucrecia; Cardineau, Guy A
2010-01-01
Yersinia pestis, the causative agent of bubonic and pneumonic plague, is an extremely virulent bacterium but there are currently no approved vaccines for protection against this organism. Plants represent an economical and safer alternative to fermentation-based expression systems for the production of therapeutic proteins. The recombinant plague vaccine candidates produced in plants are based on the two most immunogenic antigens of Y. pestis: the fraction-1 capsular antigen (F1) and the low calcium response virulent antigen (V) either in combination or as a fusion protein (F1-V). These antigens have been expressed in plants using all three known possible strategies: nuclear transformation, chloroplast transformation and plant-virus-based expression vectors. These plant-derived plague vaccine candidates were successfully tested in animal models using parenteral, oral, or prime/boost immunization regimens. This review focuses on the recent research accomplishments towards the development of safe and effective pneumonic and bubonic plague vaccines using plants as bioreactors.
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RAMP: a computer system for mapping regional areas
Bradley B. Nickey
1975-01-01
Until 1972, the U.S. Forest Service's Individual Fire Reports recorded locations by the section-township-range system..These earlier fire reports, therefore, lacked congruent locations. RAMP (Regional Area Mapping Procedure) was designed to make the reports more useful for quantitative analysis. This computer-based technique converts locations expressed in...
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Zhang, Xinjie; He, Peng; Tao, Yong; Yang, Yi
2013-11-04
High-level expression system of heterologous protein mediated by internal ribosome entry site (IRES) in Saccharomyces cerevisiae was constructed, which could be used for other applications of S. cerevisiae in metabolic engineering. We constructed co-expression cassette (promoter-mCherry-TIF4631 IRES-URA3) containing promoters Pilv5, Padh2 and Ptdh3 and recombined the co-expression cassette into the genome of W303-1B-A. The URA3+ transformants were selected. By comparing the difference in the mean florescence value of mCherry in transformants, the effect of three promoters was detected in the co-expression cassette. The copy numbers of the interested genes in the genome were determined by Real-Time PCR. We analyzed genetic stability by continuous subculturing transformants in the absence of selection pressure. To verify the application of co-expression cassette, the ORF of mCherry was replaced by beta-galactosidase (LACZ) and xylose reductase (XYL1). The enzyme activities and production of beta-galactosidase and xylose reductase were detected. mCherry has been expressed in the highest-level in transformants with co-expression cassette containing Pilv5 promoter. The highest copy number of DNA fragment integrating in the genome was 47 in transformants containing Pilv5. The engineering strains showed good genetic stability. Xylose reductase was successfully expressed in the co-expression cassette containing Pilv5 promoter and TIF4631 IRES. The highest enzyme activity was 0. 209 U/mg crude protein in the transformants WIX-10. Beta-galactosidase was also expressed successfully. The transformants that had the highest enzyme activity was WIL-1 and the enzyme activity was 12.58 U/mg crude protein. The system mediated by Pilv5 promoter and TIF4631 IRES could express heterologous protein efficiently in S. cerevisiae. This study offered a new strategy for expression of heterologous protein in S. cerevisiae and provided sufficient experimental evidence for metabolic engineering application of this system in yeast.
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An evaluative model of system performance in manned teleoperational systems
NASA Technical Reports Server (NTRS)
Haines, Richard F.
1989-01-01
Manned teleoperational systems are used in aerospace operations in which humans must interact with machines remotely. Manual guidance of remotely piloted vehicles, controling a wind tunnel, carrying out a scientific procedure remotely are examples of teleoperations. A four input parameter throughput (Tp) model is presented which can be used to evaluate complex, manned, teleoperations-based systems and make critical comparisons among candidate control systems. The first two parameters of this model deal with nominal (A) and off-nominal (B) predicted events while the last two focus on measured events of two types, human performance (C) and system performance (D). Digital simulations showed that the expression A(1-B)/C+D) produced the greatest homogeneity of variance and distribution symmetry. Results from a recently completed manned life science telescience experiment will be used to further validate the model. Complex, interacting teleoperational systems may be systematically evaluated using this expression much like a computer benchmark is used.
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Cao, Huojun; Amendt, Brad A
2016-11-01
Developmental dental anomalies are common forms of congenital defects. The molecular mechanisms of dental anomalies are poorly understood. Systematic approaches such as clustering genes based on similar expression patterns could identify novel genes involved in dental anomalies and provide a framework for understanding molecular regulatory mechanisms of these genes during tooth development (odontogenesis). A python package (pySAPC) of sparse affinity propagation clustering algorithm for large datasets was developed. Whole genome pair-wise similarity was calculated based on expression pattern similarity based on 45 microarrays of several stages during odontogenesis. pySAPC identified 743 gene clusters based on expression pattern similarity during mouse tooth development. Three clusters are significantly enriched for genes associated with dental anomalies (with FDR <0.1). The three clusters of genes have distinct expression patterns during odontogenesis. Clustering genes based on similar expression profiles recovered several known regulatory relationships for genes involved in odontogenesis, as well as many novel genes that may be involved with the same genetic pathways as genes that have already been shown to contribute to dental defects. By using sparse similarity matrix, pySAPC use much less memory and CPU time compared with the original affinity propagation program that uses a full similarity matrix. This python package will be useful for many applications where dataset(s) are too large to use full similarity matrix. This article is part of a Special Issue entitled "System Genetics" Guest Editor: Dr. Yudong Cai and Dr. Tao Huang. Copyright © 2016. Published by Elsevier B.V.
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Mallik, Saurav; Zhao, Zhongming
2017-12-28
For transcriptomic analysis, there are numerous microarray-based genomic data, especially those generated for cancer research. The typical analysis measures the difference between a cancer sample-group and a matched control group for each transcript or gene. Association rule mining is used to discover interesting item sets through rule-based methodology. Thus, it has advantages to find causal effect relationships between the transcripts. In this work, we introduce two new rule-based similarity measures-weighted rank-based Jaccard and Cosine measures-and then propose a novel computational framework to detect condensed gene co-expression modules ( C o n G E M s) through the association rule-based learning system and the weighted similarity scores. In practice, the list of evolved condensed markers that consists of both singular and complex markers in nature depends on the corresponding condensed gene sets in either antecedent or consequent of the rules of the resultant modules. In our evaluation, these markers could be supported by literature evidence, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway and Gene Ontology annotations. Specifically, we preliminarily identified differentially expressed genes using an empirical Bayes test. A recently developed algorithm-RANWAR-was then utilized to determine the association rules from these genes. Based on that, we computed the integrated similarity scores of these rule-based similarity measures between each rule-pair, and the resultant scores were used for clustering to identify the co-expressed rule-modules. We applied our method to a gene expression dataset for lung squamous cell carcinoma and a genome methylation dataset for uterine cervical carcinogenesis. Our proposed module discovery method produced better results than the traditional gene-module discovery measures. In summary, our proposed rule-based method is useful for exploring biomarker modules from transcriptomic data.
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Hayashi, Teruyuki; Shimoda, Yoshikazu; Sato, Shusei; Tabata, Satoshi; Imaizumi-Anraku, Haruko; Hayashi, Makoto
2014-01-01
For the establishment of an effective root nodule symbiosis, a coordinated regulation of the infection processes between the epidermis and cortex is required. However, it remains unclear whether the symbiotic genes identified so far are involved in epidermal and/or cortical infection, e.g. epidermal and cortical infection thread formation or cortical cell division. To analyze the symbiotic gene requirements of the infection process, we have developed an epidermis-specific expression system (pEpi expression system) and examined the symbiotic genes NFR1, NFR5, NUP85, NUP133, CASTOR, POLLUX, CCaMK, CYCLOPS, NSP1 and NSP2 for involvement in the infection process in the epidermis and cortex. Our study shows that expression of the upstream common symbiosis genes CASTOR, POLLUX, NUP85 and NUP133 in the epidermis is sufficient to induce formation of infection threads and cortical cell division, leading to the development of fully effective nodules. Our system also shows a requirement of CCaMK, CYCLOPS, NSP1 and NSP2 for the entire nodulation process, and the different contributions of NFR1 and NFR5 to cortical infection thread formation. Based on these analyses using the pEpi expression system, we propose a functional model of symbiotic genes for epidermal and cortical infection. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.
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Vrahatis, Aristidis G; Rapti, Angeliki; Sioutas, Spyros; Tsakalidis, Athanasios
2017-01-01
In the era of Systems Biology and growing flow of omics experimental data from high throughput techniques, experimentalists are in need of more precise pathway-based tools to unravel the inherent complexity of diseases and biological processes. Subpathway-based approaches are the emerging generation of pathway-based analysis elucidating the biological mechanisms under the perspective of local topologies onto a complex pathway network. Towards this orientation, we developed PerSub, a graph-based algorithm which detects subpathways perturbed by a complex disease. The perturbations are imprinted through differentially expressed and co-expressed subpathways as recorded by RNA-seq experiments. Our novel algorithm is applied on data obtained from a real experimental study and the identified subpathways provide biological evidence for the brain aging.
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Convergent Evolution at the Gametophytic Self-Incompatibility System in Malus and Prunus
Cunha, Ana E.; Fonseca, Nuno A.; Iezzoni, Amy; van Nocker, Steve; Vieira, Cristina P.
2015-01-01
S-RNase-based gametophytic self-incompatibility (GSI) has evolved once before the split of the Asteridae and Rosidae. This conclusion is based on the phylogenetic history of the S-RNase that determines pistil specificity. In Rosaceae, molecular characterizations of Prunus species, and species from the tribe Pyreae (i.e., Malus, Pyrus, Sorbus) revealed different numbers of genes determining S-pollen specificity. In Prunus only one pistil and pollen gene determine GSI, while in Pyreae there is one pistil but multiple pollen genes, implying different specificity recognition mechanisms. It is thus conceivable that within Rosaceae the genes involved in GSI in the two lineages are not orthologous but possibly paralogous. To address this hypothesis we characterised the S-RNase lineage and S-pollen lineage genes present in the genomes of five Rosaceae species from three genera: M. × domestica (apple, self-incompatible (SI); tribe Pyreae), P. persica (peach, self-compatible (SC); Amygdaleae), P. mume (mei, SI; Amygdaleae), Fragaria vesca (strawberry, SC; Potentilleae), and F. nipponica (mori-ichigo, SI; Potentilleae). Phylogenetic analyses revealed that the Malus and Prunus S-RNase and S-pollen genes belong to distinct gene lineages, and that only Prunus S-RNase and SFB-lineage genes are present in Fragaria. Thus, S-RNase based GSI system of Malus evolved independently from the ancestral system of Rosaceae. Using expression patterns based on RNA-seq data, the ancestral S-RNase lineage gene is inferred to be expressed in pistils only, while the ancestral S-pollen lineage gene is inferred to be expressed in tissues other than pollen. PMID:25993016
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Plant-derived virus-like particles as vaccines
Chen, Qiang; Lai, Huafang
2013-01-01
Virus-like particles (VLPs) are self-assembled structures derived from viral antigens that mimic the native architecture of viruses but lack the viral genome. VLPs have emerged as a premier vaccine platform due to their advantages in safety, immunogenicity, and manufacturing. The particulate nature and high-density presentation of viral structure proteins on their surface also render VLPs as attractive carriers for displaying foreign epitopes. Consequently, several VLP-based vaccines have been licensed for human use and achieved significant clinical and economical success. The major challenge, however, is to develop novel production platforms that can deliver VLP-based vaccines while significantly reducing production times and costs. Therefore, this review focuses on the essential role of plants as a novel, speedy and economical production platform for VLP-based vaccines. The advantages of plant expression systems are discussed in light of their distinctive posttranslational modifications, cost-effectiveness, production speed, and scalability. Recent achievements in the expression and assembly of VLPs and their chimeric derivatives in plant systems as well as their immunogenicity in animal models are presented. Results of human clinical trials demonstrating the safety and efficacy of plant-derived VLPs are also detailed. Moreover, the promising implications of the recent creation of “humanized” glycosylation plant lines as well as the very recent approval of the first plant-made biologics by the U. S. Food and Drug Administration (FDA) for plant production and commercialization of VLP-based vaccines are discussed. It is speculated that the combined potential of plant expression systems and VLP technology will lead to the emergence of successful vaccines and novel applications of VLPs in the near future. PMID:22995837
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Convergent evolution at the gametophytic self-incompatibility system in Malus and Prunus.
Aguiar, Bruno; Vieira, Jorge; Cunha, Ana E; Fonseca, Nuno A; Iezzoni, Amy; van Nocker, Steve; Vieira, Cristina P
2015-01-01
S-RNase-based gametophytic self-incompatibility (GSI) has evolved once before the split of the Asteridae and Rosidae. This conclusion is based on the phylogenetic history of the S-RNase that determines pistil specificity. In Rosaceae, molecular characterizations of Prunus species, and species from the tribe Pyreae (i.e., Malus, Pyrus, Sorbus) revealed different numbers of genes determining S-pollen specificity. In Prunus only one pistil and pollen gene determine GSI, while in Pyreae there is one pistil but multiple pollen genes, implying different specificity recognition mechanisms. It is thus conceivable that within Rosaceae the genes involved in GSI in the two lineages are not orthologous but possibly paralogous. To address this hypothesis we characterised the S-RNase lineage and S-pollen lineage genes present in the genomes of five Rosaceae species from three genera: M. × domestica (apple, self-incompatible (SI); tribe Pyreae), P. persica (peach, self-compatible (SC); Amygdaleae), P. mume (mei, SI; Amygdaleae), Fragaria vesca (strawberry, SC; Potentilleae), and F. nipponica (mori-ichigo, SI; Potentilleae). Phylogenetic analyses revealed that the Malus and Prunus S-RNase and S-pollen genes belong to distinct gene lineages, and that only Prunus S-RNase and SFB-lineage genes are present in Fragaria. Thus, S-RNase based GSI system of Malus evolved independently from the ancestral system of Rosaceae. Using expression patterns based on RNA-seq data, the ancestral S-RNase lineage gene is inferred to be expressed in pistils only, while the ancestral S-pollen lineage gene is inferred to be expressed in tissues other than pollen.
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A microprocessor based high speed packet switch for satellite communications
NASA Technical Reports Server (NTRS)
Arozullah, M.; Crist, S. C.
1980-01-01
The architectures of a single processor, a three processor, and a multiple processor system are described. The hardware circuits, and software routines required for implementing the three and multiple processor designs are presented. A bit-slice microprocessor was designed and microprogrammed. Maximum throughput was calculated for all three designs. Queue theoretic models for these three designs were developed and utilized to obtain analytical expressions for the average waiting times, overall average response times and average queue sizes. From these expressions, graphs were obtained showing the effect on the system performance of a number of design parameters.
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Regulatable Transgene Expression for Prevention of Chemotherapy-Induced Peripheral Neuropathy.
Kawata, Daisuke; Wu, Zetang
2017-09-15
Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating complication associated with drug treatment of cancer for which there are no effective strategies of prevention or treatment. In this study, we examined the effect of intermittent expression of neurotophin-3 (NT-3) or interleukin-10 (IL-10) from replication-defective herpes simplex virus (HSV)-based regulatable vectors delivered by subcutaneous inoculation to the dorsal root ganglion (DRG) on the development of paclitaxel-induced peripheral neuropathy. We constructed two different tetracycline (tet)-on-based regulatable HSV vectors, one expressing NT-3 and the other expressing IL-10, in which the transactivator expression in the tet-on system was under the control of HSV latency-associated promoter 2 (LAP-2), and expression of the transgene was controlled by doxycycline (DOX). We examined the therapeutic effect of intermittent expression of the transgene in animals with paclitaxel-induced peripheral neuropathy modeled by intraperitoneal injection of paclitaxel (16 mg/kg) once a week for 5 weeks. Intermittent expression of either NT-3 or IL-10 3 days before and 1 day after paclitaxel administration protected animals against paclitaxel-induced peripheral neuropathy over the course of 5 weeks. These results suggest the potential of regulatable vectors for prevention of chemotherapy-induced peripheral neuropathy.
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Rajamani, Deepa; Bhasin, Manoj K
2016-05-03
Pancreatic cancer is an aggressive cancer with dismal prognosis, urgently necessitating better biomarkers to improve therapeutic options and early diagnosis. Traditional approaches of biomarker detection that consider only one aspect of the biological continuum like gene expression alone are limited in their scope and lack robustness in identifying the key regulators of the disease. We have adopted a multidimensional approach involving the cross-talk between the omics spaces to identify key regulators of disease progression. Multidimensional domain-specific disease signatures were obtained using rank-based meta-analysis of individual omics profiles (mRNA, miRNA, DNA methylation) related to pancreatic ductal adenocarcinoma (PDAC). These domain-specific PDAC signatures were integrated to identify genes that were affected across multiple dimensions of omics space in PDAC (genes under multiple regulatory controls, GMCs). To further pin down the regulators of PDAC pathophysiology, a systems-level network was generated from knowledge-based interaction information applied to the above identified GMCs. Key regulators were identified from the GMC network based on network statistics and their functional importance was validated using gene set enrichment analysis and survival analysis. Rank-based meta-analysis identified 5391 genes, 109 miRNAs and 2081 methylation-sites significantly differentially expressed in PDAC (false discovery rate ≤ 0.05). Bimodal integration of meta-analysis signatures revealed 1150 and 715 genes regulated by miRNAs and methylation, respectively. Further analysis identified 189 altered genes that are commonly regulated by miRNA and methylation, hence considered GMCs. Systems-level analysis of the scale-free GMCs network identified eight potential key regulator hubs, namely E2F3, HMGA2, RASA1, IRS1, NUAK1, ACTN1, SKI and DLL1, associated with important pathways driving cancer progression. Survival analysis on individual key regulators revealed that higher expression of IRS1 and DLL1 and lower expression of HMGA2, ACTN1 and SKI were associated with better survival probabilities. It is evident from the results that our hierarchical systems-level multidimensional analysis approach has been successful in isolating the converging regulatory modules and associated key regulatory molecules that are potential biomarkers for pancreatic cancer progression.
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The daily timing of gene expression and physiology in mammals
Schibler, Ueli
2007-01-01
Mammalian behavior and physiology undergo daily rhythms that are coordinated by an endogenous circadian timing system. This system has a hierarchical structure, in that a master pacemaker, residing in the suprachiasmatic nucleus of the ventral hypothalamus, synchronizes peripheral oscillators in virtually all body cells. While the basic molecular mechanisms generating the daily rhythms are similar in aIl cells, most clock out-puts are cell-specific. This conclusion is based on genomewide transcriptome profiling studies in several tissues that have revealed hundreds of rhythmically expressed genes. Cyclic gene expression in the various organs governs overt rhythms in behavior and physiology, encompassing sleep-wake cycles, metabolism, xenobiotic detoxification, and cellularproliferation. As a consequence, chronic perturbation of this temporal organization may lead to increased morbidity and reduced lifespan. PMID:17969863
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Independent bases on the spatial wavefunction of four-identical-particle systems
DOE Office of Scientific and Technical Information (OSTI.GOV)
Xiao, Shuyuan; Deng, Zhixuan; Chen, Hong
2013-12-15
We construct the independent bases on the spatial wavefunction of four-identical-particle systems classified under the rotational group SO(3) and the permutation group S{sub 4} with the usage of transformation coefficients that relate wavefunctions described in one set of internal coordinates with those in another. The basis functions for N⩽ 2 are presented in the explicit expressions based on the harmonic oscillator model. Such independent bases are supposed to play a key role in the construction of the wavefunctions of the five-quark states and the variation calculation of four-body systems. Our prescription avoids the spurious states and can be programmed formore » arbitrary N.« less
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SVEP1 is a novel marker of activated pre-determined skeletal muscle satellite cells.
Shefer, Gabi; Benayahu, Dafna
2010-03-01
In this study we explored the expression pattern of SVEP1, a novel cell adhesion molecule (CAM), in bona fide satellite cells and their immediate progeny. We show that SVEP1 is expressed in activated satellite cells prior to their determination to the myogenic lineage. SVEP1 was also expressed during early phases of myogenic differentiation through the initial stage of myoblast fusion and myotube formation. The expression of SVEP1 was shown by immunostaining two cell culture systems: freshly isolated myofibers and primary myoblasts. Pax7 was used to pinpoint satellite cells situated in their niche on myofibers, and activated satellite cells were determined based on BrdU incorporation (Pax7(+)/BrdU(+)cells). MyoD marked satellite cells fated to undergo myogenesis as well as proliferating and differentiating myoblasts. Differentiating myoblasts and myotubes were identified based on their sarcomeric myosin expression. We showed that SVEP1 was specifically expressed in pre-determined activated satellite cells (Pax7(+)/ BrdU(+) /MyoD(-)) accounting for about 24% of total satellite cells. On the other hand, SVEP1 expression was absent in quiescent satellite cells (Pax7(+)/BrdU(-)/MyoD(-)). Moreover, based on MyoD/sarcomeric myosin co-expression SVEP1 was shown to be expressed throughout the early phases of myogenesis up until myoblast fusion and myotube formation. A decline in SVEP1 expression occurred upon myotube maturation. We suggest SVEP1 as a potential biomarker for pre-fated satellite cells. The impact of this finding is that it may allow scrutinizing signals that affect differentiation commitment. Thus, holds a therapeutic potential for maladies that involve deregulated stem cell fate-decision.
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Bekele, Esubalew; Zheng, Zhi; Swanson, Amy; Crittendon, Julie; Warren, Zachary; Sarkar, Nilanjan
2013-01-01
Autism Spectrum Disorders (ASD) are characterized by atypical patterns of behaviors and impairments in social communication. Among the fundamental social impairments in the ASD population are challenges in appropriately recognizing and responding to facial expressions. Traditional intervention approaches often require intensive support and well-trained therapists to address core deficits, with many with ASD having tremendous difficulty accessing such care due to lack of available trained therapists as well as intervention costs. As a result, emerging technology such as virtual reality (VR) has the potential to offer useful technology-enabled intervention systems. In this paper, an innovative VR-based facial emotional expression presentation system was developed that allows monitoring of eye gaze and physiological signals related to emotion identification to explore new efficient therapeutic paradigms. A usability study of this new system involving ten adolescents with ASD and ten typically developing adolescents as a control group was performed. The eye tracking and physiological data were analyzed to determine intragroup and intergroup variations of gaze and physiological patterns. Performance data, eye tracking indices and physiological features indicated that there were differences in the way adolescents with ASD process and recognize emotional faces compared to their typically developing peers. These results will be used in the future for an online adaptive VR-based multimodal social interaction system to improve emotion recognition abilities of individuals with ASD. PMID:23428456
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Bekele, Esubalew; Zheng, Zhi; Swanson, Amy; Crittendon, Julie; Warren, Zachary; Sarkar, Nilanjan
2013-04-01
Autism Spectrum Disorders (ASD) are characterized by atypical patterns of behaviors and impairments in social communication. Among the fundamental social impairments in the ASD population are challenges in appropriately recognizing and responding to facial expressions. Traditional intervention approaches often require intensive support and well-trained therapists to address core deficits, with many with ASD having tremendous difficulty accessing such care due to lack of available trained therapists as well as intervention costs. As a result, emerging technology such as virtual reality (VR) has the potential to offer useful technology-enabled intervention systems. In this paper, an innovative VR-based facial emotional expression presentation system was developed that allows monitoring of eye gaze and physiological signals related to emotion identification to explore new efficient therapeutic paradigms. A usability study of this new system involving ten adolescents with ASD and ten typically developing adolescents as a control group was performed. The eye tracking and physiological data were analyzed to determine intragroup and intergroup variations of gaze and physiological patterns. Performance data, eye tracking indices and physiological features indicated that there were differences in the way adolescents with ASD process and recognize emotional faces compared to their typically developing peers. These results will be used in the future for an online adaptive VR-based multimodal social interaction system to improve emotion recognition abilities of individuals with ASD.
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Massively parallel nanowell-based single-cell gene expression profiling.
Goldstein, Leonard D; Chen, Ying-Jiun Jasmine; Dunne, Jude; Mir, Alain; Hubschle, Hermann; Guillory, Joseph; Yuan, Wenlin; Zhang, Jingli; Stinson, Jeremy; Jaiswal, Bijay; Pahuja, Kanika Bajaj; Mann, Ishminder; Schaal, Thomas; Chan, Leo; Anandakrishnan, Sangeetha; Lin, Chun-Wah; Espinoza, Patricio; Husain, Syed; Shapiro, Harris; Swaminathan, Karthikeyan; Wei, Sherry; Srinivasan, Maithreyan; Seshagiri, Somasekar; Modrusan, Zora
2017-07-07
Technological advances have enabled transcriptome characterization of cell types at the single-cell level providing new biological insights. New methods that enable simple yet high-throughput single-cell expression profiling are highly desirable. Here we report a novel nanowell-based single-cell RNA sequencing system, ICELL8, which enables processing of thousands of cells per sample. The system employs a 5,184-nanowell-containing microchip to capture ~1,300 single cells and process them. Each nanowell contains preprinted oligonucleotides encoding poly-d(T), a unique well barcode, and a unique molecular identifier. The ICELL8 system uses imaging software to identify nanowells containing viable single cells and only wells with single cells are processed into sequencing libraries. Here, we report the performance and utility of ICELL8 using samples of increasing complexity from cultured cells to mouse solid tissue samples. Our assessment of the system to discriminate between mixed human and mouse cells showed that ICELL8 has a low cell multiplet rate (< 3%) and low cross-cell contamination. We characterized single-cell transcriptomes of more than a thousand cultured human and mouse cells as well as 468 mouse pancreatic islets cells. We were able to identify distinct cell types in pancreatic islets, including alpha, beta, delta and gamma cells. Overall, ICELL8 provides efficient and cost-effective single-cell expression profiling of thousands of cells, allowing researchers to decipher single-cell transcriptomes within complex biological samples.
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Peng, Jian-Hong; Fang, Yu-Jing; Li, Cai-Xia; Ou, Qing-Jian; Jiang, Wu; Lu, Shi-Xun; Lu, Zhen-Hai; Li, Pei-Xing; Yun, Jing-Ping; Zhang, Rong-Xin; Pan, Zhi-Zhong; Wan, De Sen
2016-04-19
Nearly 20% patients with stage II A colon cancer will develop recurrent disease post-operatively. The present study aims to develop a scoring system based on Artificial Neural Network (ANN) model for predicting 10-year survival outcome. The clinical and molecular data of 117 stage II A colon cancer patients from Sun Yat-sen University Cancer Center were used for training set and test set; poor pathological grading (score 49), reduced expression of TGFBR2 (score 33), over-expression of TGF-β (score 45), MAPK (score 32), pin1 (score 100), β-catenin in tumor tissue (score 50) and reduced expression of TGF-β in normal mucosa (score 22) were selected as the prognostic risk predictors. According to the developed scoring system, the patients were divided into 3 subgroups, which were supposed with higher, moderate and lower risk levels. As a result, for the 3 subgroups, the 10-year overall survival (OS) rates were 16.7%, 62.9% and 100% (P < 0.001); and the 10-year disease free survival (DFS) rates were 16.7%, 61.8% and 98.8% (P < 0.001) respectively. It showed that this scoring system for stage II A colon cancer could help to predict long-term survival and screen out high-risk individuals for more vigorous treatment.
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Wang, Chia-Hung; Naik, Nenavath Gopal; Liao, Lin-Li; Wei, Sung-Chan; Chao, Yu-Chan
2017-09-15
Although baculovirus has been used as a safe and convenient gene delivery vector in mammalian cells, baculovirus-mediated transgene expression is less effective in various mammalian cell lines. Identification of the negative regulators in host cells is necessary to improve baculovirus-based expression systems. Here, we performed high-throughput shRNA library screening, targeting 176 antiviral innate immune genes, and identified 43 host restriction factor genes in a human A549 lung carcinoma cell line. Among them, suppression of receptor interaction protein kinase 1 (RIP1, also known as RIPK1) significantly increased baculoviral transgene expression without resulting in significant cell death. Silencing of RIP1 did not affect viral entry or cell viability, but it did inhibit nuclear translocation of the IRF3 and NF-κB transcription factors. Also, activation of downstream signaling mediators (such as TBK1 and IRF7) was affected, and subsequent interferon and cytokine gene expression levels were abolished. Further, Necrostatin-1 (Nec-1)-an inhibitor of RIP1 kinase activity-dramatically increased baculoviral transgene expression in RIP1-silenced cells. Using baculovirus as a model system, this study presents an initial investigation of large numbers of human cell antiviral innate immune response factors against a "nonadaptive virus." In addition, our study has made baculovirus a more efficient gene transfer vector for some of the most frequently used mammalian cell systems.
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Overview of expression of hepatitis B surface antigen in transgenic plants.
Guan, Zheng-jun; Guo, Bin; Huo, Yan-lin; Guan, Zheng-ping; Wei, Ya-hui
2010-10-28
Hepatitis B virus (HBV), a pathogen for chronic liver infection, afflicts more than 350 million people world-wide. The effective way to control the virus is to take HBV vaccine. Hepatitis B surface antigen (HBsAg) is an effective protective antigen suitable for vaccine development. At present, "edible" vaccine based on transgenic plants is one of the most promising directions in novel types of vaccines. HBsAg production from transgenic plants has been carried out, and the transgenic plant expression systems have developed from model plants (such as tobacco, potato and tomato) to other various plant platforms. Crude or purified extracts of transformed plants have been found to conduct immunological responses and clinical trials for hepatitis B, which gave the researches of plant-based HBsAg production a big boost. The aim of this review was to summarize the recent data about plant-based HBsAg development including molecular biology of HBsAg gene, selection of expression vector, the expression of HBsAg gene in plants, as well as corresponding immunological responses in animal models or human. Copyright © 2010 Elsevier Ltd. All rights reserved.
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RILES, a novel method for temporal analysis of the in vivo regulation of miRNA expression
Ezzine, Safia; Vassaux, Georges; Pitard, Bruno; Barteau, Benoit; Malinge, Jean-Marc; Midoux, Patrick; Pichon, Chantal; Baril, Patrick
2013-01-01
Novel methods are required to investigate the complexity of microRNA (miRNA) biology and particularly their dynamic regulation under physiopathological conditions. Herein, a novel plasmid-based RNAi-Inducible Luciferase Expression System (RILES) was engineered to monitor the activity of endogenous RNAi machinery. When RILES is transfected in a target cell, the miRNA of interest suppresses the expression of a transcriptional repressor and consequently switch-ON the expression of the luciferase reporter gene. Hence, miRNA expression in cells is signed by the emission of bioluminescence signals that can be monitored using standard bioluminescence equipment. We validated this approach by monitoring in mice the expression of myomiRs-133, −206 and −1 in skeletal muscles and miRNA-122 in liver. Bioluminescence experiments demonstrated robust qualitative and quantitative data that correlate with the miRNA expression pattern detected by quantitative RT-PCR (qPCR). We further demonstrated that the regulation of miRNA-206 expression during the development of muscular atrophy is individual-dependent, time-regulated and more complex than the information generated by qPCR. As RILES is simple and versatile, we believe that this methodology will contribute to a better understanding of miRNA biology and could serve as a rationale for the development of a novel generation of regulatable gene expression systems with potential therapeutic applications. PMID:24013565
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RILES, a novel method for temporal analysis of the in vivo regulation of miRNA expression.
Ezzine, Safia; Vassaux, Georges; Pitard, Bruno; Barteau, Benoit; Malinge, Jean-Marc; Midoux, Patrick; Pichon, Chantal; Baril, Patrick
2013-11-01
Novel methods are required to investigate the complexity of microRNA (miRNA) biology and particularly their dynamic regulation under physiopathological conditions. Herein, a novel plasmid-based RNAi-Inducible Luciferase Expression System (RILES) was engineered to monitor the activity of endogenous RNAi machinery. When RILES is transfected in a target cell, the miRNA of interest suppresses the expression of a transcriptional repressor and consequently switch-ON the expression of the luciferase reporter gene. Hence, miRNA expression in cells is signed by the emission of bioluminescence signals that can be monitored using standard bioluminescence equipment. We validated this approach by monitoring in mice the expression of myomiRs-133, -206 and -1 in skeletal muscles and miRNA-122 in liver. Bioluminescence experiments demonstrated robust qualitative and quantitative data that correlate with the miRNA expression pattern detected by quantitative RT-PCR (qPCR). We further demonstrated that the regulation of miRNA-206 expression during the development of muscular atrophy is individual-dependent, time-regulated and more complex than the information generated by qPCR. As RILES is simple and versatile, we believe that this methodology will contribute to a better understanding of miRNA biology and could serve as a rationale for the development of a novel generation of regulatable gene expression systems with potential therapeutic applications.
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Dark proteins: effect of inclusion body formation on quantification of protein expression.
Iafolla, Marco A J; Mazumder, Mostafizur; Sardana, Vandit; Velauthapillai, Tharsan; Pannu, Karanbir; McMillen, David R
2008-09-01
Plasmid-borne gene expression systems have found wide application in the emerging fields of systems biology and synthetic biology, where plasmids are used to implement simple network architectures, either to test systems biology hypotheses about issues such as gene expression noise or as a means of exerting artificial control over a cell's dynamics. In both these cases, fluorescent proteins are commonly applied as a means of monitoring the expression of genes in the living cell, and efforts have been made to quantify protein expression levels through fluorescence intensity calibration and by monitoring the partitioning of proteins among the two daughter cells after division; such quantification is important in formulating the predictive models desired in systems and synthetic biology research. A potential pitfall of using plasmid-based gene expression systems is that the high protein levels associated with expression from plasmids can lead to the formation of inclusion bodies, insoluble aggregates of misfolded, nonfunctional proteins that will not generate fluorescence output; proteins caught in these inclusion bodies are thus "dark" to fluorescence-based detection methods. If significant numbers of proteins are incorporated into inclusion bodies rather than becoming biologically active, quantitative results obtained by fluorescent measurements will be skewed; we investigate this phenomenon here. We have created two plasmid constructs with differing average copy numbers, both incorporating an unregulated promoter (P(LtetO-1) in the absence of TetR) expressing the GFP derivative enhanced green fluorescent protein (EGFP), and inserted them into Escherichia coli bacterial cells (a common model organism for work on the dynamics of prokaryotic gene expression). We extracted the inclusion bodies, denatured them, and refolded them to render them active, obtaining a measurement of the average number of EGFP per cell locked into these aggregates; at the same time, we used calibrated fluorescent intensity measurements to determine the average number of active EGFP present per cell. Both measurements were carried out as a function of cellular doubling time, over a range of 45-75 min. We found that the ratio of inclusion body EGFP to active EGFP varied strongly as a function of the cellular growth rate, and that the number of "dark" proteins in the aggregates could in fact be substantial, reaching ratios as high as approximately five proteins locked into inclusion bodies for every active protein (at the fastest growth rate), and dropping to ratios well below 1 (for the slowest growth rate). Our results suggest that efforts to compare computational models to protein numbers derived from fluorescence measurements should take inclusion body loss into account, especially when working with rapidly growing cells. 2008 Wiley-Liss, Inc.
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2012-01-01
Background Baeyer-Villiger monooxygenases (BVMOs) represent a group of enzymes of considerable biotechnological relevance as illustrated by their growing use as biocatalyst in a variety of synthetic applications. However, due to their increased use the reproducible expression of BVMOs and other biotechnologically relevant enzymes has become a pressing matter while knowledge about the factors governing their reproducible expression is scattered. Results Here, we have used phenylacetone monooxygenase (PAMO) from Thermobifida fusca, a prototype Type I BVMO, as a model enzyme to develop a stepwise strategy to optimize the biotransformation performance of recombinant E. coli expressing PAMO in 96-well microtiter plates in a reproducible fashion. Using this system, the best expression conditions of PAMO were investigated first, including different host strains, temperature as well as time and induction period for PAMO expression. This optimized system was used next to improve biotransformation conditions, the PAMO-catalyzed conversion of phenylacetone, by evaluating the best electron donor, substrate concentration, and the temperature and length of biotransformation. Combining all optimized parameters resulted in a more than four-fold enhancement of the biocatalytic performance and, importantly, this was highly reproducible as indicated by the relative standard deviation of 1% for non-washed cells and 3% for washed cells. Furthermore, the optimized procedure was successfully adapted for activity-based mutant screening. Conclusions Our optimized procedure, which provides a comprehensive overview of the key factors influencing the reproducible expression and performance of a biocatalyst, is expected to form a rational basis for the optimization of miniaturized biotransformations and for the design of novel activity-based screening procedures suitable for BVMOs and other NAD(P)H-dependent enzymes as well. PMID:22720747
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Automatic Methods and Tools for the Verification of Real Time Systems
1997-07-31
real - time systems . This was accomplished by extending techniques, based on automata theory and temporal logic, that have been successful for the verification of time-independent reactive systems. As system specification lanmaage for embedded real - time systems , we introduced hybrid automata, which equip traditional discrete automata with real-numbered clock variables and continuous environment variables. As requirements specification languages, we introduced temporal logics with clock variables for expressing timing constraints.
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40 CFR 85.2108 - Dealer certification.
Code of Federal Regulations, 2013 CFR
2013-07-01
... that: (1) Based upon written notification furnished by the manufacturer, the dealer has knowledge that... representation or a warranty, express or implied, by the dealer that the emission control system or any part...
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40 CFR 85.2108 - Dealer certification.
Code of Federal Regulations, 2011 CFR
2011-07-01
... that: (1) Based upon written notification furnished by the manufacturer, the dealer has knowledge that... representation or a warranty, express or implied, by the dealer that the emission control system or any part...
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40 CFR 85.2108 - Dealer certification.
Code of Federal Regulations, 2010 CFR
2010-07-01
... that: (1) Based upon written notification furnished by the manufacturer, the dealer has knowledge that... representation or a warranty, express or implied, by the dealer that the emission control system or any part...
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40 CFR 85.2108 - Dealer certification.
Code of Federal Regulations, 2012 CFR
2012-07-01
... that: (1) Based upon written notification furnished by the manufacturer, the dealer has knowledge that... representation or a warranty, express or implied, by the dealer that the emission control system or any part...
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40 CFR 85.2108 - Dealer certification.
Code of Federal Regulations, 2014 CFR
2014-07-01
... that: (1) Based upon written notification furnished by the manufacturer, the dealer has knowledge that... representation or a warranty, express or implied, by the dealer that the emission control system or any part...
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Back to basics: pBR322 and protein expression systems in E. coli.
Balbás, Paulina; Bolívar, Francisco
2004-01-01
The extensive variety of plasmid-based expression systems in E. coli resulted from the fact that there is no single strategy for achieving maximal expression of every cloned gene. Although a number of strategies have been implemented to deal with problems associated to gene transcription and translation, protein folding, secretion, location, posttranslational modifications, particularities of different strains, and the like and more integrated processes have been developed, the basic plasmid-borne elements and their interaction with the particular host strain will influence the overall expression system and final productivity. Plasmid vector pBR322 is a well-established multipurpose cloning vector in laboratories worldwide, and a large number of derivatives have been created for specific applications and research purposes, including gene expression in its natural host, E. coli, and few other bacteria. The early characterization of the molecule, including its nucleotide sequence, replication and maintenance mechanisms, and determination of its coding regions, accounted for its success, not only as a universal cloning vector, but also as a provider of genes and an origin of replication for other intraspecies vectors. Since the publication of the aforementioned reviews, novel discoveries pertaining to these issues have appeared in the literature that deepen the understanding of the plasmid's features, behavior, and impact in gene expression systems, as well as some important strain characteristics that affect plasmid replication and stability. The objectives of this review include updating and discussing the new information about (1) the replication and maintenance of pBR322; (2) the host-related modulation mechanisms of plasmid replication; (3) the effects of growth rate on replication control, stability, and recombinant gene expression; (4) ways for plasmid amplification and elimination. Finally, (5) a summary of novel ancillary studies about pBR322 is presented.
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Saberi, Fatemeh; Kamali, Mehdi; Najafi, Ali; Yazdanparast, Alavieh; Moghaddam, Mehrdad Moosazadeh
2016-01-01
Naturally occurring antisense RNAs are small, diffusible, untranslated transcripts that pair to target RNAs at specific regions of complementarity to control their biological function by regulating gene expression at the post-transcriptional level. This review focuses on known cases of antisense RNA control in prokaryotes and provides an overview of some natural RNA-based mechanisms that bacteria use to modulate gene expression, such as mRNA sensors, riboswitches and antisense RNAs. We also highlight recent advances in RNA-based technology. The review shows that studies on both natural and synthetic systems are reciprocally beneficial.
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NASA Astrophysics Data System (ADS)
Li, Husheng; Betz, Sharon M.; Poor, H. Vincent
2007-05-01
This paper examines the performance of decision feedback based iterative channel estimation and multiuser detection in channel coded aperiodic DS-CDMA systems operating over multipath fading channels. First, explicit expressions describing the performance of channel estimation and parallel interference cancellation based multiuser detection are developed. These results are then combined to characterize the evolution of the performance of a system that iterates among channel estimation, multiuser detection and channel decoding. Sufficient conditions for convergence of this system to a unique fixed point are developed.
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Efficient gene editing in Corynebacterium glutamicum using the CRISPR/Cas9 system.
Peng, Feng; Wang, Xinyue; Sun, Yang; Dong, Guibin; Yang, Yankun; Liu, Xiuxia; Bai, Zhonghu
2017-11-14
Corynebacterium glutamicum (C. glutamicum) has traditionally been used as a microbial cell factory for the industrial production of many amino acids and other industrially important commodities. C. glutamicum has recently been established as a host for recombinant protein expression; however, some intrinsic disadvantages could be improved by genetic modification. Gene editing techniques, such as deletion, insertion, or replacement, are important tools for modifying chromosomes. In this research, we report a CRISPR/Cas9 system in C. glutamicum for rapid and efficient genome editing, including gene deletion and insertion. The system consists of two plasmids: one containing a target-specific guide RNA and a homologous sequence to a target gene, the other expressing Cas9 protein. With high efficiency (up to 100%), this system was used to disrupt the porB, mepA, clpX and Ncgl0911 genes, which affect the ability to express proteins. The porB- and mepA-deletion strains had enhanced expression of green fluorescent protein, compared with the wild-type stain. This system can also be used to engineer point mutations and gene insertions. In this study, we adapted the CRISPR/Cas9 system from S. pyogens to gene deletion, point mutations and insertion in C. glutamicum. Compared with published genome modification methods, methods based on the CRISPR/Cas9 system can rapidly and efficiently achieve genome editing. Our research provides a powerful tool for facilitating the study of gene function, metabolic pathways, and enhanced productivity in C. glutamicum.
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Ando, Tatsuya; Suguro, Miyuki; Kobayashi, Takeshi; Seto, Masao; Honda, Hiroyuki
2003-10-01
A fuzzy neural network (FNN) using gene expression profile data can select combinations of genes from thousands of genes, and is applicable to predict outcome for cancer patients after chemotherapy. However, wide clinical heterogeneity reduces the accuracy of prediction. To overcome this problem, we have proposed an FNN system based on majoritarian decision using multiple noninferior models. We used transcriptional profiling data, which were obtained from "Lymphochip" DNA microarrays (http://llmpp.nih.gov/DLBCL), reported by Rosenwald (N Engl J Med 2002; 346: 1937-47). When the data were analyzed by our FNN system, accuracy (73.4%) of outcome prediction using only 1 FNN model with 4 genes was higher than that (68.5%) of the Cox model using 17 genes. Higher accuracy (91%) was obtained when an FNN system with 9 noninferior models, consisting of 35 independent genes, was used. The genes selected by the system included genes that are informative in the prognosis of Diffuse large B-cell lymphoma (DLBCL), such as genes showing an expression pattern similar to that of CD10 and BCL-6 or similar to that of IRF-4 and BCL-4. We classified 220 DLBCL patients into 5 groups using the prediction results of 9 FNN models. These groups may correspond to DLBCL subtypes. In group A containing half of the 220 patients, patients with poor outcome were found to satisfy 2 rules, i.e., high expression of MAX dimerization with high expression of unknown A (LC_26146), or high expression of MAX dimerization with low expression of unknown B (LC_33144). The present paper is the first to describe the multiple noninferior FNN modeling system. This system is a powerful tool for predicting outcome and classifying patients, and is applicable to other heterogeneous diseases.
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In vivo delivery of miRNAs for cancer therapy: Challenges and strategies⋆
Chen, Yunching; Gao, Dong-Yu; Huang, Leaf
2016-01-01
MicroRNAs (miRNAs), small non-coding RNAs, can regulate post-transcriptional gene expressions and silence a broad set of target genes. miRNAs, aberrantly expressed in cancer cells, play an important role in modulating gene expressions, thereby regulating downstream signaling pathways and affecting cancer formation and progression. Oncogenes or tumor suppressor genes regulated by miRNAs mediate cell cycle progression, metabolism, cell death, angiogenesis, metastasis and immunosuppression in cancer. Recently, miRNAs have emerged as therapeutic targets or tools and biomarkers for diagnosis and therapy monitoring in cancer. Since miRNAs can regulate multiple cancer-related genes simultaneously, using miRNAs as a therapeutic approach plays an important role in cancer therapy. However, one of the major challenges of miRNA-based cancer therapy is to achieve specific, efficient and safe systemic delivery of therapeutic miRNAs In vivo. This review discusses the key challenges to the development of the carriers for miRNA-based therapy and explores current strategies to systemically deliver miRNAs to cancer without induction of toxicity. PMID:24859533
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Lim, Hyoun-Sub; Vaira, Anna Maria; Domier, Leslie L; Lee, Sung Chul; Kim, Hong Gi; Hammond, John
2010-06-20
We have developed plant virus-based vectors for virus-induced gene silencing (VIGS) and protein expression, based on Alternanthera mosaic virus (AltMV), for infection of a wide range of host plants including Nicotiana benthamiana and Arabidopsis thaliana by either mechanical inoculation of in vitro transcripts or via agroinfiltration. In vivo transcripts produced by co-agroinfiltration of bacteriophage T7 RNA polymerase resulted in T7-driven AltMV infection from a binary vector in the absence of the Cauliflower mosaic virus 35S promoter. An artificial bipartite viral vector delivery system was created by separating the AltMV RNA-dependent RNA polymerase and Triple Gene Block (TGB)123-Coat protein (CP) coding regions into two constructs each bearing the AltMV 5' and 3' non-coding regions, which recombined in planta to generate a full-length AltMV genome. Substitution of TGB1 L(88)P, and equivalent changes in other potexvirus TGB1 proteins, affected RNA silencing suppression efficacy and suitability of the vectors from protein expression to VIGS. Published by Elsevier Inc.
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A Drosophila LexA Enhancer-Trap Resource for Developmental Biology and Neuroendocrine Research
Kockel, Lutz; Huq, Lutfi M.; Ayyar, Anika; Herold, Emma; MacAlpine, Elle; Logan, Madeline; Savvides, Christina; Kim, Grace E. S.; Chen, Jiapei; Clark, Theresa; Duong, Trang; Fazel-Rezai, Vahid; Havey, Deanna; Han, Samuel; Jagadeesan, Ravi; Kim, Eun Soo Jackie; Lee, Diane; Lombardo, Kaelina; Piyale, Ida; Shi, Hansen; Stahr, Lydia; Tung, Dana; Tayvah, Uriel; Wang, Flora; Wang, Ja-Hon; Xiao, Sarah; Topper, Sydni M.; Park, Sangbin; Rotondo, Cheryl; Rankin, Anne E.; Chisholm, Townley W.; Kim, Seung K.
2016-01-01
Novel binary gene expression tools like the LexA-LexAop system could powerfully enhance studies of metabolism, development, and neurobiology in Drosophila. However, specific LexA drivers for neuroendocrine cells and many other developmentally relevant systems remain limited. In a unique high school biology course, we generated a LexA-based enhancer trap collection by transposon mobilization. The initial collection provides a source of novel LexA-based elements that permit targeted gene expression in the corpora cardiaca, cells central for metabolic homeostasis, and other neuroendocrine cell types. The collection further contains specific LexA drivers for stem cells and other enteric cells in the gut, and other developmentally relevant tissue types. We provide detailed analysis of nearly 100 new LexA lines, including molecular mapping of insertions, description of enhancer-driven reporter expression in larval tissues, and adult neuroendocrine cells, comparison with established enhancer trap collections and tissue specific RNAseq. Generation of this open-resource LexA collection facilitates neuroendocrine and developmental biology investigations, and shows how empowering secondary school science can achieve research and educational goals. PMID:27527793
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A Drosophila LexA Enhancer-Trap Resource for Developmental Biology and Neuroendocrine Research.
Kockel, Lutz; Huq, Lutfi M; Ayyar, Anika; Herold, Emma; MacAlpine, Elle; Logan, Madeline; Savvides, Christina; Kim, Grace E S; Chen, Jiapei; Clark, Theresa; Duong, Trang; Fazel-Rezai, Vahid; Havey, Deanna; Han, Samuel; Jagadeesan, Ravi; Kim, Eun Soo Jackie; Lee, Diane; Lombardo, Kaelina; Piyale, Ida; Shi, Hansen; Stahr, Lydia; Tung, Dana; Tayvah, Uriel; Wang, Flora; Wang, Ja-Hon; Xiao, Sarah; Topper, Sydni M; Park, Sangbin; Rotondo, Cheryl; Rankin, Anne E; Chisholm, Townley W; Kim, Seung K
2016-10-13
Novel binary gene expression tools like the LexA-LexAop system could powerfully enhance studies of metabolism, development, and neurobiology in Drosophila However, specific LexA drivers for neuroendocrine cells and many other developmentally relevant systems remain limited. In a unique high school biology course, we generated a LexA-based enhancer trap collection by transposon mobilization. The initial collection provides a source of novel LexA-based elements that permit targeted gene expression in the corpora cardiaca, cells central for metabolic homeostasis, and other neuroendocrine cell types. The collection further contains specific LexA drivers for stem cells and other enteric cells in the gut, and other developmentally relevant tissue types. We provide detailed analysis of nearly 100 new LexA lines, including molecular mapping of insertions, description of enhancer-driven reporter expression in larval tissues, and adult neuroendocrine cells, comparison with established enhancer trap collections and tissue specific RNAseq. Generation of this open-resource LexA collection facilitates neuroendocrine and developmental biology investigations, and shows how empowering secondary school science can achieve research and educational goals. Copyright © 2016 Kockel et al.
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Lamb, Joleah B; van Oppen, Madeleine J H; Willis, Bette L; Bourne, David G
2015-01-01
Abstract Unravelling the contributions of local anthropogenic and seasonal environmental factors in suppressing the coral immune system is important for prioritizing management actions at reefs exposed to high levels of human activities. Here, we monitor health of the model coral Acropora millepora adjacent to a high-use and an unused reef-based tourist platform, plus a nearby control site without a platform, over 7 months spanning a typical austral summer. Comparisons of temporal patterns in a range of biochemical and genetic immune parameters (Toll-like receptor signalling pathway, lectin–complement system, prophenoloxidase-activating system and green fluorescent protein-like proteins) among healthy, injured and diseased corals revealed that corals exhibit a diverse array of immune responses to environmental and anthropogenic stressors. In healthy corals at the control site, expression of genes involved in the Toll-like receptor signalling pathway (MAPK p38, MEKK1, cFos and ATF4/5) and complement system (C3 and Bf) was modulated by seasonal environmental factors in summer months. Corals at reef platform sites experienced additional stressors over the summer, as evidenced by increased expression of various immune genes, including MAPK p38 and MEKK1. Despite increased expression of immune genes, signs of white syndromes were detected in 31% of study corals near tourist platforms in the warmest summer month. Evidence that colonies developing disease showed reduced expression of genes involved in the complement pathway prior to disease onset suggests that their immune systems may have been compromised. Responses to disease and physical damage primarily involved the melanization cascade and GFP-like proteins, and appeared to be sufficient for recovery when summer heat stress subsided. Overall, seasonal and anthropogenic factors may have interacted synergistically to overwhelm the immune systems of corals near reef platforms, leading to increased disease prevalence in summer at these sites. PMID:27293717
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Cross-layer Design for MIMO Systems with Transmit Antenna Selection and Imperfect CSI
NASA Astrophysics Data System (ADS)
Yu, Xiangbin; Liu, Yan; Rui, Yun; Zhou, Tingting; Yin, Xin
2013-04-01
In this paper, by combining adaptive modulation and automatic repeat request (ARQ), a cross-layer design (CLD) scheme for multiple-input and multiple-output (MIMO) system with transmit antenna selection (TAS) and imperfect channel state information (CSI) is presented. Based on the imperfect CSI, the probability density function of the effective signal to noise ratio (SNR) is derived, and the fading gain switching thresholds are also derived subject to a target packet loss rate and fixed power constraint. According to these results, we further derive the average spectrum efficiency (SE) and packet error rate (PER) of the system. As a result, closed-form expressions of the average SE and PER are obtained, respectively. The derived expressions include the expressions under perfect CSI as special cases, and can provide good performance evaluation for the CLD system with imperfect CSI. Simulation results verify the validity of the theoretical analysis. The results show that the CLD system with TAS provides better SE than that with space-time block coding, but the SE and PER performance of the system with imperfect CSI are worse than those with perfect CSI due to the estimation error.
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NASA Astrophysics Data System (ADS)
Ventouras, E.-C.; Lardi, I.; Dimitriou, S.; Margariti, A.; Chondraki, P.; Kalatzis, I.; Economou, N.-T.; Tsekou, H.; Paparrigopoulos, T.; Ktonas, P. Y.
2015-09-01
Primitive expression (PE) is a form of dance therapy (DT) that involves an interaction of ethologically and socially based forms which are supplied for re-enactment. Brain connectivity has been measured in electroencephalographic (EEG) data of patients with schizophrenia undergoing PE DT, using the correlation coefficient and mutual information. These parameters do not measure the existence or absence of directionality in the connectivity. The present study investigates the use of the G-autonomy measure of EEG electrode voltages of the same group of schizophrenic patients. G-autonomy is a measure of the “autonomy” of a system. It indicates the degree by which prediction of the system's future evolution is enhanced by taking into account its own past states, in comparison to predictions based on past states of a set of external variables. In the present research, “own” past states refer to voltage values in the time series recorded at a specific electrode and “external” variables refer to the voltage values recorded at other electrodes. Indication is provided for an acute effect of early-stage PE DT expressed by the augmentation of G-autonomy in the delta rhythm and an acute effect of late- stage PE DT expressed by the reduction of G-autonomy in the theta and alpha rhythms.
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Expression regulation by a methyl-CpG binding domain in an E. coli based, cell-free TX-TL system
NASA Astrophysics Data System (ADS)
Schenkelberger, M.; Shanak, S.; Finkler, M.; Worst, E. G.; Noireaux, V.; Helms, V.; Ott, A.
2017-04-01
Cytosine methylation plays an important role in the epigenetic regulation of eukaryotic gene expression. The methyl-CpG binding domain (MBD) is common to a family of eukaryotic transcriptional regulators. How MBD, a stretch of about 80 amino acids, recognizes CpGs in a methylation dependent manner, and as a function of sequence, is only partly understood. Here we show, using an Escherichia coli cell-free expression system, that MBD from the human transcriptional regulator MeCP2 performs as a specific, methylation-dependent repressor in conjunction with the BDNF (brain-derived neurotrophic factor) promoter sequence. Mutation of either base flanking the central CpG pair changes the expression level of the target gene. However, the relative degree of repression as a function of MBD concentration remains unaltered. Molecular dynamics simulations that address the DNA B fiber ratio and the handedness reveal cooperative transitions in the promoter DNA upon MBD binding that correlate well with our experimental observations. We suggest that not only steric hindrance, but also conformational changes of the BDNF promoter as a result of MBD binding are required for MBD to act as a specific inhibitory element. Our work demonstrates that the prokaryotic transcription machinery can reproduce features of epigenetic mammalian transcriptional regulatory elements.
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NASA Astrophysics Data System (ADS)
Niederman, Thomas M. J.; Ghogawala, Zoher; Carter, Bob S.; Tompkins, Hillary S.; Russell, Margaret M.; Mulligan, Richard C.
2002-05-01
The demonstration that angiogenesis is required for the growth of solid tumors has fueled an intense interest in the development of new therapeutic strategies that target the tumor vasculature. Here we report the development of an immune-based antiangiogenic strategy that is based on the generation of T lymphocytes that possess a killing specificity for cells expressing vascular endothelial growth factor receptors (VEGFRs). To target VEGFR-expressing cells, recombinant retroviral vectors were generated that encoded a chimeric T cell receptor comprised of VEGF sequences linked to intracellular signaling sequences derived from the chain of the T cell receptor. After transduction of primary murine CD8 lymphocytes by such vectors, the transduced cells were shown to possess an efficient killing specificity for cells expressing the VEGF receptor, Flk-1, as measured by in vitro cytotoxicity assays. After adoptive transfer into tumor-bearing mice, the genetically modified cytotoxic T lymphocytes strongly inhibited the growth of a variety of syngeneic murine tumors and human tumor xenografts. An increased effect on in vivo tumor growth inhibition was seen when this therapy was combined with the systemic administration of TNP-470, a conventional angiogenesis inhibitor. The utilization of the immune system to target angiogenic markers expressed on tumor vasculature may prove to be a powerful means for controlling tumor growth.
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Chen, Young-Bin; Lan, Ying-Wei; Hung, Tsai-Hsien; Chen, Lih-Geeng; Choo, Kong-Bung; Cheng, Winston T K; Lee, Hsuan-Shu; Chong, Kowit-Yu
2015-07-01
Several studies of stem cell-based gene therapy have indicated that long-lasting regeneration following vessel ischemia may be stimulated through VEGFA gene therapy and/or MSC transplantation for reduction of ischemic injury in limb ischemia and heart failure. The therapeutic potential of MSC transplantation can be further improved by genetically modifying MSCs with genes which enhance angiogenesis following ischemic injury. In the present study, we aimed to develop an approach in MSC-based therapy for repair and mitigation of ischemic injury and regeneration of damaged tissues in ischemic disease. HSP70 promoter-driven VEGFA expression was induced by resveratrol (RSV) in MSCs, and in combination with known RSV biological functions, the protective effects of our approach were investigated by using ex vivo aortic ring coculture system and a 3D scaffolds in vivo model. Results of this investigation demonstrated that HSP promoter-driven VEGFA expression in MSC increased approximately 2-fold over the background VEGFA levels upon HSP70 promoter induction by RSV. Exposure of HUVEC cells to medium containing MSC in which VEGFA had been induced by cis-RSV enhanced tube formation in the treated HUVEC cells. RSV-treated MSC cells differentiated into endothelial-like phenotypes, exhibiting markedly elevated expression of endothelial cell markers. These MSCs also induced aortic ring sprouting, characteristic of neovascular formation from pre-existing vessels, and additionally promoted neovascularization at the MSC transplantation site in a mouse model. These observations support a hypothesis that VEGFA expression induced by cis-RSV acting on the HSP70 promoter in transplanted MSC augments the angiogenic effects of stem cell gene therapy. The use of an inducible system also vastly reduces possible clinical risks associated with constitutive VEGFA expression.
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Definition of Contravariant Velocity Components
NASA Technical Reports Server (NTRS)
Hung, Ching-Mao; Kwak, Dochan (Technical Monitor)
2002-01-01
This is an old issue in computational fluid dynamics (CFD). What is the so-called contravariant velocity or contravariant velocity component? In the article, we review the basics of tensor analysis and give the contravariant velocity component a rigorous explanation. For a given coordinate system, there exist two uniquely determined sets of base vector systems - one is the covariant and another is the contravariant base vector system. The two base vector systems are reciprocal. The so-called contravariant velocity component is really the contravariant component of a velocity vector for a time-independent coordinate system, or the contravariant component of a relative velocity between fluid and coordinates, for a time-dependent coordinate system. The contravariant velocity components are not physical quantities of the velocity vector. Their magnitudes, dimensions, and associated directions are controlled by their corresponding covariant base vectors. Several 2-D (two-dimensional) linear examples and 2-D mass-conservation equation are used to illustrate the details of expressing a vector with respect to the covariant and contravariant base vector systems, respectively.
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Aranda, Alejandro; Bezunartea, Jaione; Casales, Erkuden; Rodriguez-Madoz, Juan R; Larrea, Esther; Prieto, Jesus; Smerdou, Cristian
2014-12-01
We report a new method to generate high-expressing mammalian cell lines in a quick and efficient way. For that purpose, we developed a master cell line (MCL) containing an inducible alphavirus vector expressing GFP integrated into the genome. In the MCL, recombinant RNA levels increased >4,600-fold after induction, due to a doxycycline-dependent RNA amplification loop. The MCL maintained inducibility and expression during 50 passages, being more efficient for protein expression than a conventional cell line. To generate new cell lines, mutant LoxP sites were inserted into the MCL, allowing transgene and selection gene exchange by Cre-directed recombination, leading to quick generation of inducible cell lines expressing proteins of therapeutic interest, like human cardiotrophin-1 and oncostatin-M at several mg/l/24 h. These proteins contained posttranslational modifications, showed bioactivity, and were efficiently purified. Remarkably, this system allowed production of toxic proteins, like oncostatin-M, since cells able to express it could be grown to the desired amount before induction. These cell lines were easily adapted to growth in suspension, making this methodology very attractive for therapeutic protein production.
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Bröker, M; Bäuml, O; Göttig, A; Ochs, J; Bodenbenner, M; Amann, E
1991-03-01
The human blood coagulation protein Factor XIIIa (FXIIIa) was expressed in Saccharomyces cerevisiae employing Escherichia coli-yeast shuttle vectors based on a 2-mu plasmid. Several factors affecting high production yield of recombinant FXIIIa were analysed. The use of the regulatable GAL-CYC1 hybrid promoter resulted in higher FXIIIa expression when compared with the constitutive ADCI promoter. Screening for suitable yeast strains for expression of FXIIIa under the transcriptional control of the GAL-CYC1 hybrid promoter revealed a broad spectrum of productivity. No obvious correlation between the expression rate and the genetic markers of the strains could be identified. The medium composition markedly influenced the FXIIIa expression rates. The expression of FXIIIa was strictly regulated by the carbon source. Glucose as the only sugar and energy source repressed the synthesis of FXIIIa, whereas addition of galactose induced FXIIIa expression. Special feeding schemes resulted in a productivity of up to 100 mg FXIIIa/l in shake flasks.
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A novel expression vector for the secretion of abaecin in Bacillus subtilis.
Li, Li; Mu, Lan; Wang, Xiaojuan; Yu, Jingfeng; Hu, Ruiping; Li, Zhen
This study aimed to describe a Bacillus subtilis expression system based on genetically modified B. subtilis. Abaecin, an antimicrobial peptide obtained from Apis mellifera, can enhance the effect of pore-forming peptides from other species on the inhibition of bacterial growth. For the exogenous expression, the abaecin gene was fused with a tobacco etch virus protease cleavage site, a promoter Pglv, and a mature beta-glucanase signal peptide. Also, a B. subtilis expression system was constructed. The recombinant abaecin gene was expressed and purified as a recombinant protein in the culture supernatant. The purified abaecin did not inhibit the growth of Escherichia coli strain K88. Cecropin A and hymenoptaecin exhibited potent bactericidal activities at concentrations of 1 and 1.5μM. Combinatorial assays revealed that cecropin A and hymenoptaecin had sublethal concentrations of 0.3 and 0.5μM. This potentiating functional interaction represents a promising therapeutic strategy. It provides an opportunity to address the rising threat of multidrug-resistant pathogens that are recalcitrant to conventional antibiotics. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.
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Generation of a Tet-On Expression System to Study Transactivation Ability of Tax-2.
Bignami, Fabio; Sozzi, Riccardo Alessio; Pilotti, Elisabetta
2017-01-01
HTLV Tax proteins (Tax-1 and Tax-2) are known to be able to transactivate several host cellular genes involved in complex molecular pathways. Here, we describe a stable and regulated high-level expression model based on Tet-On system, to study the capacity of Tax-2 to transactivate host genes. In particular, the Jurkat Tet-On cell line suitable for evaluating the ability of Tax-2 to stimulate transactivation of a specific host gene, CCL3L1 (C-C motif chemokine ligand 3 like 1 gene), was selected. Then, a plasmid expressing tax-2 gene under control of a tetracycline-response element was constructed. To avoid the production of a fusion protein between the report gene and the inserted gene, a bidirectional plasmid was designed. Maximum expression and fast response time were achieved by using nucleofection technology as transfection method. After developing an optimized protocol for efficiently transferring tax-2 gene in Jurkat Tet-On cellular model and exposing transfected cells to Dox (doxycycline, a tetracycline derivate), a kinetics of tax-2 expression through TaqMan Real-time PCR assay was determined.
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LaPelle, Nancy R; Luckmann, Roger; Simpson, E Hatheway; Martin, Elaine R
2006-01-01
Background Movement towards evidence-based practices in many fields suggests that public health (PH) challenges may be better addressed if credible information about health risks and effective PH practices is readily available. However, research has shown that many PH information needs are unmet. In addition to reviewing relevant literature, this study performed a comprehensive review of existing information resources and collected data from two representative PH groups, focusing on identifying current practices, expressed information needs, and ideal systems for information access. Methods Nineteen individual interviews were conducted among employees of two domains in a state health department – communicable disease control and community health promotion. Subsequent focus groups gathered additional data on preferences for methods of information access and delivery as well as information format and content. Qualitative methods were used to identify themes in the interview and focus group transcripts. Results Informants expressed similar needs for improved information access including single portal access with a good search engine; automatic notification regarding newly available information; access to best practice information in many areas of interest that extend beyond biomedical subject matter; improved access to grey literature as well as to more systematic reviews, summaries, and full-text articles; better methods for indexing, filtering, and searching for information; and effective ways to archive information accessed. Informants expressed a preference for improving systems with which they were already familiar such as PubMed and listservs rather than introducing new systems of information organization and delivery. A hypothetical ideal model for information organization and delivery was developed based on informants' stated information needs and preferred means of delivery. Features of the model were endorsed by the subjects who reviewed it. Conclusion Many critical information needs of PH practitioners are not being met efficiently or at all. We propose a dual strategy of: 1) promoting incremental improvements in existing information delivery systems based on the expressed preferences of the PH users of the systems and 2) the concurrent development and rigorous evaluation of new models of information organization and delivery that draw on successful resources already operating to deliver information to clinical medical practitioners. PMID:16597331
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Protein interaction networks from literature mining
NASA Astrophysics Data System (ADS)
Ihara, Sigeo
2005-03-01
The ability to accurately predict and understand physiological changes in the biological network system in response to disease or drug therapeutics is of crucial importance in life science. The extensive amount of gene expression data generated from even a single microarray experiment often proves difficult to fully interpret and comprehend the biological significance. An increasing knowledge of protein interactions stored in the PubMed database, as well as the advancement of natural language processing, however, makes it possible to construct protein interaction networks from the gene expression information that are essential for understanding the biological meaning. From the in house literature mining system we have developed, the protein interaction network for humans was constructed. By analysis based on the graph-theoretical characterization of the total interaction network in literature, we found that the network is scale-free and semantic long-ranged interactions (i.e. inhibit, induce) between proteins dominate in the total interaction network, reducing the degree exponent. Interaction networks generated based on scientific text in which the interaction event is ambiguously described result in disconnected networks. In contrast interaction networks based on text in which the interaction events are clearly stated result in strongly connected networks. The results of protein-protein interaction networks obtained in real applications from microarray experiments are discussed: For example, comparisons of the gene expression data indicative of either a good or a poor prognosis for acute lymphoblastic leukemia with MLL rearrangements, using our system, showed newly discovered signaling cross-talk.
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Chichester, Jessica A; Manceva, Slobodanka D; Rhee, Amy; Coffin, Megan V; Musiychuk, Konstantin; Mett, Vadim; Shamloul, Moneim; Norikane, Joey; Streatfield, Stephen J; Yusibov, Vidadi
2013-03-01
The potential use of Bacillus anthracis as a bioterrorism weapon threatens the security of populations globally, requiring the immediate availability of safe, efficient and easily delivered anthrax vaccine for mass vaccination. Extensive research efforts have been directed toward the development of recombinant subunit vaccines based on protective antigen (PA), the principal virulence factor of B. anthracis. Among the emerging technologies for the production of these vaccine antigens is our launch vector-based plant transient expression system. Using this system, we have successfully engineered, expressed, purified and characterized full-length PA (pp-PA83) in Nicotiana benthamiana plants using agroinfiltration. This plant-produced antigen elicited high toxin neutralizing antibody titers in mice and rabbits after two vaccine administrations with Alhydrogel. In addition, immunization with this vaccine candidate protected 100% of rabbits from a lethal aerosolized B. anthracis challenge. The vaccine effects were dose-dependent and required the presence of Alhydrogel adjuvant. In addition, the vaccine antigen formulated with Alhydrogel was stable and retained immunogenicity after two-week storage at 4°C, the conditions intended for clinical use. These results support the testing of this vaccine candidate in human volunteers and the utility of our plant expression system for the production of a recombinant anthrax vaccine.
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Cardinal, Marie-Josée; Kaur, Rajvinder; Singh, Jaswinder
2016-10-01
Domestication and intensive selective breeding of plants has triggered erosion of genetic diversity of important stress-related alleles. Researchers highlight the potential of using wild accessions as a gene source for improvement of cereals such as barley, which has major economic and social importance worldwide. Previously, we have successfully introduced the maize Ac/Ds transposon system for gene identification in cultivated barley. The objective of current research was to investigate the response of Hordeum vulgare ssp. spontaneum wild barley accessions in tissue culture to standardize parameters for introduction of Ac/Ds transposons through genetic transformation. We investigated the response of ten wild barley genotypes for callus induction, regenerative green callus induction and regeneration of fertile plants. The activity of exogenous Ac/Ds elements was observed through a transient assay on immature wild barley embryos/callus whereby transformed embryos/calli were identified by the expression of GUS. Transient Ds expression bombardment experiments were performed on 352 pieces of callus (3-5 mm each) or immature embryos in 4 genotypes of wild barley. The transformation frequency of putative transgenic callus lines based on transient GUS expression ranged between 72 and100 % in wild barley genotypes. This is the first report of a transformation system in H. vulgare ssp. spontaneum.
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Yamada, Shigehiro; Hotta, Kohji; Yamamoto, Takamasa S; Ueno, Naoto; Satoh, Nori; Takahashi, Hiroki
2009-04-01
The midline organ the notochord and its overlying dorsal neural tube are the most prominent features of the chordate body plan. Although the molecular mechanisms involved in the formation of the central nervous system (CNS) have been studied extensively in vertebrate embryos, none of the genes that are expressed exclusively in notochord cells has been shown to function in this process. Here, we report a gene in the urochordate Ciona intestinalis encoding a fibrinogen-like protein that plays a pivotal role in the notochord-dependent positioning of neuronal cells. While this gene (Ci-fibrn) is expressed exclusively in notochord cells, its protein product is not confined to these cells but is distributed underneath the CNS as fibril-like protrusions. We demonstrated that Ci-fibrn interacts physically and functionally with Ci-Notch that is expressed in the central nervous system, and that the correct distribution of Ci-fibrn protein is dependent on Notch signaling. Disturbance of the Ci-fibrn distribution caused an abnormal positioning of neuronal cells and an abnormal track of axon extension. Therefore, it is highly likely that the interaction between the notochord-based fibrinogen-like protein and the neural tube-based Notch signaling plays an essential role in the proper patterning of CNS.
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Computational design of a Zn2+ receptor that controls bacterial gene expression
NASA Astrophysics Data System (ADS)
Dwyer, M. A.; Looger, L. L.; Hellinga, H. W.
2003-09-01
The control of cellular physiology and gene expression in response to extracellular signals is a basic property of living systems. We have constructed a synthetic bacterial signal transduction pathway in which gene expression is controlled by extracellular Zn2+. In this system a computationally designed Zn2+-binding periplasmic receptor senses the extracellular solute and triggers a two-component signal transduction pathway via a chimeric transmembrane protein, resulting in transcriptional up-regulation of a -galactosidase reporter gene. The Zn2+-binding site in the designed receptor is based on a four-coordinate, tetrahedral primary coordination sphere consisting of histidines and glutamates. In addition, mutations were introduced in a secondary coordination sphere to satisfy the residual hydrogen-bonding potential of the histidines coordinated to the metal. The importance of the secondary shell interactions is demonstrated by their effect on metal affinity and selectivity, as well as protein stability. Three designed protein sequences, comprising two distinct metal-binding positions, were all shown to bind Zn2+ and to function in the cell-based assay, indicating the generality of the design methodology. These experiments demonstrate that biological systems can be manipulated with computationally designed proteins that have drastically altered ligand-binding specificities, thereby extending the repertoire of genetic control by extracellular signals.
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Rice-based mucosal vaccine as a global strategy for cold-chain- and needle-free vaccination
Nochi, Tomonori; Takagi, Hidenori; Yuki, Yoshikazu; Yang, Lijun; Masumura, Takehiro; Mejima, Mio; Nakanishi, Ushio; Matsumura, Akiko; Uozumi, Akihiro; Hiroi, Takachika; Morita, Shigeto; Tanaka, Kunisuke; Takaiwa, Fumio; Kiyono, Hiroshi
2007-01-01
Capable of inducing antigen-specific immune responses in both systemic and mucosal compartments without the use of syringe and needle, mucosal vaccination is considered ideal for the global control of infectious diseases. In this study, we developed a rice-based oral vaccine expressing cholera toxin B subunit (CTB) under the control of the endosperm-specific expression promoter 2.3-kb glutelin GluB-1 with codon usage optimization for expression in rice seed. An average of 30 μg of CTB per seed was stored in the protein bodies, which are storage organelles in rice. When mucosally fed, rice seeds expressing CTB were taken up by the M cells covering the Peyer's patches and induced CTB-specific serum IgG and mucosal IgA antibodies with neutralizing activity. When expressed in rice, CTB was protected from pepsin digestion in vitro. Rice-expressed CTB also remained stable and thus maintained immunogenicity at room temperature for >1.5 years, meaning that antigen-specific mucosal immune responses were induced at much lower doses than were necessary with purified recombinant CTB. Because they require neither refrigeration (cold-chain management) nor a needle, these rice-based mucosal vaccines offer a highly practical and cost-effective strategy for orally vaccinating large populations against mucosal infections, including those that may result from an act of bioterrorism. PMID:17573530
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Cognitive penetrability and emotion recognition in human facial expressions
Marchi, Francesco
2015-01-01
Do our background beliefs, desires, and mental images influence our perceptual experience of the emotions of others? In this paper, we will address the possibility of cognitive penetration (CP) of perceptual experience in the domain of social cognition. In particular, we focus on emotion recognition based on the visual experience of facial expressions. After introducing the current debate on CP, we review examples of perceptual adaptation for facial expressions of emotion. This evidence supports the idea that facial expressions are perceptually processed as wholes. That is, the perceptual system integrates lower-level facial features, such as eyebrow orientation, mouth angle etc., into facial compounds. We then present additional experimental evidence showing that in some cases, emotion recognition on the basis of facial expression is sensitive to and modified by the background knowledge of the subject. We argue that such sensitivity is best explained as a difference in the visual experience of the facial expression, not just as a modification of the judgment based on this experience. The difference in experience is characterized as the result of the interference of background knowledge with the perceptual integration process for faces. Thus, according to the best explanation, we have to accept CP in some cases of emotion recognition. Finally, we discuss a recently proposed mechanism for CP in the face-based recognition of emotion. PMID:26150796
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2013-01-01
Background Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. Results We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit’s component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. Conclusions We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior modifications. Using this technology, any existing Gateway destination expression vector with its model-specific properties could be easily adapted for expressing fusion proteins. PMID:23957834
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Buj, Raquel; Iglesias, Noa; Planas, Anna M; Santalucía, Tomàs
2013-08-20
Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit's component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome collections to be used without prior modifications. Using this technology, any existing Gateway destination expression vector with its model-specific properties could be easily adapted for expressing fusion proteins.
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NASA Technical Reports Server (NTRS)
Sledd, Annette; Danford, Mike; Key, Brian
2002-01-01
The EXpedite the PRocessing of Experiments to Space Station or EXPRESS Rack System was developed to provide Space Station accommodations for subrack payloads. The EXPRESS Rack accepts Space Shuttle middeck locker type payloads and International Subrack Interface Standard (ISIS) Drawer payloads, allowing previously flown payloads an opportunity to transition to the International Space Station. The EXPRESS Rack provides power, data command and control, video, water cooling, air cooling, vacuum exhaust, and Nitrogen supply to payloads. The EXPRESS Rack system also includes transportation racks to transport payloads to and from the Space Station, Suitcase Simulators to allow a payload developer to verify data interfaces at the development site, Functional Checkout Units to allow payload checkout at KSC prior to launch, and trainer racks for the astronauts to learn how to operate the EXPRESS Racks prior to flight. Standard hardware and software interfaces provided by the EXPRESS Rack simplify the integration processes, and facilitate simpler ISS payload development. Whereas most ISS Payload facilities are designed to accommodate one specific type of science, the EXPRESS Rack is designed to accommodate multi-discipline research within the same rack allowing for the independent operation of each subrack payload. On-orbit operations began with the EXPRESS Rack Project on April 24, 2001, with one rack operating continuously to support long-running payloads. The other on-orbit EXPRESS Racks operate based on payload need and resource availability. Sustaining Engineering and Logistics and Maintenance functions are in place to maintain operations and to provide software upgrades.
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A Micro Fluorescent Activated Cell Sorter for Astrobiology Applications
NASA Technical Reports Server (NTRS)
Platt, Donald W.; Hoover, Richard B.
2009-01-01
A micro-scale Fluorescent Activated Cell Sorter (microFACS) for astrobiology applications is under development. This device is designed to have a footprint of 7 cm x 7 cm x 4 cm and allow live-dead counts and sorting of cells that have fluorescent characteristics from staining. The FACS system takes advantage of microfluidics to create a cell sorter that can fit in the palm of the hand. A micron-scale channel allows cells to pass by a blue diode which causes emission of marker-expressed cells which are detected by a filtered photodetector. A small microcontroller then counts cells and operates high speed valves to select which chamber the cell is collected in (a collection chamber or a waste chamber). Cells with the expressed characteristic will be collected in the collection chamber. This system has been built and is currently being tested. We are also designing a system with integrated MEMS-based pumps and valves for a small and compact unit to fly on small satellite-based biology experiments.
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Szebényi, Kornélia; Füredi, András; Kolacsek, Orsolya; Pergel, Enikő; Bősze, Zsuzsanna; Bender, Balázs; Vajdovich, Péter; Tóvári, József; Homolya, László; Szakács, Gergely; Héja, László; Enyedi, Ágnes; Sarkadi, Balázs; Apáti, Ágota; Orbán, Tamás I
2015-08-03
In drug discovery, prediction of selectivity and toxicity require the evaluation of cellular calcium homeostasis. The rat is a preferred laboratory animal for pharmacology and toxicology studies, while currently no calcium indicator protein expressing rat model is available. We established a transgenic rat strain stably expressing the GCaMP2 fluorescent calcium sensor by a transposon-based methodology. Zygotes were co-injected with mRNA of transposase and a CAG-GCaMP2 expressing construct, and animals with one transgene copy were pre-selected by measuring fluorescence in blood cells. A homozygous rat strain was generated with high sensor protein expression in the heart, kidney, liver, and blood cells. No pathological alterations were found in these animals, and fluorescence measurements in cardiac tissue slices and primary cultures demonstrated the applicability of this system for studying calcium signaling. We show here that the GCaMP2 expressing rat cardiomyocytes allow the prediction of cardiotoxic drug side-effects, and provide evidence for the role of Na(+)/Ca(2+) exchanger and its beneficial pharmacological modulation in cardiac reperfusion. Our data indicate that drug-induced alterations and pathological processes can be followed by using this rat model, suggesting that transgenic rats expressing a calcium-sensitive protein provide a valuable system for pharmacological and toxicological studies.
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A common anchor facilitated GO-DNA nano-system for multiplex microRNA analysis in live cells.
Yu, Jiantao; He, Sihui; Shao, Chen; Zhao, Haoran; Li, Jing; Tian, Leilei
2018-04-19
The design of a nano-system for the detection of intracellular microRNAs is challenging as it must fulfill complex requirements, i.e., it must have a high sensitivity to determine the dynamic expression level, a good reliability for multiplex and simultaneous detection, and a satisfactory biostability to work in biological environments. Instead of employing a commonly used physisorption or a full-conjugation strategy, here, a GO-DNA nano-system was developed under graft/base-pairing construction. The common anchor sequence was chemically grafted to GO to base-pair with various microRNA probes; and the hybridization with miRNAs drives the dyes on the probes to leave away from GO, resulting in "turned-on" fluorescence. This strategy not only simplifies the synthesis but also efficiently balances the loading yields of different probes. Moreover, the conjugation yield of GO with a base-paired hybrid has been improved by more than two-fold compared to that of the conjugation with a single strand. We demonstrated that base-paired DNA probes could be efficiently delivered into cells along with GO and are properly stabilized by the conjugated anchor sequence. The resultant GO-DNA nano-system exhibited high stability in a complex biological environment and good resistance to nucleases, and was able to accurately discriminate various miRNAs without cross-reaction. With all of these positive features, the GO-DNA nano-system can simultaneously detect three miRNAs and monitor their dynamic expression levels.
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Analytical Optimization of the Net Residual Dispersion in SPM-Limited Dispersion-Managed Systems
NASA Astrophysics Data System (ADS)
Xiao, Xiaosheng; Gao, Shiming; Tian, Yu; Yang, Changxi
2006-05-01
Dispersion management is an effective technique to suppress the nonlinear impairment in fiber transmission systems, which includes tuning the amounts of precompensation, residual dispersion per span (RDPS), and net residual dispersion (NRD) of the systems. For self-phase modulation (SPM)-limited systems, optimizing the NRD is necessary because it can greatly improve the system performance. In this paper, an analytical method is presented to optimize NRD for SPM-limited dispersion-managed systems. The method is based on the correlation between the nonlinear impairment and the output pulse broadening of SPM-limited systems; therefore, dispersion-managed systems can be optimized through minimizing the output single-pulse broadening. A set of expressions is derived to calculate the output pulse broadening of the SPM-limited dispersion-managed system, from which the analytical result of optimal NRD is obtained. Furthermore, with the expressions of pulse broadening, how the nonlinear impairment depends on the amounts of precompensation and RDPS can be revealed conveniently.
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Expression-based clustering of CAZyme-encoding genes of Aspergillus niger.
Gruben, Birgit S; Mäkelä, Miia R; Kowalczyk, Joanna E; Zhou, Miaomiao; Benoit-Gelber, Isabelle; De Vries, Ronald P
2017-11-23
The Aspergillus niger genome contains a large repertoire of genes encoding carbohydrate active enzymes (CAZymes) that are targeted to plant polysaccharide degradation enabling A. niger to grow on a wide range of plant biomass substrates. Which genes need to be activated in certain environmental conditions depends on the composition of the available substrate. Previous studies have demonstrated the involvement of a number of transcriptional regulators in plant biomass degradation and have identified sets of target genes for each regulator. In this study, a broad transcriptional analysis was performed of the A. niger genes encoding (putative) plant polysaccharide degrading enzymes. Microarray data focusing on the initial response of A. niger to the presence of plant biomass related carbon sources were analyzed of a wild-type strain N402 that was grown on a large range of carbon sources and of the regulatory mutant strains ΔxlnR, ΔaraR, ΔamyR, ΔrhaR and ΔgalX that were grown on their specific inducing compounds. The cluster analysis of the expression data revealed several groups of co-regulated genes, which goes beyond the traditionally described co-regulated gene sets. Additional putative target genes of the selected regulators were identified, based on their expression profile. Notably, in several cases the expression profile puts questions on the function assignment of uncharacterized genes that was based on homology searches, highlighting the need for more extensive biochemical studies into the substrate specificity of enzymes encoded by these non-characterized genes. The data also revealed sets of genes that were upregulated in the regulatory mutants, suggesting interaction between the regulatory systems and a therefore even more complex overall regulatory network than has been reported so far. Expression profiling on a large number of substrates provides better insight in the complex regulatory systems that drive the conversion of plant biomass by fungi. In addition, the data provides additional evidence in favor of and against the similarity-based functions assigned to uncharacterized genes.
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Recombinant adeno-associated virus targets passenger gene expression to cones in primate retina
NASA Astrophysics Data System (ADS)
Mancuso, Katherine; Hendrickson, Anita E.; Connor, Thomas B., Jr.; Mauck, Matthew C.; Kinsella, James J.; Hauswirth, William W.; Neitz, Jay; Neitz, Maureen
2007-05-01
Recombinant adeno-associated virus (rAAV) is a promising vector for gene therapy of photoreceptor-based diseases. Previous studies have demonstrated that rAAV serotypes 2 and 5 can transduce both rod and cone photoreceptors in rodents and dogs, and it can target rods, but not cones in primates. Here we report that using a human cone-specific enhancer and promoter to regulate expression of a green fluorescent protein (GFP) reporter gene in an rAAV-5 vector successfully targeted expression of the reporter gene to primate cones, and the time course of GFP expression was able to be monitored in a living animal using the RetCam II digital imaging system.
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A unified probabilistic framework for spontaneous facial action modeling and understanding.
Tong, Yan; Chen, Jixu; Ji, Qiang
2010-02-01
Facial expression is a natural and powerful means of human communication. Recognizing spontaneous facial actions, however, is very challenging due to subtle facial deformation, frequent head movements, and ambiguous and uncertain facial motion measurements. Because of these challenges, current research in facial expression recognition is limited to posed expressions and often in frontal view. A spontaneous facial expression is characterized by rigid head movements and nonrigid facial muscular movements. More importantly, it is the coherent and consistent spatiotemporal interactions among rigid and nonrigid facial motions that produce a meaningful facial expression. Recognizing this fact, we introduce a unified probabilistic facial action model based on the Dynamic Bayesian network (DBN) to simultaneously and coherently represent rigid and nonrigid facial motions, their spatiotemporal dependencies, and their image measurements. Advanced machine learning methods are introduced to learn the model based on both training data and subjective prior knowledge. Given the model and the measurements of facial motions, facial action recognition is accomplished through probabilistic inference by systematically integrating visual measurements with the facial action model. Experiments show that compared to the state-of-the-art techniques, the proposed system yields significant improvements in recognizing both rigid and nonrigid facial motions, especially for spontaneous facial expressions.
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Lee, Jeunghun; Shishido-Hara, Yukiko; Suzuki, Kaori; Shimizu, Saki; Kobayashi, Keiichi; Kamma, Hiroshi; Shiokawa, Yoshiaki; Nagane, Motoo
2017-10-01
Primary central nervous system lymphoma (PCNSL) remains an aggressive and refractory tumor despite high-dose methotrexate-based chemo-radiotherapy. Age and performance status have been shown to be important clinical prognostic factors, however others, especially molecular factors, affecting the prognosis are still uncertain. We investigate clinical, neuroimaging and immunohistochemical data in tissue from 41 PCNSL patients treated primarily with methotrexate-based chemo-radiotherapy and evaluate the influence of potential prognostic factors on clinical outcome as well as correlation among these factors. Median progression-free survival (PFS) and overall survival (OS) were 29 and 73 months, respectively. Expression of the mismatch repair (MMR) proteins, MLH1, MSH2, MSH6 and PMS2, correlated tightly with each other and high expression of MSH2 was significantly associated with better OS and PFS (P = 0.005 and P = 0.007), while methotrexate metabolism-related proteins did not affect survival. In addition, low expression of PMS2 was an independent predictor of methotrexate resistance (P = 0.039). Among neuroimaging findings, involvement of the fornix and tegmentum/velum were significantly associated with poorer OS (P < 0.001 and P = 0.013) and PFS (P = 0.014 and P = 0.043, respectively). Germinal center B cell (GCB)-PCNSL subtype as opposed to non-GCB subtype, tended toward better survival. Regarding oncogenes, cMYC-positive cases showed unfavorable OS (P = 0.046). By multivariate analysis, MSH2 and involvement of the fornix were independent predictors for both OS and PFS, whereas tegmentum/velum location and cMYC expression were significantly associated with OS. Although further studies are needed, these results suggest that MMR protein expression, as well as specific deep locations and cMYC expression, may be a novel prognostic and predictive markers for PCNSL. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
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Kim, Jaehee; Ogden, Robert Todd; Kim, Haseong
2013-10-18
Time course gene expression experiments are an increasingly popular method for exploring biological processes. Temporal gene expression profiles provide an important characterization of gene function, as biological systems are both developmental and dynamic. With such data it is possible to study gene expression changes over time and thereby to detect differential genes. Much of the early work on analyzing time series expression data relied on methods developed originally for static data and thus there is a need for improved methodology. Since time series expression is a temporal process, its unique features such as autocorrelation between successive points should be incorporated into the analysis. This work aims to identify genes that show different gene expression profiles across time. We propose a statistical procedure to discover gene groups with similar profiles using a nonparametric representation that accounts for the autocorrelation in the data. In particular, we first represent each profile in terms of a Fourier basis, and then we screen out genes that are not differentially expressed based on the Fourier coefficients. Finally, we cluster the remaining gene profiles using a model-based approach in the Fourier domain. We evaluate the screening results in terms of sensitivity, specificity, FDR and FNR, compare with the Gaussian process regression screening in a simulation study and illustrate the results by application to yeast cell-cycle microarray expression data with alpha-factor synchronization.The key elements of the proposed methodology: (i) representation of gene profiles in the Fourier domain; (ii) automatic screening of genes based on the Fourier coefficients and taking into account autocorrelation in the data, while controlling the false discovery rate (FDR); (iii) model-based clustering of the remaining gene profiles. Using this method, we identified a set of cell-cycle-regulated time-course yeast genes. The proposed method is general and can be potentially used to identify genes which have the same patterns or biological processes, and help facing the present and forthcoming challenges of data analysis in functional genomics.
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2017-01-01
Although in recent years the study of gene expression variation in the absence of genetic or environmental cues or gene expression heterogeneity has intensified considerably, many basic and applied biological fields still remain unaware of how useful the study of gene expression heterogeneity patterns might be for the characterization of biological systems and/or processes. Largely based on the modulator effect chromatin compaction has for gene expression heterogeneity and the extensive changes in chromatin compaction known to occur for specialized cells that are naturally or artificially induced to revert to less specialized states or dedifferentiate, I recently hypothesized that processes that concur with cell dedifferentiation would show an extensive reduction in gene expression heterogeneity. The confirmation of the existence of such trend could be of wide interest because of the biomedical and biotechnological relevance of cell dedifferentiation-based processes, i.e., regenerative development, cancer, human induced pluripotent stem cells, or plant somatic embryogenesis. Here, I report the first empirical evidence consistent with the existence of an extensive reduction in gene expression heterogeneity for processes that concur with cell dedifferentiation by analyzing transcriptome dynamics along forearm regenerative development in Ambystoma mexicanum or axolotl. Also, I briefly discuss on the utility of the study of gene expression heterogeneity dynamics might have for the characterization of cell dedifferentiation-based processes, and the engineering of tools that afforded better monitoring and modulating such processes. Finally, I reflect on how a transitional reduction in gene expression heterogeneity for dedifferentiated cells can promote a long-term increase in phenotypic heterogeneity following cell dedifferentiation with potential adverse effects for biomedical and biotechnological applications. PMID:29134148
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Scheili, A; Rodriguez, Manuel J; Sadiq, R
2015-11-01
The aim of this study was to produce a drinking water assessment tool for operators of small distribution systems. A drinking water quality index (DWQI) was developed and applied to small systems based on the water quality index of the Canadian Council of Ministers of Environment. The drinking water quality index was adapted to specific needs by creating four drinking water quality scenarios. First, the temporal and spatial dimensions of drinking water quality variability were taken into account. The DWQI was designed to express global drinking water quality according to different monitoring frequencies. Daily, monthly, and seasonal assessment was also considered. With the data made available, it was possible to use the index as a spatial monitoring tool and express water quality in different points in the distribution system. Moreover, adjustments were made to prioritize the type of contaminant to monitor. For instance, monitoring contaminants with acute health effects led to a scenario based on daily measures, including easily accessible and affordable water quality parameters. On the other hand, contaminants with chronic effects, especially disinfection by-products, were considered in a seasonal monitoring scenario where disinfection by-product reference values were redefined according to their seasonal variability. A sensitivity analysis was also carried out to validate the index. Globally, the DWQI developed is adapted to the needs of small systems. In fact, expressing drinking water quality using the DWQI contributes to the identification of problematic periods and segments in the distribution system. Further work may include this index in the development of a customized decision-making tool for small-system operators and managers.
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Luu, Van-Trinh; Moon, Hye Yun; Hwang, Jee Youn; Kang, Bo-Kyu; Kang, Hyun Ah
2017-08-01
Nervous necrosis virus (NNV) causes viral encephalopathy and retinopathy, a devastating disease of many species of cultured marine fish worldwide. In this study, we used the dimorphic non-pathogenic yeast Yarrowia lipolytica as a host to express the capsid protein of red-spotted grouper nervous necrosis virus (RGNNV-CP) and evaluated its potential as a platform for vaccine production. An initial attempt was made to express the codon-optimized synthetic genes encoding intact and N-terminal truncated forms of RGNNV-CP under the strong constitutive TEF1 promoter using autonomously replicating sequence (ARS)-based vectors. The full-length recombinant capsid proteins expressed in Y. lipolytica were detected not only as monomers and but also as trimers, which is a basic unit for formation of NNV virus-like particles (VLPs). Oral immunization of mice with whole recombinant Y. lipolytica harboring the ARS-based plasmids was shown to efficiently induce the formation of IgG against RGNNV-CP. To increase the number of integrated copies of the RGNNV-CP expression cassette, a set of 26S ribosomal DNA-based multiple integrative vectors was constructed in combination with a series of defective Ylura3 with truncated promoters as selection markers, resulting in integrants harboring up to eight copies of the RGNNV-CP cassette. Sucrose gradient centrifugation and transmission electron microscopy of this high-copy integrant were carried out to confirm the expression of RGNNV-CPs as VLPs. This is the first report on efficient expression of viral capsid proteins as VLPs in Y. lipolytica, demonstrating high potential for the Y. lipolytica expression system as a platform for recombinant vaccine production based on VLPs.
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Lv, Yufeng; Wei, Wenhao; Huang, Zhong; Chen, Zhichao; Fang, Yuan; Pan, Lili; Han, Xueqiong; Xu, Zihai
2018-06-20
The aim of this study was to develop a novel long non-coding RNA (lncRNA) expression signature to accurately predict early recurrence for patients with hepatocellular carcinoma (HCC) after curative resection. Using expression profiles downloaded from The Cancer Genome Atlas database, we identified multiple lncRNAs with differential expression between early recurrence (ER) group and non-early recurrence (non-ER) group of HCC. Least absolute shrinkage and selection operator (LASSO) for logistic regression models were used to develop a lncRNA-based classifier for predicting ER in the training set. An independent test set was used to validated the predictive value of this classifier. Futhermore, a co-expression network based on these lncRNAs and its highly related genes was constructed and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of genes in the network were performed. We identified 10 differentially expressed lncRNAs, including 3 that were upregulated and 7 that were downregulated in ER group. The lncRNA-based classifier was constructed based on 7 lncRNAs (AL035661.1, PART1, AC011632.1, AC109588.1, AL365361.1, LINC00861 and LINC02084), and its accuracy was 0.83 in training set, 0.87 in test set and 0.84 in total set. And ROC curve analysis showed the AUROC was 0.741 in training set, 0.824 in the test set and 0.765 in total set. A functional enrichment analysis suggested that the genes of which is highly related to 4 lncRNAs were involved in immune system. This 7-lncRNA expression profile can effectively predict the early recurrence after surgical resection for HCC. This article is protected by copyright. All rights reserved.
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Genetic and epigenetic control of gene expression by CRISPR–Cas systems
Lo, Albert; Qi, Lei
2017-01-01
The discovery and adaption of bacterial clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) systems has revolutionized the way researchers edit genomes. Engineering of catalytically inactivated Cas variants (nuclease-deficient or nuclease-deactivated [dCas]) combined with transcriptional repressors, activators, or epigenetic modifiers enable sequence-specific regulation of gene expression and chromatin state. These CRISPR–Cas-based technologies have contributed to the rapid development of disease models and functional genomics screening approaches, which can facilitate genetic target identification and drug discovery. In this short review, we will cover recent advances of CRISPR–dCas9 systems and their use for transcriptional repression and activation, epigenome editing, and engineered synthetic circuits for complex control of the mammalian genome. PMID:28649363
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2014-11-03
Members of the media watch a demonstration of the Regolith Advanced Surface System Operations Robot, or RASSOR, during a media event at the automated landing and hazard avoidance technology, or ALHAT, hazard field at the north end of the Shuttle Landing Facility at NASA's Kennedy Space Center in Florida. Tom Engler, center, in the suit, deputy director of Kennedy's Center Planning and Development, announced Moon Express Inc., of Moffett Field, California is selected to utilize Kennedy facilities for NASA's Lunar Cargo Transportation and Landing by Soft Touchdown, or Lunar CATALYST, initiative. Moon Express is developing a lander with capabilities that will enable delivery of payloads to the surface of the moon, as well as new science and exploration missions of interest to NASA and scientific and academic communities. Moon Express will base its activities at Kennedy and utilize the Morpheus ALHAT field and a hangar nearby for CATALYST testing. The Advanced Exploration Systems Division of NASA's Human Exploration and Operations Mission Directorate manages Lunar CATALYST.
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[Eukaryotic expression and application of HCV Hebei strain E2 extracellular core region].
Ye, Chuantao; Bian, Peiyu; Weng, Daihui; Zhang, Hui; Yang, Jing; Zhang, Ying; Lei, Yingfeng; Jia, Zhansheng
2016-06-01
Objective To express core region of HCV1b (Hebei strain) E2 protein (E2c) by eukaryotic system, and establish the detection method of specific anti-HCV E2 antibody in the sera from hepatitis C patients. Methods Based on the literature, the E2c gene was modified from the HCV1b gene and synthesized via overlapping PCR. Thereafter, the E2c gene including tissue-type plasminogen activator (tPA) signal peptide was cloned into the pCI-neo eukaryotic expression vector, and the product was named pCI-tpa-1bE2c. After HEK293T cells were transfected with pCI-tpa-1bE2c, the supernatant was collected, condensed and purified. Its specificity was identified by Western blotting. Galanthus nivalis agglutinin (GNA)-based ELISA was used to detect the antibody against HCVE2 in the sera from hepatitis C patients. Results Modified HCV E2c protein was successfully expressed in HEK293T cells and the GNA-based ELISA was developed for detecting the antibody against HCV E2 in the sera from hepatitis C patients. Conclusion HCV-1bE2c protein can be effectively expressed in HEK293T cells and applied clinically.
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Neurochemical background and approaches in the understanding of motion sickness
NASA Technical Reports Server (NTRS)
Kohl, R. L.
1982-01-01
The problems and nature of space motion sickness were defined. The neurochemical and neurophysiological bases of vestibular system function and of the expression of motion sickness wre reviewed. Emphasis was given to the elucidation of the neuropharmacological mechanisms underlying the effects of scopolamine and amphetamine on motion sickness. Characterization of the ascending reticular activating system and the limbic system provided clues to the etiology of the side effects of scopolamine. The interrelationship between central cholinergic pathways and the peripheral (autonomic) expression of motion sickness was described. A correlation between the stress of excessive motion and a variety of hormonal responses to that stress was also detailed. The cholinergic system is involved in the efferent modulation of the vestibular hair cells, as an afferent modulator of the vestibular nuclei, in the activation of cortical and limbic structures, in the expression of motion sickness symptoms and most likely underscores a number of the hormonal changes that occur in stressful motion environments. The role of lecithin in the regulation of the levels of neurotransmitters was characterized as a possible means by which cholinergic neurochemistry can be modulated.
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Li, Jingtao; Sun, Xinhua; Liu, Yanzhi; Wang, Xueliang; Zhang, Hao; Pan, Hongyu
2017-01-01
Plant productivity is limited by salinity stress, both in natural and agricultural systems. Identification of salt stress-related genes from halophyte can provide insights into mechanisms of salt stress tolerance in plants. Atriplex canescens is a xero-halophyte that exhibits optimum growth in the presence of 400 mM NaCl. A cDNA library derived from highly salt-treated A. canescens plants was constructed based on a yeast expression system. A total of 53 transgenic yeast clones expressing enhanced salt tolerance were selected from 105 transformants. Their plasmids were sequenced and the gene characteristics were annotated using a BLASTX search. Retransformation of yeast cells with the selected plasmids conferred salt tolerance to the resulting transformants. The expression patterns of 28 of these stress-related genes were further investigated in A. canescens leaves by quantitative reverse transcription-PCR. In this study, we provided a rapid and robust assay system for large-scale screening of genes for varied abiotic stress tolerance with high efficiency in A. canescens. PMID:29149055
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FPGA-accelerated algorithm for the regular expression matching system
NASA Astrophysics Data System (ADS)
Russek, P.; Wiatr, K.
2015-01-01
This article describes an algorithm to support a regular expressions matching system. The goal was to achieve an attractive performance system with low energy consumption. The basic idea of the algorithm comes from a concept of the Bloom filter. It starts from the extraction of static sub-strings for strings of regular expressions. The algorithm is devised to gain from its decomposition into parts which are intended to be executed by custom hardware and the central processing unit (CPU). The pipelined custom processor architecture is proposed and a software algorithm explained accordingly. The software part of the algorithm was coded in C and runs on a processor from the ARM family. The hardware architecture was described in VHDL and implemented in field programmable gate array (FPGA). The performance results and required resources of the above experiments are given. An example of target application for the presented solution is computer and network security systems. The idea was tested on nearly 100,000 body-based viruses from the ClamAV virus database. The solution is intended for the emerging technology of clusters of low-energy computing nodes.
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Scalable and expressive medical terminologies.
Mays, E; Weida, R; Dionne, R; Laker, M; White, B; Liang, C; Oles, F J
1996-01-01
The K-Rep system, based on description logic, is used to represent and reason with large and expressive controlled medical terminologies. Expressive concept descriptions incorporate semantically precise definitions composed using logical operators, together with important non-semantic information such as synonyms and codes. Examples are drawn from our experience with K-Rep in modeling the InterMed laboratory terminology and also developing a large clinical terminology now in production use at Kaiser-Permanente. System-level scalability of performance is achieved through an object-oriented database system which efficiently maps persistent memory to virtual memory. Equally important is conceptual scalability-the ability to support collaborative development, organization, and visualization of a substantial terminology as it evolves over time. K-Rep addresses this need by logically completing concept definitions and automatically classifying concepts in a taxonomy via subsumption inferences. The K-Rep system includes a general-purpose GUI environment for terminology development and browsing, a custom interface for formulary term maintenance, a C+2 application program interface, and a distributed client-server mode which provides lightweight clients with efficient run-time access to K-Rep by means of a scripting language.
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Shimono, Kazumi; Goto, Mie; Kikukawa, Takashi; Miyauchi, Seiji; Shirouzu, Mikako; Kamo, Naoki; Yokoyama, Shigeyuki
2009-10-01
Cell-free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis-based Escherichia coli cell-free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light-driven proton pump bacteriorhodopsin, consisting of seven transmembrane alpha-helices. The cell-free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lipid, phosphatidylcholine from egg yolk, in only the reaction mixture. By examining a variety of detergents, we found that the combination of a steroid detergent (digitonin, cholate, or CHAPS) and egg phosphatidylcholine yielded a large amount (0.3-0.7 mg/mL reaction mixture) of the fully functional bacteriorhodopsin. We also analyzed the process of functional expression in our system. The synthesized polypeptide was well protected from aggregation by the detergent-lipid mixed micelles and/or lipid disks, and was integrated into liposomes upon detergent removal by dialysis. This approach might be useful for the high yield production of functional membrane proteins.
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Algoe, Sara B; Way, Baldwin M
2014-12-01
Oxytocin is thought to play a central role in promoting close social bonds via influence on social interactions. The current investigation targeted interactions involving expressed gratitude between members of romantic relationships because recent evidence suggests gratitude and its expression provides behavioral and psychological 'glue' to bind individuals closer together. Specifically, we took a genetic approach to test the hypothesis that social interactions involving expressed gratitude would be associated with variation in a gene, CD38, which has been shown to affect oxytocin secretion. A polymorphism (rs6449182) that affects CD38 expression was significantly associated with global relationship satisfaction, perceived partner responsiveness and positive emotions (particularly love) after lab-based interactions, observed behavioral expression of gratitude toward a romantic partner in the lab, and frequency of expressed gratitude in daily life. A separate polymorphism in CD38 (rs3796863) previously associated with plasma oxytocin levels and social engagement was also associated with perceived responsiveness in the benefactor after an expression of gratitude. The combined influence of the two polymorphisms was associated with a broad range of gratitude-related behaviors and feelings. The consistent pattern of findings suggests that the oxytocin system is associated with solidifying the glue that binds adults into meaningful and important relationships. © The Author (2014). Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.
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Gacerez, Albert T; Hua, Casey K; Ackerman, Margaret E; Sentman, Charles L
2018-05-01
B7H6 is emerging as a promising tumor antigen that is known to be expressed on a wide array of tumors and is reported to stimulate anti-tumor responses from the immune system. As such, B7H6 presents a good target for tumor-specific immunotherapies. B7H6-specific chimeric antigen receptors (CAR) based on a murine antibody showed successful targeting and elimination of tumors expressing B7H6. However, mouse single chain variable fragments (scFvs) have the potential to induce host anti-CAR responses that may limit efficacy, so human scFvs specific for B7H6 were selected by yeast surface display. In this study, we validate the functionality of these human scFvs when formatted into chimeric antigen receptors. The data indicate that T cells expressing these B7H6-specific human scFvs as CARs induced potent anti-tumor activity in vitro and in vivo against tumors expressing high amounts of B7H6. Importantly, these human scFv-based CARs are sensitive to changes in B7H6 expression which may potentially spare non-tumor cells that express B7H6 and provides the foundation for future clinical development.
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Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector
Kabadi, Ami M.; Ousterout, David G.; Hilton, Isaac B.; Gersbach, Charles A.
2014-01-01
Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types. PMID:25122746
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The development of automated behavior analysis software
NASA Astrophysics Data System (ADS)
Jaana, Yuki; Prima, Oky Dicky A.; Imabuchi, Takashi; Ito, Hisayoshi; Hosogoe, Kumiko
2015-03-01
The measurement of behavior for participants in a conversation scene involves verbal and nonverbal communications. The measurement validity may vary depending on the observers caused by some aspects such as human error, poorly designed measurement systems, and inadequate observer training. Although some systems have been introduced in previous studies to automatically measure the behaviors, these systems prevent participants to talk in a natural way. In this study, we propose a software application program to automatically analyze behaviors of the participants including utterances, facial expressions (happy or neutral), head nods, and poses using only a single omnidirectional camera. The camera is small enough to be embedded into a table to allow participants to have spontaneous conversation. The proposed software utilizes facial feature tracking based on constrained local model to observe the changes of the facial features captured by the camera, and the Japanese female facial expression database to recognize expressions. Our experiment results show that there are significant correlations between measurements observed by the observers and by the software.
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2014-11-03
CAPE CANAVERAL, Fla. - Rob Mueller, NASA senior technologist in the Surface Systems Office in Kennedy Space Center's Engineering and Technology Directorate, demonstrates the Regolith Advanced Surface System Operations Robot, or RASSOR, during a media event at Kennedy's automated landing and hazard avoidance technology, or ALHAT, hazard field at the north end of the Shuttle Landing Facility. The event was held to announce Moon Express Inc., of Moffett Field, California is selected to utilize Kennedy facilities for NASA's Lunar Cargo Transportation and Landing by Soft Touchdown, or Lunar CATALYST, initiative. Moon Express is developing a lander with capabilities that will enable delivery of payloads to the surface of the moon, as well as new science and exploration missions of interest to NASA and scientific and academic communities. Moon Express will base its activities at Kennedy and utilize the Morpheus ALHAT field and a hangar nearby for CATALYST testing. The Advanced Exploration Systems Division of NASA's Human Exploration and Operations Mission Directorate manages Lunar CATALYST. Photo credit: NASA/Ben Smegelsky
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Overview of the Purification of Recombinant Proteins
Wingfield, Paul T.
2015-01-01
When the first version of this unit was written in 1995 protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches many of which were described and mentioned in this unit and elsewhere in the book. In the interim there has been a shift towards an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein and whether to engineer a self cleavage system or simply leave them on. We will briefly address some of these issues. Also although this overview focuses on E.coli, protein expression and purification from the other commonly used expression systems are mentioned and apart from cell breakage methods, the protein purification methods and strategies are essentially the same. PMID:25829302
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Overview of the purification of recombinant proteins.
Wingfield, Paul T
2015-04-01
When the first version of this unit was written in 1995, protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches, many of which were described and mentioned throughout Current Protocols in Protein Science. In the interim, there has been a shift toward an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein, and whether to engineer a self-cleavage system or simply leave them on. We will briefly address some of these issues. Also, although this overview focuses on E.coli, protein expression and purification, other commonly used expression systems are mentioned and, apart from cell-breakage methods, protein purification methods and strategies are essentially the same. Copyright © 2015 John Wiley & Sons, Inc.
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Production of membrane proteins without cells or detergents.
Rajesh, Sundaresan; Knowles, Timothy; Overduin, Michael
2011-04-30
The production of membrane proteins in cellular systems is besieged by several problems due to their hydrophobic nature which often causes misfolding, protein aggregation and cytotoxicity, resulting in poor yields of stable proteins. Cell-free expression has emerged as one of the most versatile alternatives for circumventing these obstacles by producing membrane proteins directly into designed hydrophobic environments. Efficient optimisation of expression and solubilisation conditions using a variety of detergents, membrane mimetics and lipids has yielded structurally and functionally intact membrane proteins, with yields several fold above the levels possible from cell-based systems. Here we review recently developed techniques available to produce functional membrane proteins, and discuss amphipols, nanodisc and styrene maleic acid lipid particle (SMALP) technologies that can be exploited alongside cell-free expression of membrane proteins. Copyright © 2010 Elsevier B.V. All rights reserved.
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NASA Technical Reports Server (NTRS)
Havelund, Klaus; Smith, Margaret H.; Barringer, Howard; Groce, Alex
2012-01-01
LogScope is a software package for analyzing log files. The intended use is for offline post-processing of such logs, after the execution of the system under test. LogScope can, however, in principle, also be used to monitor systems online during their execution. Logs are checked against requirements formulated as monitors expressed in a rule-based specification language. This language has similarities to a state machine language, but is more expressive, for example, in its handling of data parameters. The specification language is user friendly, simple, and yet expressive enough for many practical scenarios. The LogScope software was initially developed to specifically assist in testing JPL s Mars Science Laboratory (MSL) flight software, but it is very generic in nature and can be applied to any application that produces some form of logging information (which almost any software does).
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Zheng, Nuoyan; Huang, Xiahe; Yin, Bojiao; Wang, Dan; Xie, Qi
2012-12-01
Detection of protein-protein interaction can provide valuable information for investigating the biological function of proteins. The current methods that applied in protein-protein interaction, such as co-immunoprecipitation and pull down etc., often cause plenty of working time due to the burdensome cloning and purification procedures. Here we established a system that characterization of protein-protein interaction was accomplished by co-expression and simply purification of target proteins from one expression cassette within E. coli system. We modified pET vector into co-expression vector pInvivo which encoded PPV NIa protease, two cleavage site F and two multiple cloning sites that flanking cleavage sites. The target proteins (for example: protein A and protein B) were inserted at multiple cloning sites and translated into polyprotein in the order of MBP tag-protein A-site F-PPV NIa protease-site F-protein B-His(6) tag. PPV NIa protease carried out intracellular cleavage along expression, then led to the separation of polyprotein components, therefore, the interaction between protein A-protein B can be detected through one-step purification and analysis. Negative control for protein B was brought into this system for monitoring interaction specificity. We successfully employed this system to prove two cases of reported protien-protein interaction: RHA2a/ANAC and FTA/FTB. In conclusion, a convenient and efficient system has been successfully developed for detecting protein-protein interaction.
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An Evaluation Methodology for Natural Language Processing Systems
1992-12-01
8217DT".3 "_Griffiss Air Force Base U , . d J _!::! • •, .>:--------. ..._ _. ...........• E v¢ .................. ......... Av,:iabihty Codes Avv L...each " trial " or evaluation item. This approach to assessing reliability has some similarity to the reliability study per- formed by Hix and Schulman for...the clinic . Criteria: Demonstrated understanding that the object or entity expressed by the first noun benefits from the object expressed by the second
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Kuriakose, Selvia; Lahiri, Uttama
2015-07-01
Individuals with Autism are characterized by deficits in socialization and communication. In recent years several assistive technologies, e.g., Virtual Reality (VR), have been investigated to address the socialization deficits in these individuals. Presently available VR-based systems address various aspects of social communication in an isolated manner and without monitoring one's affective state such as, anxiety. However, in conventional observation-based therapy, a therapist adjusts the intervention paradigm by monitoring one's anxiety level. But, often these individuals have an inherent inability to explicitly express their anxiety thereby inducing limitations on conventional techniques. Physiological signals being continuously available and not directly impacted by these communication difficulties can be alternatively used as markers of one's anxiety level. In our research we aim at designing a Virtual-reality bAsed Social-communication Task (VAST) system that can address the various aspects of social communication, e.g., social context, subtle social cues, emotional expression, etc., in a cumulative and structured way. In addition, we augment this with a capability to use one's physiological signals as markers of one's anxiety level. In our preliminary feasibility study we investigate the potential of VAST to cause variations in one's performance and anxiety level that can be mapped from one's physiological indices.
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Regulation of melanopsin expression.
Hannibal, Jens
2006-01-01
Circadian rhythms in mammals are adjusted daily to the environmental day/night cycle by photic input via the retinohypothalamic tract (RHT). Retinal ganglion cells (RGCs) of the RHT constitute a separate light-detecting system in the mammalian retina used for irradiance detection and for transmission to the circadian system and other non-imaging forming processes in the brain. The RGCs of the RHT are intrinsically photosensitive due to the expression of melanopsin, an opsin-like photopigment. This notion is based on anatomical and functional data and on studies of mice lacking melanopsin. Furthermore, heterologous expression of melanopsin in non-neuronal mammalian cell lines was found sufficient to render these cells photosensitive. Even though solid evidence regarding the function of melanopsin exists, little is known about the regulation of melanopsin gene expression. Studies in albino Wistar rats showed that the expression of melanopsin is diurnal at both the mRNA and protein levels. The diurnal changes in melanopsin expression seem, however, to be overridden by prolonged exposure to light or darkness. Significant increase in melanopsin expression was observed from the first day in constant darkness and the expression continued to increase during prolonged exposure in constant darkness. Prolonged exposure to constant light, on the other hand, decreased melanopsin expression to an almost undetectable level after 5 days of constant light. The induction of melanopsin by darkness was even more pronounced if darkness was preceded by light suppression for 5 days. These observations show that dual mechanisms regulate melanopsin gene expression and that the intrinsic light-responsive RGCs in the albino Wistar rat adapt their expression of melanopsin to environmental light and darkness.
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Forecasting overhaul or replacement intervals based on estimated system failure intensity
NASA Astrophysics Data System (ADS)
Gannon, James M.
1994-12-01
System reliability can be expressed in terms of the pattern of failure events over time. Assuming a nonhomogeneous Poisson process and Weibull intensity function for complex repairable system failures, the degree of system deterioration can be approximated. Maximum likelihood estimators (MLE's) for the system Rate of Occurrence of Failure (ROCOF) function are presented. Evaluating the integral of the ROCOF over annual usage intervals yields the expected number of annual system failures. By associating a cost of failure with the expected number of failures, budget and program policy decisions can be made based on expected future maintenance costs. Monte Carlo simulation is used to estimate the range and the distribution of the net present value and internal rate of return of alternative cash flows based on the distributions of the cost inputs and confidence intervals of the MLE's.
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Recognition of chemical entities: combining dictionary-based and grammar-based approaches.
Akhondi, Saber A; Hettne, Kristina M; van der Horst, Eelke; van Mulligen, Erik M; Kors, Jan A
2015-01-01
The past decade has seen an upsurge in the number of publications in chemistry. The ever-swelling volume of available documents makes it increasingly hard to extract relevant new information from such unstructured texts. The BioCreative CHEMDNER challenge invites the development of systems for the automatic recognition of chemicals in text (CEM task) and for ranking the recognized compounds at the document level (CDI task). We investigated an ensemble approach where dictionary-based named entity recognition is used along with grammar-based recognizers to extract compounds from text. We assessed the performance of ten different commercial and publicly available lexical resources using an open source indexing system (Peregrine), in combination with three different chemical compound recognizers and a set of regular expressions to recognize chemical database identifiers. The effect of different stop-word lists, case-sensitivity matching, and use of chunking information was also investigated. We focused on lexical resources that provide chemical structure information. To rank the different compounds found in a text, we used a term confidence score based on the normalized ratio of the term frequencies in chemical and non-chemical journals. The use of stop-word lists greatly improved the performance of the dictionary-based recognition, but there was no additional benefit from using chunking information. A combination of ChEBI and HMDB as lexical resources, the LeadMine tool for grammar-based recognition, and the regular expressions, outperformed any of the individual systems. On the test set, the F-scores were 77.8% (recall 71.2%, precision 85.8%) for the CEM task and 77.6% (recall 71.7%, precision 84.6%) for the CDI task. Missed terms were mainly due to tokenization issues, poor recognition of formulas, and term conjunctions. We developed an ensemble system that combines dictionary-based and grammar-based approaches for chemical named entity recognition, outperforming any of the individual systems that we considered. The system is able to provide structure information for most of the compounds that are found. Improved tokenization and better recognition of specific entity types is likely to further improve system performance.
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Recognition of chemical entities: combining dictionary-based and grammar-based approaches
2015-01-01
Background The past decade has seen an upsurge in the number of publications in chemistry. The ever-swelling volume of available documents makes it increasingly hard to extract relevant new information from such unstructured texts. The BioCreative CHEMDNER challenge invites the development of systems for the automatic recognition of chemicals in text (CEM task) and for ranking the recognized compounds at the document level (CDI task). We investigated an ensemble approach where dictionary-based named entity recognition is used along with grammar-based recognizers to extract compounds from text. We assessed the performance of ten different commercial and publicly available lexical resources using an open source indexing system (Peregrine), in combination with three different chemical compound recognizers and a set of regular expressions to recognize chemical database identifiers. The effect of different stop-word lists, case-sensitivity matching, and use of chunking information was also investigated. We focused on lexical resources that provide chemical structure information. To rank the different compounds found in a text, we used a term confidence score based on the normalized ratio of the term frequencies in chemical and non-chemical journals. Results The use of stop-word lists greatly improved the performance of the dictionary-based recognition, but there was no additional benefit from using chunking information. A combination of ChEBI and HMDB as lexical resources, the LeadMine tool for grammar-based recognition, and the regular expressions, outperformed any of the individual systems. On the test set, the F-scores were 77.8% (recall 71.2%, precision 85.8%) for the CEM task and 77.6% (recall 71.7%, precision 84.6%) for the CDI task. Missed terms were mainly due to tokenization issues, poor recognition of formulas, and term conjunctions. Conclusions We developed an ensemble system that combines dictionary-based and grammar-based approaches for chemical named entity recognition, outperforming any of the individual systems that we considered. The system is able to provide structure information for most of the compounds that are found. Improved tokenization and better recognition of specific entity types is likely to further improve system performance. PMID:25810767
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Shi, Chenguang; Wang, Fei; Salous, Sana; Zhou, Jianjiang
2017-10-18
In this study, the modified Cramér-Rao lower bounds (MCRLBs) on the joint estimation of target position and velocity is investigated for a universal mobile telecommunication system (UMTS)-based passive multistatic radar system with antenna arrays. First, we analyze the log-likelihood redfunction of the received signal for a complex Gaussian extended target. Then, due to the non-deterministic transmitted data symbols, the analytically closed-form expressions of the MCRLBs on the Cartesian coordinates of target position and velocity are derived for a multistatic radar system with N t UMTS-based transmit station of L t antenna elements and N r receive stations of L r antenna elements. With the aid of numerical simulations, it is shown that increasing the number of receiving elements in each receive station can reduce the estimation errors. In addition, it is demonstrated that the MCRLB is not only a function of signal-to-noise ratio (SNR), the number of receiving antenna elements and the properties of the transmitted UMTS signals, but also a function of the relative geometric configuration between the target and the multistatic radar system.The analytical expressions for MCRLB will open up a new dimension for passive multistatic radar system by aiding the optimal placement of receive stations to improve the target parameter estimation performance.
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Wang, Fei; Salous, Sana; Zhou, Jianjiang
2017-01-01
In this study, the modified Cramér-Rao lower bounds (MCRLBs) on the joint estimation of target position and velocity is investigated for a universal mobile telecommunication system (UMTS)-based passive multistatic radar system with antenna arrays. First, we analyze the log-likelihood redfunction of the received signal for a complex Gaussian extended target. Then, due to the non-deterministic transmitted data symbols, the analytically closed-form expressions of the MCRLBs on the Cartesian coordinates of target position and velocity are derived for a multistatic radar system with Nt UMTS-based transmit station of Lt antenna elements and Nr receive stations of Lr antenna elements. With the aid of numerical simulations, it is shown that increasing the number of receiving elements in each receive station can reduce the estimation errors. In addition, it is demonstrated that the MCRLB is not only a function of signal-to-noise ratio (SNR), the number of receiving antenna elements and the properties of the transmitted UMTS signals, but also a function of the relative geometric configuration between the target and the multistatic radar system.The analytical expressions for MCRLB will open up a new dimension for passive multistatic radar system by aiding the optimal placement of receive stations to improve the target parameter estimation performance. PMID:29057805
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Howe, Kevin; Karsi, Attila; Germon, Pierre; Wills, Robert W; Lawrence, Mark L; Bailey, Richard H
2010-07-23
Salmonellosis may be a food safety problem when raw food products are mishandled and not fully cooked. In previous work, we developed bioluminescent Salmonella enterica serotypes using a plasmid-based reporting system that can be used for real-time monitoring of the pathogen's growth on food products in short term studies. In this study, we report the use of a Tn7-based transposon system for subcloning of luxCDABE genes into the chromosome of eleven Salmonella enterica serotypes isolated from the broiler production continuum. We found that the lux operon is constitutively expressed from the chromosome post-transposition and the lux cassette is stable without external pressure, i.e. antibiotic selection, for all Salmonella enterica serotypes used. Bioluminescence expression is based on an active electron transport chain and is directly related with metabolic activity. This relationship was quantified by measuring bioluminescence against a temperature gradient in aqueous solution using a luminometer. In addition, bioluminescent monitoring of two serotypes confirmed that our chicken skin model has the potential to be used to evaluate pathogen mitigation strategies. This study demonstrated that our new stable reporting system eliminates bioluminescence variation due to plasmid instability and provides a reliable real-time experimental system to study application of preventive measures for Salmonella on food products in real-time for both short and long term studies.
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CEBS object model for systems biology data, SysBio-OM.
Xirasagar, Sandhya; Gustafson, Scott; Merrick, B Alex; Tomer, Kenneth B; Stasiewicz, Stanley; Chan, Denny D; Yost, Kenneth J; Yates, John R; Sumner, Susan; Xiao, Nianqing; Waters, Michael D
2004-09-01
To promote a systems biology approach to understanding the biological effects of environmental stressors, the Chemical Effects in Biological Systems (CEBS) knowledge base is being developed to house data from multiple complex data streams in a systems friendly manner that will accommodate extensive querying from users. Unified data representation via a single object model will greatly aid in integrating data storage and management, and facilitate reuse of software to analyze and display data resulting from diverse differential expression or differential profile technologies. Data streams include, but are not limited to, gene expression analysis (transcriptomics), protein expression and protein-protein interaction analysis (proteomics) and changes in low molecular weight metabolite levels (metabolomics). To enable the integration of microarray gene expression, proteomics and metabolomics data in the CEBS system, we designed an object model, Systems Biology Object Model (SysBio-OM). The model is comprehensive and leverages other open source efforts, namely the MicroArray Gene Expression Object Model (MAGE-OM) and the Proteomics Experiment Data Repository (PEDRo) object model. SysBio-OM is designed by extending MAGE-OM to represent protein expression data elements (including those from PEDRo), protein-protein interaction and metabolomics data. SysBio-OM promotes the standardization of data representation and data quality by facilitating the capture of the minimum annotation required for an experiment. Such standardization refines the accuracy of data mining and interpretation. The open source SysBio-OM model, which can be implemented on varied computing platforms is presented here. A universal modeling language depiction of the entire SysBio-OM is available at http://cebs.niehs.nih.gov/SysBioOM/. The Rational Rose object model package is distributed under an open source license that permits unrestricted academic and commercial use and is available at http://cebs.niehs.nih.gov/cebsdownloads. The database and interface are being built to implement the model and will be available for public use at http://cebs.niehs.nih.gov.
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Candidate innate immune system gene expression in the ecological model Daphnia
Decaestecker, Ellen; Labbé, Pierrick; Ellegaard, Kirsten; Allen, Judith E.; Little, Tom J.
2011-01-01
The last ten years have witnessed increasing interest in host–pathogen interactions involving invertebrate hosts. The invertebrate innate immune system is now relatively well characterised, but in a limited range of genetic model organisms and under a limited number of conditions. Immune systems have been little studied under real-world scenarios of environmental variation and parasitism. Thus, we have investigated expression of candidate innate immune system genes in the water flea Daphnia, a model organism for ecological genetics, and whose capacity for clonal reproduction facilitates an exceptionally rigorous control of exposure dose or the study of responses at many time points. A unique characteristic of the particular Daphnia clones and pathogen strain combinations used presently is that they have been shown to be involved in specific host–pathogen coevolutionary interactions in the wild. We choose five genes, which are strong candidates to be involved in Daphnia–pathogen interactions, given that they have been shown to code for immune effectors in related organisms. Differential expression of these genes was quantified by qRT-PCR following exposure to the bacterial pathogen Pasteuria ramosa. Constitutive expression levels differed between host genotypes, and some genes appeared to show correlated expression. However, none of the genes appeared to show a major modification of expression level in response to Pasteuria exposure. By applying knowledge from related genetic model organisms (e.g. Drosophila) to models for the study of evolutionary ecology and coevolution (i.e. Daphnia), the candidate gene approach is temptingly efficient. However, our results show that detection of only weak patterns is likely if one chooses target genes for study based on previously identified genome sequences by comparison to homologues from other related organisms. Future work on the Daphnia–Pasteuria system will need to balance a candidate gene approach with more comprehensive approaches to de novo identify immune system genes specific to the Daphnia–Pasteuria interaction. PMID:21550363
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Candidate innate immune system gene expression in the ecological model Daphnia.
Decaestecker, Ellen; Labbé, Pierrick; Ellegaard, Kirsten; Allen, Judith E; Little, Tom J
2011-10-01
The last ten years have witnessed increasing interest in host-pathogen interactions involving invertebrate hosts. The invertebrate innate immune system is now relatively well characterised, but in a limited range of genetic model organisms and under a limited number of conditions. Immune systems have been little studied under real-world scenarios of environmental variation and parasitism. Thus, we have investigated expression of candidate innate immune system genes in the water flea Daphnia, a model organism for ecological genetics, and whose capacity for clonal reproduction facilitates an exceptionally rigorous control of exposure dose or the study of responses at many time points. A unique characteristic of the particular Daphnia clones and pathogen strain combinations used presently is that they have been shown to be involved in specific host-pathogen coevolutionary interactions in the wild. We choose five genes, which are strong candidates to be involved in Daphnia-pathogen interactions, given that they have been shown to code for immune effectors in related organisms. Differential expression of these genes was quantified by qRT-PCR following exposure to the bacterial pathogen Pasteuria ramosa. Constitutive expression levels differed between host genotypes, and some genes appeared to show correlated expression. However, none of the genes appeared to show a major modification of expression level in response to Pasteuria exposure. By applying knowledge from related genetic model organisms (e.g. Drosophila) to models for the study of evolutionary ecology and coevolution (i.e. Daphnia), the candidate gene approach is temptingly efficient. However, our results show that detection of only weak patterns is likely if one chooses target genes for study based on previously identified genome sequences by comparison to homologues from other related organisms. Future work on the Daphnia-Pasteuria system will need to balance a candidate gene approach with more comprehensive approaches to de novo identify immune system genes specific to the Daphnia-Pasteuria interaction. Copyright © 2011 Elsevier Ltd. All rights reserved.
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MindDigger: Feature Identification and Opinion Association for Chinese Movie Reviews
NASA Astrophysics Data System (ADS)
Zhao, Lili; Li, Chunping
In this paper, we present a prototype system called MindDigger, which can be used to analyze the opinions in Chinese movie reviews. Different from previous research that employed techniques on product reviews, we focus on Chinese movie reviews, in which opinions are expressed in subtle and varied ways. The system designed in this work aims to extract the opinion expressions and assign them to the corresponding features. The core tasks include feature and opinion extraction, and feature-opinion association. To deal with Chinese effectively, several novel approaches based on syntactic analysis are proposed in this paper. Running results show the performance is satisfactory.