Universal light-switchable gene promoter system

DOEpatents

Quail, Peter H.; Huq, Enamul; Tepperman, James; Sato, Sae

2005-02-22

An artificial promoter system that can be fused upstream of any desired gene enabling reversible induction or repression of the expression of the gene at will in any suitable host cell or organisms by light is described. The design of the system is such that a molecule of the plant photoreceptor phytochrome is targeted to the specific DNA binding site in the promoter by a protein domain that is fused to the phytochrome and that specifically recognizes this binding site. This bound phytochrome, upon activation by light, recruits a second fusion protein consisting of a protein that binds to phytochrome only upon light activation and a transcriptional activation domain that activates expression of the gene downstream of the promoter.

Engine out of the Chassis: Cell-Free Protein Synthesis and its Uses

PubMed Central

Rosenblum, Gabriel; Cooperman, Barry S.

2013-01-01

The translation machinery is the engine of life. Extracting the cytoplasmic milieu from a cell affords a lysate capable of producing proteins in concentrations reaching tens of micromolar. Such lysates, derivable from a variety of cells, allow the facile addition and subtraction of components that are directly or indirectly related to the translation machinery and/or the over-expressed protein. The flexible nature of such cell-free expression systems, when coupled with high throughput monitoring, can be especially suitable for protein engineering studies, allowing one to bypass multiple steps typically required using conventional in vivo protein expression. PMID:24161673

Conformational antibody binding to a native, cell-free expressed GPCR in block copolymer membranes.

PubMed

de Hoog, Hans-Peter M; Lin JieRong, Esther M; Banerjee, Sourabh; Décaillot, Fabien M; Nallani, Madhavan

2014-01-01

G-protein coupled receptors (GPCRs) play a key role in physiological processes and are attractive drug targets. Their biophysical characterization is, however, highly challenging because of their innate instability outside a stabilizing membrane and the difficulty of finding a suitable expression system. We here show the cell-free expression of a GPCR, CXCR4, and its direct embedding in diblock copolymer membranes. The polymer-stabilized CXCR4 is readily immobilized onto biosensor chips for label-free binding analysis. Kinetic characterization using a conformationally sensitive antibody shows the receptor to exist in the correctly folded conformation, showing binding behaviour that is commensurate with heterologously expressed CXCR4.

Conformational Antibody Binding to a Native, Cell-Free Expressed GPCR in Block Copolymer Membranes

PubMed Central

de Hoog, Hans-Peter M.; Lin JieRong, Esther M.; Banerjee, Sourabh; Décaillot, Fabien M.; Nallani, Madhavan

2014-01-01

G-protein coupled receptors (GPCRs) play a key role in physiological processes and are attractive drug targets. Their biophysical characterization is, however, highly challenging because of their innate instability outside a stabilizing membrane and the difficulty of finding a suitable expression system. We here show the cell-free expression of a GPCR, CXCR4, and its direct embedding in diblock copolymer membranes. The polymer-stabilized CXCR4 is readily immobilized onto biosensor chips for label-free binding analysis. Kinetic characterization using a conformationally sensitive antibody shows the receptor to exist in the correctly folded conformation, showing binding behaviour that is commensurate with heterologously expressed CXCR4. PMID:25329156

Fungal Gene Expression on Demand: an Inducible, Tunable, and Metabolism-Independent Expression System for Aspergillus niger▿†

PubMed Central

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

Efficient scheme for parametric fitting of data in arbitrary dimensions.

PubMed

Pang, Ning-Ning; Tzeng, Wen-Jer; Kao, Hisen-Ching

2008-07-01

We propose an efficient scheme for parametric fitting expressed in terms of the Legendre polynomials. For continuous systems, our scheme is exact and the derived explicit expression is very helpful for further analytical studies. For discrete systems, our scheme is almost as accurate as the method of singular value decomposition. Through a few numerical examples, we show that our algorithm costs much less CPU time and memory space than the method of singular value decomposition. Thus, our algorithm is very suitable for a large amount of data fitting. In addition, the proposed scheme can also be used to extract the global structure of fluctuating systems. We then derive the exact relation between the correlation function and the detrended variance function of fluctuating systems in arbitrary dimensions and give a general scaling analysis.

A reporter model to visualize imprinting stability at the Dlk1 locus during mouse development and in pluripotent cells.

PubMed

Swanzey, Emily; Stadtfeld, Matthias

2016-11-15

Genomic imprinting results in the monoallelic expression of genes that encode important regulators of growth and proliferation. Dysregulation of imprinted genes, such as those within the Dlk1-Dio3 locus, is associated with developmental syndromes and specific diseases. Our ability to interrogate causes of imprinting instability has been hindered by the absence of suitable model systems. Here, we describe a Dlk1 knock-in reporter mouse that enables single-cell visualization of allele-specific expression and prospective isolation of cells, simultaneously. We show that this 'imprinting reporter mouse' can be used to detect tissue-specific Dlk1 expression patterns in developing embryos. We also apply this system to pluripotent cell culture and demonstrate that it faithfully indicates DNA methylation changes induced upon cellular reprogramming. Finally, the reporter system reveals the role of elevated oxygen levels in eroding imprinted Dlk1 expression during prolonged culture and in vitro differentiation. The possibility to study allele-specific expression in different contexts makes our reporter system a useful tool to dissect the regulation of genomic imprinting in normal development and disease. © 2016. Published by The Company of Biologists Ltd.

Validation of miRNA genes suitable as reference genes in qPCR analyses of miRNA gene expression in Atlantic salmon (Salmo salar).

PubMed

Johansen, Ilona; Andreassen, Rune

2014-12-23

MicroRNAs (miRNAs) are an abundant class of endogenous small RNA molecules that downregulate gene expression at the post-transcriptional level. They play important roles by regulating genes that control multiple biological processes, and recent years there has been an increased interest in studying miRNA genes and miRNA gene expression. The most common method applied to study gene expression of single genes is quantitative PCR (qPCR). However, before expression of mature miRNAs can be studied robust qPCR methods (miRNA-qPCR) must be developed. This includes identification and validation of suitable reference genes. We are particularly interested in Atlantic salmon (Salmo salar). This is an economically important aquaculture species, but no reference genes dedicated for use in miRNA-qPCR methods has been validated for this species. Our aim was, therefore, to identify suitable reference genes for miRNA-qPCR methods in Salmo salar. We used a systematic approach where we utilized similar studies in other species, some biological criteria, results from deep sequencing of small RNAs and, finally, experimental validation of candidate reference genes by qPCR to identify the most suitable reference genes. Ssa-miR-25-3p was identified as most suitable single reference gene. The best combinations of two reference genes were ssa-miR-25-3p and ssa-miR-455-5p. These two genes were constitutively and stably expressed across many different tissues. Furthermore, infectious salmon anaemia did not seem to affect their expression levels. These genes were amplified with high specificity, good efficiency and the qPCR assays showed a good linearity when applying a simple cybergreen miRNA-PCR method using miRNA gene specific forward primers. We have identified suitable reference genes for miRNA-qPCR in Atlantic salmon. These results will greatly facilitate further studies on miRNA genes in this species. The reference genes identified are conserved genes that are identical in their mature sequence in many aquaculture species. Therefore, they may also be suitable as reference genes in other teleosts. Finally, the systematic approach used in our study successfully identified suitable reference genes, suggesting that this may be a useful strategy to apply in similar validation studies in other aquaculture species.

Production of functional human insulin-like growth factor binding proteins (IGFBPs) using recombinant expression in HEK293 cells.

PubMed

Wanscher, Anne Sofie Molsted; Williamson, Michael; Ebersole, Tasja Wainani; Streicher, Werner; Wikström, Mats; Cazzamali, Giuseppe

2015-04-01

Insulin-like growth factor binding proteins (IGFBPs) display many functions in humans including regulation of the insulin-like growth factor (IGF) signaling pathway. The various roles of human IGFBPs make them attractive protein candidates in drug discovery. Structural and functional knowledge on human proteins with therapeutic relevance is needed to design and process the next generation of protein therapeutics. In order to conduct structural and functional investigations large quantities of recombinant proteins are needed. However, finding a suitable recombinant production system for proteins such as full-length human IGFBPs, still remains a challenge. Here we present a mammalian HEK293 expression method suitable for over-expression of secretory full-length human IGFBP-1 to -7. Protein purification of full-length human IGFBP-1, -2, -3 and -5 was conducted using a two-step chromatography procedure and the final protein yields were between 1 and 12mg protein per liter culture media. The recombinant IGFBPs contained PTMs and exhibited high-affinity interactions with their natural ligands IGF-1 and IGF-2. Copyright © 2014 Elsevier Inc. All rights reserved.

Evaluation of Reference Genes for RT qPCR Analyses of Structure-Specific and Hormone Regulated Gene Expression in Physcomitrella patens Gametophytes

PubMed Central

Le Bail, Aude; Scholz, Sebastian; Kost, Benedikt

2013-01-01

The use of the moss Physcomitrella patens as a model system to study plant development and physiology is rapidly expanding. The strategic position of P. patens within the green lineage between algae and vascular plants, the high efficiency with which transgenes are incorporated by homologous recombination, advantages associated with the haploid gametophyte representing the dominant phase of the P. patens life cycle, the simple structure of protonemata, leafy shoots and rhizoids that constitute the haploid gametophyte, as well as a readily accessible high-quality genome sequence make this moss a very attractive experimental system. The investigation of the genetic and hormonal control of P. patens development heavily depends on the analysis of gene expression patterns by real time quantitative PCR (RT qPCR). This technique requires well characterized sets of reference genes, which display minimal expression level variations under all analyzed conditions, for data normalization. Sets of suitable reference genes have been described for most widely used model systems including e.g. Arabidopsis thaliana, but not for P. patens. Here, we present a RT qPCR based comparison of transcript levels of 12 selected candidate reference genes in a range of gametophytic P. patens structures at different developmental stages, and in P. patens protonemata treated with hormones or hormone transport inhibitors. Analysis of these RT qPCR data using GeNorm and NormFinder software resulted in the identification of sets of P. patens reference genes suitable for gene expression analysis under all tested conditions, and suggested that the two best reference genes are sufficient for effective data normalization under each of these conditions. PMID:23951063

Suitable Reference Genes for Accurate Gene Expression Analysis in Parsley (Petroselinum crispum) for Abiotic Stresses and Hormone Stimuli

PubMed Central

Li, Meng-Yao; Song, Xiong; Wang, Feng; Xiong, Ai-Sheng

2016-01-01

Parsley, one of the most important vegetables in the Apiaceae family, is widely used in the food, medicinal, and cosmetic industries. Recent studies on parsley mainly focus on its chemical composition, and further research involving the analysis of the plant's gene functions and expressions is required. qPCR is a powerful method for detecting very low quantities of target transcript levels and is widely used to study gene expression. To ensure the accuracy of results, a suitable reference gene is necessary for expression normalization. In this study, four software, namely geNorm, NormFinder, BestKeeper, and RefFinder were used to evaluate the expression stabilities of eight candidate reference genes of parsley (GAPDH, ACTIN, eIF-4α, SAND, UBC, TIP41, EF-1α, and TUB) under various conditions, including abiotic stresses (heat, cold, salt, and drought) and hormone stimuli treatments (GA, SA, MeJA, and ABA). Results showed that EF-1α and TUB were the most stable genes for abiotic stresses, whereas EF-1α, GAPDH, and TUB were the top three choices for hormone stimuli treatments. Moreover, EF-1α and TUB were the most stable reference genes among all tested samples, and UBC was the least stable one. Expression analysis of PcDREB1 and PcDREB2 further verified that the selected stable reference genes were suitable for gene expression normalization. This study can guide the selection of suitable reference genes in gene expression in parsley. PMID:27746803

Suitable Reference Genes for Accurate Gene Expression Analysis in Parsley (Petroselinum crispum) for Abiotic Stresses and Hormone Stimuli.

PubMed

Li, Meng-Yao; Song, Xiong; Wang, Feng; Xiong, Ai-Sheng

2016-01-01

Parsley, one of the most important vegetables in the Apiaceae family, is widely used in the food, medicinal, and cosmetic industries. Recent studies on parsley mainly focus on its chemical composition, and further research involving the analysis of the plant's gene functions and expressions is required. qPCR is a powerful method for detecting very low quantities of target transcript levels and is widely used to study gene expression. To ensure the accuracy of results, a suitable reference gene is necessary for expression normalization. In this study, four software, namely geNorm, NormFinder, BestKeeper, and RefFinder were used to evaluate the expression stabilities of eight candidate reference genes of parsley ( GAPDH, ACTIN, eIF-4 α, SAND, UBC, TIP41, EF-1 α, and TUB ) under various conditions, including abiotic stresses (heat, cold, salt, and drought) and hormone stimuli treatments (GA, SA, MeJA, and ABA). Results showed that EF-1 α and TUB were the most stable genes for abiotic stresses, whereas EF-1 α, GAPDH , and TUB were the top three choices for hormone stimuli treatments. Moreover, EF-1 α and TUB were the most stable reference genes among all tested samples, and UBC was the least stable one. Expression analysis of PcDREB1 and PcDREB2 further verified that the selected stable reference genes were suitable for gene expression normalization. This study can guide the selection of suitable reference genes in gene expression in parsley.

Ausdruckskraft und Regelmaessigkeit: Was Esperanto fuer automatische Uebersetzung geeignet macht (Expressiveness and Formal Regularity: What Makes Esperanto Suitable for Machine Translation).

ERIC Educational Resources Information Center

Schubert, Klaus

1988-01-01

Describes DLT, the multilingual machine translation system that uses Esperanto as an intermediate language in which substantial portions of the translation subprocesses are carried out. The criteria for choosing an intermediate language and the reasons for preferring Esperanto over other languages are explained. (Author/DJD)

The Minkowski metric in non-inertial observer radar coordinates

NASA Astrophysics Data System (ADS)

Minguzzi, E.

2005-12-01

We give a closed expression for the Minkowski (1+1)-dimensional metric in the radar coordinates of an arbitrary non-inertial observer O in terms of O's proper acceleration. Knowledge of the metric allows the non-inertial observer to perform experiments in spacetime without making reference to inertial frames. To clarify the relation between inertial and non-inertial observers the coordinate transformation between radar and inertial coordinates also is given. We show that every conformally flat coordinate system can be regarded as the radar coordinate system of a suitable observer for a suitable parametrization of the observer worldline. Therefore, the coordinate transformation between arbitrarily moving observers is a conformal transformation and conformally invariant (1+1)-dimensional theories lead to the same physics for all observers, independently of their relative motion.

Ising game: Nonequilibrium steady states of resource-allocation systems

NASA Astrophysics Data System (ADS)

Xin, C.; Yang, G.; Huang, J. P.

2017-04-01

Resource-allocation systems are ubiquitous in the human society. But how external fields affect the state of such systems remains poorly explored due to the lack of a suitable model. Because the behavior of spins pursuing energy minimization required by physical laws is similar to that of humans chasing payoff maximization studied in game theory, here we combine the Ising model with the market-directed resource-allocation game, yielding an Ising game. Based on the Ising game, we show theoretical, simulative and experimental evidences for a formula, which offers a clear expression of nonequilibrium steady states (NESSs). Interestingly, the formula also reveals a convertible relationship between the external field (exogenous factor) and resource ratio (endogenous factor), and a class of saturation as the external field exceeds certain limits. This work suggests that the Ising game could be a suitable model for studying external-field effects on resource-allocation systems, and it could provide guidance both for seeking more relations between NESSs and equilibrium states and for regulating human systems by choosing NESSs appropriately.

A versatile and efficient high-throughput cloning tool for structural biology.

PubMed

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.

A Novel Tightly Regulated Gene Expression System for the Human Intestinal Symbiont Bacteroides thetaiotaomicron.

PubMed

Horn, Nikki; Carvalho, Ana L; Overweg, Karin; Wegmann, Udo; Carding, Simon R; Stentz, Régis

2016-01-01

There is considerable interest in studying the function of Bacteroides species resident in the human gastrointestinal (GI)-tract and the contribution they make to host health. Reverse genetics and protein expression techniques, such as those developed for well-characterized Escherichia coli cannot be applied to Bacteroides species as they and other members of the Bacteriodetes phylum have unique promoter structures. The availability of useful Bacteroides-specific genetic tools is therefore limited. Here we describe the development of an effective mannan-controlled gene expression system for Bacteroides thetaiotaomicron containing the mannan-inducible promoter-region of an α-1,2-mannosidase gene (BT_3784), a ribosomal binding site designed to modulate expression, a multiple cloning site to facilitate the cloning of genes of interest, and a transcriptional terminator. Using the Lactobacillus pepI as a reporter gene, mannan induction resulted in an increase of reporter activity in a time- and concentration-dependent manner with a wide range of activity. The endogenous BtcepA cephalosporinase gene was used to demonstrate the suitability of this novel expression system, enabling the isolation of a His-tagged version of BtCepA. We have also shown with experiments performed in mice that the system can be induced in vivo in the presence of an exogenous source of mannan. By enabling the controlled expression of endogenous and exogenous genes in B. thetaiotaomicron this novel inducer-dependent expression system will aid in defining the physiological role of individual genes and the functional analyses of their products.

Co-expression of human cytochrome P4501A1 (CYP1A1) variants and human NADPH-cytochrome P450 reductase in the baculovirus/insect cell system.

PubMed

Schwarz, D; Kisselev, P; Honeck, H; Cascorbi, I; Schunck, W H; Roots, I

2001-06-01

1. Three human cytochrome P4501A1 (CYP1A1) variants, wild-type (CYP1A1.1), CYP1A1.2 (1462V) and CYP1A1.4 (T461N), were co-expressed with human NADPH-P450 reductase (OR) in Spodoptera frugiperda (Sf9) insect cells by baculovirus co-infection to elaborate a suitable system for studying the role of CYPA1 polymorphism in the metabolism of exogenous and endogenous substrates. 2. A wide range of conditions was examined to optimize co-expression with regard to such parameters as relative multiplicity of infection (MOI), time of harvest, haem precursor supplementation and post-translational stabilization. tinder optimized conditions, almost identical expression levels and molar OR/CYP1A1 ratios (20:1) were attained for all CYP1A1 variants. 3. Microsomes isolated from co-infected cells demonstrated ethoxyresorufin deethlylase activities (nmol/min(-1) nmol(-1) CYP1A1) of 16.0 (CYP1A1.1), 20.5 (CYP1A1.2) and 22.5 (CYP1A1.4). Pentoxyresorufin was dealkylated approximately 10-20 times slower with all enzyme variants. 4. All three CYP1A1 variants were active in metabolizing the precarcinogen benzo[a]pyrene (B[a]P), with wild-type enzyme showing the highest activity, followed by CYP1A1.4 (60%) and CYP1A1.2 (40%). Each variant produced all major metabolites including B[a]P-7,8-dihydrodiol, the precursor of the ultimate carcinogenic species. 5. These studies demonstrate that the baculovirus-mediated co-expression-by-co-infection approach all CYP1A1 variants yields functionally active enzyme systems with similar molar OR/CYP1A1 ratios, thus providing suitable preconditions to examine the metabolism of and environmental chemicals by the different CY1A1 variants.

Utilizing high-throughput experimentation to enhance specific productivity of an E.coli T7 expression system by phosphate limitation.

PubMed

Huber, Robert; Roth, Simon; Rahmen, Natalie; Büchs, Jochen

2011-03-17

The specific productivity of cultivation processes can be optimized, amongst others, by using genetic engineering of strains, choice of suitable host/vector systems or process optimization (e.g. choosing the right induction time). A further possibility is to reduce biomass buildup in favor of an enhanced product formation, e.g. by limiting secondary substrates in the medium, such as phosphate. However, with conventional techniques (e.g. small scale cultivations in shake flasks), it is very tedious to establish optimal conditions for cell growth and protein expression, as the start of protein expression (induction time) and the degree of phosphate limitation have to be determined in numerous concerted, manually conducted experiments. We investigated the effect of different induction times and a concurrent phosphate limitation on the specific productivity of the T7 expression system E.coli BL21(DE3) pRhotHi-2-EcFbFP, which produces the model fluorescence protein EcFbFP upon induction. Therefore, specific online-monitoring tools for small scale cultivations (RAMOS, BioLector) as well as a novel cultivation platform (Robo-Lector) were used for rapid process optimization. The RAMOS system monitored the oxygen transfer rate in shake flasks, whereas the BioLector device allowed to monitor microbial growth and the production of EcFbFP in microtiter plates. The Robo-Lector is a combination of a BioLector and a pipetting robot and can conduct high-throughput experiments fully automated. By using these tools, it was possible to determine the optimal induction time and to increase the specific productivity for EcFbFP from 22% (for unlimited conditions) to 31% of total protein content of the E.coli cells via a phosphate limitation. The results revealed that a phosphate limitation at the right induction time was suitable to redirect the available cellular resources during cultivation to protein expression rather than in biomass production. To our knowledge, such an effect was shown for the first time for an IPTG-inducible expression system. Finally, this finding and the utilization of the introduced high-throughput experimentation approach could help to find new targets to further enhance the production capacity of recombinant E.coli-strains.

Pichia pastoris is a Suitable Host for the Heterologous Expression of Predicted Class I and Class II Hydrophobins for Discovery, Study, and Application in Biotechnology

PubMed Central

Gandier, Julie-Anne; Master, Emma R.

2018-01-01

The heterologous expression of proteins is often a crucial first step in not only investigating their function, but also in their industrial application. The functional assembly and aggregation of hydrophobins offers intriguing biotechnological applications from surface modification to drug delivery, yet make developing systems for their heterologous expression challenging. In this article, we describe the development of Pichia pastoris KM71H strains capable of solubly producing the full set of predicted Cordyceps militaris hydrophobins CMil1 (Class IA), CMil2 (Class II), and CMil3 (IM) at mg/L yields with the use of 6His-tags not only for purification but for their detection. This result further demonstrates the feasibility of using P. pastoris as a host organism for the production of hydrophobins from all Ascomycota Class I subdivisions (a classification our previous work defined) as well as Class II. We highlight the specific challenges related to the production of hydrophobins, notably the challenge in detecting the protein that will be described, in particular during the screening of transformants. Together with the literature, our results continue to show that P. pastoris is a suitable host for the soluble heterologous expression of hydrophobins with a wide range of properties. PMID:29303996

Pichia pastoris is a Suitable Host for the Heterologous Expression of Predicted Class I and Class II Hydrophobins for Discovery, Study, and Application in Biotechnology.

PubMed

Gandier, Julie-Anne; Master, Emma R

2018-01-05

The heterologous expression of proteins is often a crucial first step in not only investigating their function, but also in their industrial application. The functional assembly and aggregation of hydrophobins offers intriguing biotechnological applications from surface modification to drug delivery, yet make developing systems for their heterologous expression challenging. In this article, we describe the development of Pichia pastoris KM71H strains capable of solubly producing the full set of predicted Cordyceps militaris hydrophobins CMil1 (Class IA), CMil2 (Class II), and CMil3 (IM) at mg/L yields with the use of 6His-tags not only for purification but for their detection. This result further demonstrates the feasibility of using P. pastoris as a host organism for the production of hydrophobins from all Ascomycota Class I subdivisions (a classification our previous work defined) as well as Class II. We highlight the specific challenges related to the production of hydrophobins, notably the challenge in detecting the protein that will be described, in particular during the screening of transformants. Together with the literature, our results continue to show that P. pastoris is a suitable host for the soluble heterologous expression of hydrophobins with a wide range of properties.

Expression of the human blood coagulation protein factor XIIIa in Saccharomyces cerevisiae: dependence of the expression levels from host-vector systems and medium conditions.

PubMed

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.

Performance benchmarking of four cell-free protein expression systems.

PubMed

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.

5S rRNA Promoter for Guide RNA Expression Enabled Highly Efficient CRISPR/Cas9 Genome Editing in Aspergillus niger.

PubMed

Zheng, Xiaomei; Zheng, Ping; Zhang, Kun; Cairns, Timothy C; Meyer, Vera; Sun, Jibin; Ma, Yanhe

2018-04-30

The CRISPR/Cas9 system is a revolutionary genome editing tool. However, in eukaryotes, search and optimization of a suitable promoter for guide RNA expression is a significant technical challenge. Here we used the industrially important fungus, Aspergillus niger, to demonstrate that the 5S rRNA gene, which is both highly conserved and efficiently expressed in eukaryotes, can be used as a guide RNA promoter. The gene editing system was established with 100% rates of precision gene modifications among dozens of transformants using short (40-bp) homologous donor DNA. This system was also applicable for generation of designer chromosomes, as evidenced by deletion of a 48 kb gene cluster required for biosynthesis of the mycotoxin fumonisin B1. Moreover, this system also facilitated simultaneous mutagenesis of multiple genes in A. niger. We anticipate that the use of the 5S rRNA gene as guide RNA promoter can broadly be applied for engineering highly efficient eukaryotic CRISPR/Cas9 toolkits. Additionally, the system reported here will enable development of designer chromosomes in model and industrially important fungi.

A Critical Review of the Concept of Transgenic Plants: Insights into Pharmaceutical Biotechnology and Molecular Farming.

PubMed

Abiri, Rambod; Valdiani, Alireza; Maziah, Mahmood; Shaharuddin, Noor Azmi; Sahebi, Mahbod; Yusof, Zetty Norhana Balia; Atabaki, Narges; Talei, Daryush

2016-01-01

Using transgenic plants for the production of high-value recombinant proteins for industrial and clinical applications has become a promising alternative to using conventional bioproduction systems, such as bacteria, yeast, and cultured insect and animal cells. This novel system offers several advantages over conventional systems in terms of safety, scale, cost-effectiveness, and the ease of distribution and storage. Currently, plant systems are being utilised as recombinant bio-factories for the expression of various proteins, including potential vaccines and pharmaceuticals, through employing several adaptations of recombinant processes and utilizing the most suitable tools and strategies. The level of protein expression is a critical factor in plant molecular farming, and this level fluctuates according to the plant species and the organs involved. The production of recombinant native and engineered proteins is a complicated procedure that requires an inter- and multi-disciplinary effort involving a wide variety of scientific and technological disciplines, ranging from basic biotechnology, biochemistry, and cell biology to advanced production systems. This review considers important plant resources, affecting factors, and the recombinant-protein expression techniques relevant to the plant molecular farming process.

Expression profiling of G-protein-coupled receptors in human urothelium and related cell lines.

PubMed

Ochodnický, Peter; Humphreys, Sian; Eccles, Rachel; Poljakovic, Mirjana; Wiklund, Peter; Michel, Martin C

2012-09-01

What's known on the subject? and What does the study add? Urothelium emerged as a crucial integrator of sensory inputs and outputs in the bladder wall, and urothelial G-protein-coupled receptors (GPCRs) may represent plausible targets for treatment of various bladder pathologies. Urothelial cell lines provide a useful tool to study urothelial receptor function, but their validity as models for native human urothelium remains unclear. We characterize the mRNA expression of genes coding for GPCRs in human freshly isolated urothelium and compare the expression pattern with those in human urothelial cell lines. To characterize the mRNA expression pattern of genes coding for G-protein-coupled receptors (GPCRs) in human freshly isolated urothelium. To compare GPCR expression in human urothelium-derived cell lines to explore the suitability of these cell lines as model systems to study urothelial function. Native human urothelium (commercially sourced) and human urothelium-derived non-cancer (UROtsa and TERT-NHUC) and cancer (J82) cell lines were used. For mRNA expression profiling we used custom-designed real-time polymerase chain reaction array for 40 receptors and several related genes. Native urothelium expressed a wide variety of GPCRs, including α(1A), α(1D) and all subtypes of α(2) and β adrenoceptors. In addition, M(2) and M(3) cholinergic muscarinic receptors, angiotensin II AT(1) receptor, serotonin 5-HT(2A) receptor and all subtypes of bradykinin, endothelin, cannabinoid, tachykinin and sphingosine-1-phosphate receptors were detected. Nerve growth factor and both its low- and high-affinity receptors were also expressed in urothelium. In all cell lines expression of most GPCRs was markedly downregulated, with few exceptions. In UROtsa cells, but much less in other cell lines, the expression of β(2) adrenoceptors, M(3) muscarinic receptors, B(1) and B(2) bradykinin receptors, ET(B) endothelin receptors and several subtypes of sphingosine-1-phosphate receptors was largely retained. Human urothelium expresses a wide range of receptors which enables sensing and integration of various extracellular signals. Human urothelium-derived cell lines, especially UROtsa cells, show comparable mRNA expression to native tissue for several physiologically relevant GPCRs, but lose expression of many other receptors. The use of cell lines as model systems of human urothelium requires careful validation of suitability for the genes of interest. © 2012 BJU INTERNATIONAL.

Recombinant protein production and streptomycetes.

PubMed

Anné, Jozef; Maldonado, Bárbara; Van Impe, Jan; Van Mellaert, Lieve; Bernaerts, Kristel

2012-04-30

The biopharmaceutical market has come a long way since 1982, when the first biopharmaceutical product, recombinant human insulin, was launched. Just over 200 biopharma products have already gained approval. The global market for biopharmaceuticals which is currently valued at over US$99 billion has been growing at an impressive compound annual growth rate over the previous years. To produce these biopharmaceuticals and other industrially important heterologous proteins, different prokaryotic and eukaryotic expression systems are used. All expression systems have some advantages as well as some disadvantages that should be considered in selecting which one to use. Choosing the best one requires evaluating the options--from yield to glycosylation, to proper folding, to economics of scale-up. No host cell from which all the proteins can be universally expressed in large quantities has been found so far. Therefore, it is important to provide a variety of host-vector expression systems in order to increase the opportunities to screen for the most suitable expression conditions or host cell. In this overview, we focus on Streptomyces lividans, a Gram-positive bacterium with a proven excellence in secretion capacity, as host for heterologous protein production. We will discuss its advantages and disadvantages, and how with systems biology approaches strains can be developed to better producing cell factories. Copyright © 2011 Elsevier B.V. All rights reserved.

Agent-based Decision Support System for the Third Generation Distributed Dynamic Decision-making (DDD-III) Simulator

DTIC Science & Technology

2004-06-01

suitable form of organizational adaptation is effective organizational diagnosis and analysis. The organizational diagnosis and analysis involve...related to the mission environment, organizational structure, and strategy is imperative for an effective and efficient organizational diagnosis . The...not easily articulated nor expressed otherwise. These displays are crucial to facilitate effective organizational diagnosis and analysis, and

Defining suitable reference genes for RT-qPCR analysis on human sertoli cells after 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure.

PubMed

Ribeiro, Mariana Antunes; dos Reis, Mariana Bisarro; de Moraes, Leonardo Nazário; Briton-Jones, Christine; Rainho, Cláudia Aparecida; Scarano, Wellerson Rodrigo

2014-11-01

Quantitative real-time RT-PCR (qPCR) has proven to be a valuable molecular technique to quantify gene expression. There are few studies in the literature that describe suitable reference genes to normalize gene expression data. Studies of transcriptionally disruptive toxins, like tetrachlorodibenzo-p-dioxin (TCDD), require careful consideration of reference genes. The present study was designed to validate potential reference genes in human Sertoli cells after exposure to TCDD. 32 candidate reference genes were analyzed to determine their applicability. geNorm and NormFinder softwares were used to obtain an estimation of the expression stability of the 32 genes and to identify the most suitable genes for qPCR data normalization.

A peroxidase/dual oxidase system modulates midgut epithelial immunity in Anopheles gambiae.

PubMed

Kumar, Sanjeev; Molina-Cruz, Alvaro; Gupta, Lalita; Rodrigues, Janneth; Barillas-Mury, Carolina

2010-03-26

Extracellular matrices in diverse biological systems are cross-linked by dityrosine covalent bonds catalyzed by the peroxidase/oxidase system. We show that a peroxidase, secreted by the Anopheles gambiae midgut, and dual oxidase form a dityrosine network that decreases gut permeability to immune elicitors. This network protects the microbiota by preventing activation of epithelial immunity. It also provides a suitable environment for malaria parasites to develop within the midgut lumen without inducing nitric oxide synthase expression. Disruption of this barrier results in strong and effective pathogen-specific immune responses.

Formal verification of automated teller machine systems using SPIN

NASA Astrophysics Data System (ADS)

Iqbal, Ikhwan Mohammad; Adzkiya, Dieky; Mukhlash, Imam

2017-08-01

Formal verification is a technique for ensuring the correctness of systems. This work focuses on verifying a model of the Automated Teller Machine (ATM) system against some specifications. We construct the model as a state transition diagram that is suitable for verification. The specifications are expressed as Linear Temporal Logic (LTL) formulas. We use Simple Promela Interpreter (SPIN) model checker to check whether the model satisfies the formula. This model checker accepts models written in Process Meta Language (PROMELA), and its specifications are specified in LTL formulas.

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

PubMed

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

2017-01-01

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

Rapid correction of electron microprobe data for multicomponent metallic systems

NASA Technical Reports Server (NTRS)

Gupta, K. P.; Sivakumar, R.

1973-01-01

This paper describes an empirical relation for the correction of electron microprobe data for multicomponent metallic systems. It evaluates the empirical correction parameter, a for each element in a binary alloy system using a modification of Colby's MAGIC III computer program and outlines a simple and quick way of correcting the probe data. This technique has been tested on a number of multicomponent metallic systems and the agreement with the results using theoretical expressions is found to be excellent. Limitations and suitability of this relation are discussed and a model calculation is also presented in the Appendix.

Soluble expression of an amebic cysteine protease in the cytoplasm of Escherichia coli SHuffle Express cells and purification of active enzyme.

PubMed

Jalomo-Khayrova, Ekaterina; Mares, Rosa E; Muñoz, Patricia L A; Meléndez-López, Samuel G; Rivero, Ignacio A; Ramos, Marco A

2018-04-03

Recombinant production of amebic cysteine proteases using Escherichia coli cells as the bacterial system has become a challenging effort, with protein insolubility being the most common issue. Since many of these enzymes need a native conformation stabilized by disulfide bonds, an elaborate process of oxidative folding is usually demanded to get a functional protein. The cytoplasm of E. coli SHuffle Express cells owns an enhanced ability to properly fold proteins with disulfide bonds. Because of this cellular feature, it was possible to assume that this strain represents a reliable expression system and worthwhile been considered as an efficient bacterial host for the recombinant production of amebic cysteine proteases. Using E. coli SHuffle Express cells as the bacterial system, we efficiently produce soluble recombinant EhCP1protein. Enzymatic and inhibition analyses revealed that it exhibits proper catalytic abilities, proceeds effectively over the substrate (following an apparent Michaelis-Menten kinetics), and displays a typical inhibition profile. We report the first feasibility study of the recombinant production of amebic cysteine proteases using E. coli SHuffle Express as the bacterial host. We present a simple protocol for the recombinant expression and purification of fully soluble and active EhCP1 enzyme. We confirm the suitability of recombinant EhCP1 as a therapeutic target. We propose an approachable bacterial system for the recombinant production of amebic proteins, particularly for those with a need for proper oxidative folding.

Sensitivity of housekeeping genes in the suprachiasmatic nucleus of the mouse brain to diet and the daily light-dark cycle.

PubMed

Cleal, Jane K; Shepherd, James N; Shearer, Jasmine L; Bruce, Kimberley D; Cagampang, Felino R

2014-08-05

The endogenous timing system within the suprachiasmatic nuclei (SCN) of the hypothalamus drives the cyclic expression of the clock molecules across the 24h day-night cycle controlling downstream molecular pathways and physiological processes. The developing fetal clock system is sensitive to the environment and physiology of the pregnant mother and as such disruption of this system could lead to altered physiology in the offspring. Characterizing the gene profiles of the endogenous molecular clock system by quantitative reverse transcription polymerase chain reaction is dependent on normalization by appropriate housekeeping genes (HKGs). However, many HKGs commonly used as internal controls, although stably expressed under control conditions, can vary significantly in their expression under certain experimental conditions. Here we analyzed the expression of 10 classic HKG across the 24h light-dark cycle in the SCN of mouse offspring exposed to normal chow or a high fat diet during early development and in postnatal life. We found that the HKGs glyceraldehyde-3-phosphate dehydrogenase, beta actin and adenosine triphosphate synthase subunit to be the most stably expressed genes in the SCN regardless of diet or time within the 24h light-dark cycle, and are therefore suitable to be used as internal controls. However SCN samples collected during the light and dark periods did show differences in expression and as such the timing of collection should be considered when carrying out gene expression studies. Copyright © 2014 Elsevier B.V. All rights reserved.

Evaluation of reference gene suitability for quantitative expression analysis by quantitative polymerase chain reaction in the mandibular condyle of sheep.

PubMed

Jiang, Xin; Xue, Yang; Zhou, Hongzhi; Li, Shouhong; Zhang, Zongmin; Hou, Rui; Ding, Yuxiang; Hu, Kaijin

2015-10-01

Reference genes are commonly used as a reliable approach to normalize the results of quantitative polymerase chain reaction (qPCR), and to reduce errors in the relative quantification of gene expression. Suitable reference genes belonging to numerous functional classes have been identified for various types of species and tissue. However, little is currently known regarding the most suitable reference genes for bone, specifically for the sheep mandibular condyle. Sheep are important for the study of human bone diseases, particularly for temporomandibular diseases. The present study aimed to identify a set of reference genes suitable for the normalization of qPCR data from the mandibular condyle of sheep. A total of 12 reference genes belonging to various functional classes were selected, and the expression stability of the reference genes was determined in both the normal and fractured area of the sheep mandibular condyle. RefFinder, which integrates the following currently available computational algorithms: geNorm, NormFinder, BestKeeper, and the comparative ΔCt method, was used to compare and rank the candidate reference genes. The results obtained from the four methods demonstrated a similar trend: RPL19, ACTB, and PGK1 were the most stably expressed reference genes in the sheep mandibular condyle. As determined by RefFinder comprehensive analysis, the results of the present study suggested that RPL19 is the most suitable reference gene for studies associated with the sheep mandibular condyle. In addition, ACTB and PGK1 may be considered suitable alternatives.

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

PubMed

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.

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

PubMed

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

2015-03-20

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

A Novel Tightly Regulated Gene Expression System for the Human Intestinal Symbiont Bacteroides thetaiotaomicron

PubMed Central

Horn, Nikki; Carvalho, Ana L.; Overweg, Karin; Wegmann, Udo; Carding, Simon R.; Stentz, Régis

2016-01-01

There is considerable interest in studying the function of Bacteroides species resident in the human gastrointestinal (GI)-tract and the contribution they make to host health. Reverse genetics and protein expression techniques, such as those developed for well-characterized Escherichia coli cannot be applied to Bacteroides species as they and other members of the Bacteriodetes phylum have unique promoter structures. The availability of useful Bacteroides-specific genetic tools is therefore limited. Here we describe the development of an effective mannan-controlled gene expression system for Bacteroides thetaiotaomicron containing the mannan-inducible promoter–region of an α-1,2-mannosidase gene (BT_3784), a ribosomal binding site designed to modulate expression, a multiple cloning site to facilitate the cloning of genes of interest, and a transcriptional terminator. Using the Lactobacillus pepI as a reporter gene, mannan induction resulted in an increase of reporter activity in a time- and concentration-dependent manner with a wide range of activity. The endogenous BtcepA cephalosporinase gene was used to demonstrate the suitability of this novel expression system, enabling the isolation of a His-tagged version of BtCepA. We have also shown with experiments performed in mice that the system can be induced in vivo in the presence of an exogenous source of mannan. By enabling the controlled expression of endogenous and exogenous genes in B. thetaiotaomicron this novel inducer-dependent expression system will aid in defining the physiological role of individual genes and the functional analyses of their products. PMID:27468280

The Coulomb problem on a 3-sphere and Heun polynomials

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

Bellucci, Stefano; Yeghikyan, Vahagn; Yerevan State University, Alex-Manoogian st. 1, 00025 Yerevan

2013-08-15

The paper studies the quantum mechanical Coulomb problem on a 3-sphere. We present a special parametrization of the ellipto-spheroidal coordinate system suitable for the separation of variables. After quantization we get the explicit form of the spectrum and present an algebraic equation for the eigenvalues of the Runge-Lentz vector. We also present the wave functions expressed via Heun polynomials.

Titanium dioxide nanoparticles-mediated in vitro cytotoxicity does not induce Hsp70 and Grp78 expression in human bronchial epithelial A549 cells.

PubMed

Aueviriyavit, Sasitorn; Phummiratch, Duangkamol; Kulthong, Kornphimol; Maniratanachote, Rawiwan

2012-10-01

Titanium dioxide nanoparticles (TiO(2)NPs) are increasingly being used in various industrial applications including the production of paper, plastics, cosmetics and paints. With the increasing number of nano-related products, the concern of governments and the general public about the health and environmental risks, especially with regard to occupational and other environmental exposure, are gradually increasing. However, there is insufficient knowledge about the actual affects upon human health and the environment, as well as a lack of suitable biomarkers for assessing TiO(2)NP-induced cytotoxicity. Since the respiratory tract is likely to be the main exposure route of industrial workers to TiO(2)NPs, we investigated the cytotoxicity of the anatase and rutile crystalline forms of TiO(2)NPs in A549 cells, a human alveolar type II-like epithelial cell line. In addition, we evaluated the transcript and protein expression levels of two heat shock protein (HSP) members, Grp78 and Hsp70, to ascertain their suitability as biomarkers of TiO(2)NP-induced toxicity in the respiratory system. Ultrastructural observations confirmed the presence of TiO(2)NPs inside cells. In vitro exposure of A549 cells to the anatase or rutile forms of TiO(2)NPs led to cell death and induced intracellular ROS generation in a dose-dependent manner, as determined by the MTS and dichlorofluorescein (DCF) assays, respectively. In contrast, the transcript and protein expression levels of Hsp70 and Grp78 did not change within the same TiO(2)NPs dose range (25-500 μg/ml). Thus, whilst TiO(2)NPs can cause cytotoxicity in A549 cells, and thus potentially in respiratory cells, Hsp70 and Grp78 are not suitable biomarkers for evaluating the acute toxicological effects of TiO(2)NPs in the respiratory system.

Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples

PubMed Central

2010-01-01

Background Given the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals. Results Cross-validation of expression stability of eight selected reference genes using three popular algorithms, GeNorm, NormFinder and BestKeeper found ACTB and RPII as most stable reference genes. Conclusions We recommend ACTB and RPII as stable reference genes most suitable for gene expression studies of human visceral adipose tissue. The use of these genes as a reference pair may further enhance the robustness of qRT-PCR in this model system. PMID:20492695

Validation of endogenous reference genes for qRT-PCR analysis of human visceral adipose samples.

PubMed

Mehta, Rohini; Birerdinc, Aybike; Hossain, Noreen; Afendy, Arian; Chandhoke, Vikas; Younossi, Zobair; Baranova, Ancha

2010-05-21

Given the epidemic proportions of obesity worldwide and the concurrent prevalence of metabolic syndrome, there is an urgent need for better understanding the underlying mechanisms of metabolic syndrome, in particular, the gene expression differences which may participate in obesity, insulin resistance and the associated series of chronic liver conditions. Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in different tissues and experimental conditions. However, variations in amount of starting material, enzymatic efficiency and presence of inhibitors can lead to quantification errors. Hence the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Recent studies have shown that both obesity and presence of insulin resistance influence an expression of commonly used reference genes in omental fat. In this study we validated candidate reference genes suitable for qRT-PCR profiling experiments using visceral adipose samples from obese and lean individuals. Cross-validation of expression stability of eight selected reference genes using three popular algorithms, GeNorm, NormFinder and BestKeeper found ACTB and RPII as most stable reference genes. We recommend ACTB and RPII as stable reference genes most suitable for gene expression studies of human visceral adipose tissue. The use of these genes as a reference pair may further enhance the robustness of qRT-PCR in this model system.

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

PubMed

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.

Selection of reference genes for expression analysis in the entomophthoralean fungus Pandora neoaphidis.

PubMed

Chen, Chun; Xie, Tingna; Ye, Sudan; Jensen, Annette Bruun; Eilenberg, Jørgen

2016-01-01

The selection of suitable reference genes is crucial for accurate quantification of gene expression and can add to our understanding of host-pathogen interactions. To identify suitable reference genes in Pandora neoaphidis, an obligate aphid pathogenic fungus, the expression of three traditional candidate genes including 18S rRNA(18S), 28S rRNA(28S) and elongation factor 1 alpha-like protein (EF1), were measured by quantitative polymerase chain reaction at different developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae), and under different nutritional conditions. We calculated the expression stability of candidate reference genes using four algorithms including geNorm, NormFinder, BestKeeper and Delta Ct. The analysis results revealed that the comprehensive ranking of candidate reference genes from the most stable to the least stable was 18S (1.189), 28S (1.414) and EF1 (3). The 18S was, therefore, the most suitable reference gene for real-time RT-PCR analysis of gene expression under all conditions. These results will support further studies on gene expression in P. neoaphidis. Copyright © 2015 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Establishing references for gene expression analyses by RT-qPCR in Theobroma cacao tissues.

PubMed

Pinheiro, T T; Litholdo, C G; Sereno, M L; Leal, G A; Albuquerque, P S B; Figueira, A

2011-11-17

Lack of continuous progress in Theobroma cacao (Malvaceae) breeding, especially associated with seed quality traits, requires more efficient selection methods based on genomic information. Reverse transcript quantitative PCR (RT-qPCR) has become the method of choice for gene expression analysis, but relative expression analysis requires various reference genes, which must be stable across various biological conditions. We sought suitable reference genes for various tissues of cacao, especially developing seeds. Ten potential reference genes were analyzed for stability at various stages of embryo development, leaves, stems, roots, flowers, and pod epicarp; seven of them were also evaluated in shoot tips treated either with hormones (salicylate; ethefon; methyl-jasmonate) or after inoculation with the fungus Moniliophthora perniciosa (Marasmiaceae sensu lato). For developing embryos, the three most stable genes were actin (ACT), polyubiquitin (PUB), and ribosomal protein L35 (Rpl35). In the analyses of various tissues, the most stable genes were malate dehydrogenase (MDH), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and acyl-carrier protein B (ACP B). GAPDH, MDH and tubulin (TUB) were the most appropriate for normalization when shoot apexes were treated with hormones, while ACT, TUB and Rpl35 were the most appropriate after inoculation with M. perniciosa. We conclude that for each plant system and biological or ontogenetical condition, there is a need to define suitable reference genes. This is the first report to define reference genes for expression studies in cacao.

Selection and validation of reference genes for miRNA expression studies during porcine pregnancy.

PubMed

Wessels, Jocelyn M; Edwards, Andrew K; Zettler, Candace; Tayade, Chandrakant

2011-01-01

MicroRNAs comprise a family of small non-coding RNAs that modulate several developmental and physiological processes including pregnancy. Their ubiquitous presence is confirmed in mammals, worms, flies and plants. Although rapid advances have been made in microRNA research, information on stable reference genes for validation of microRNA expression is still lacking. Real time PCR is a widely used tool to quantify gene transcripts. An appropriate reference gene must be chosen to minimize experimental error in this system. A small difference in miRNA levels between experimental samples can be biologically meaningful as these entities can affect multiple targets in a pathway. This study examined the suitability of six commercially available reference genes (RNU1A, RNU5A, RNU6B, SNORD25, SCARNA17, and SNORA73A) in maternal-fetal tissues from healthy and spontaneously arresting/dying conceptuses from sows were separately analyzed at gestation day 20. Comparisons were also made with non-pregnant endometrial tissues from sows. Spontaneous fetal loss is a prime concern to the commercial pork industry. Our laboratory has previously identified deficits in vasculature development at maternal-fetal interface as one of the major participating causes of fetal loss. Using this well-established model, we have extended our studies to identify suitable microRNA reference genes. A methodical approach to assessing suitability was adopted using standard curve and melting curve analysis, PCR product sequencing, real time PCR expression in a panel of gestational tissues, and geNorm and NormFinder analysis. Our quantitative real time PCR analysis confirmed expression of all 6 reference genes in maternal and fetal tissues. All genes were uniformly expressed in tissues from healthy and spontaneously arresting conceptus attachment sites. Comparisons between tissue types (maternal/fetal/non-pregnant) revealed significant differences for RNU5A, RNU6B, SCARNA17, and SNORA73A expression. Based on our methodical assessment of all 6 reference genes, results suggest that RNU1A is the most stable reference gene for porcine pregnancy studies.

Study of using microfiltration and reverse osmosis membrane technologies for reclaiming cooling water in the power industry.

PubMed

Li, J; Xu, Z Y; An, H G; Liu, L Q

2007-07-01

A study of using dual membrane technologies, microfiltration (MF) and reverse osmosis (RO), for reclaiming blowdown of the cooling tower was conducted at ZJK power plant, Hebei province, China. The study shows that the combined MF-RO system can effectively reduce water consumption in the power industry. The results indicate that MF process is capable of producing a filtrate suitable for RO treatment and achieving a silt density index (SDI) less than 2, turbidity of 0.2 NTU. The water quality of RO effluent is very good with an average conductivity of about 40 micros/cm and rejection of 98%. The product water is suitable for injection into the cooling tower to counteract with cooling water intrusion. After adopting this system, water-saving effectiveness as expressed in terms of cycles of concentration could be increased from 2.5-2.8 times to 5 times.

Protein and genome evolution in Mammalian cells for biotechnology applications.

PubMed

Majors, Brian S; Chiang, Gisela G; Betenbaugh, Michael J

2009-06-01

Mutation and selection are the essential steps of evolution. Researchers have long used in vitro mutagenesis, expression, and selection techniques in laboratory bacteria and yeast cultures to evolve proteins with new properties, termed directed evolution. Unfortunately, the nature of mammalian cells makes applying these mutagenesis and whole-organism evolution techniques to mammalian protein expression systems laborious and time consuming. Mammalian evolution systems would be useful to test unique mammalian cell proteins and protein characteristics, such as complex glycosylation. Protein evolution in mammalian cells would allow for generation of novel diagnostic tools and designer polypeptides that can only be tested in a mammalian expression system. Recent advances have shown that mammalian cells of the immune system can be utilized to evolve transgenes during their natural mutagenesis processes, thus creating proteins with unique properties, such as fluorescence. On a more global level, researchers have shown that mutation systems that affect the entire genome of a mammalian cell can give rise to cells with unique phenotypes suitable for commercial processes. This review examines the advances in mammalian cell and protein evolution and the application of this work toward advances in commercial mammalian cell biotechnology.

Cytochrome P450 system expression and DNA adduct formation in the liver of Zacco platypus following waterborne benzo(a)pyrene exposure: implications for biomarker determination.

PubMed

Lee, Jin Wuk; Kim, Yong Hwa; Yoon, Seokjoo; Lee, Sung Kyu

2014-09-01

Benzo(a)pyrene (BaP) is a polycyclic aromatic hydrocarbon that causes mutations and tumor formation. Zacco platypus is a sentinel species that is suitable for monitoring aquatic environments. We studied cytochrome P450 system (CYP system) expression and DNA adduct formation in the liver of Z. platypus following waterborne exposure to BaP. The results showed both dose and time dependency. The significant induction levels of CYP system mRNA and protein reached maximums at 2 days and 14 days, respectively, and hepatosomatic index was maximally induced at 4 days during 14 days BaP exposure. DNA adduct formation was significantly induced compared to corresponding controls (t-test, p < 0.01) after 4 days of exposure in 100 μg/L BaP. These results indicate that the only use of mRNA expression level of CYP system as a biomarker make us underestimate prolonged toxicity (4-14 days) of BaP and the only use of protein expression level of CYP system make us underestimate acute toxicity (1-2 days) of BaP. Therefore, we suggests that a combinational use of the mRNA expression level and protein expression level of CYP system, hepatosomatic index is a useful biomarker in risk assessment of waterborne BaP exposure. In addition, DNA adduct formation was a useful biomarker in risk assessment of waterborne BaP exposure at 4 days. CYP1A was a more sensitive biomarker than CYP reductase for BaP exposure when considering both the mRNA and protein level. Furthermore, our results show that Z. platypus is a useful species for assessing the risk of waterborne BaP exposure. Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.

Temporal logics and real time expert systems.

PubMed

Blom, J A

1996-10-01

This paper introduces temporal logics. Due to the eternal compromise between expressive adequacy and reasoning efficiency that must decided upon in any application, full (first order logic or modal logic based) temporal logics are frequently not suitable. This is especially true in real time expert systems, where a fixed (and usually small) response time must be guaranteed. One such expert system, Fagan's VM, is reviewed, and a delineation is given of how to formally describe and reason with time in medical protocols. It is shown that Petri net theory is a useful tool to check the correctness of formalised protocols.

Identification of Suitable Reference Genes for mRNA Studies in Bone Marrow in a Mouse Model of Hematopoietic Stem Cell Transplantation.

PubMed

Li, H; Chen, C; Yao, H; Li, X; Yang, N; Qiao, J; Xu, K; Zeng, L

2016-10-01

Bone marrow micro-environment changes during hematopoietic stem cell transplantation (HSCT) with subsequent alteration of genes expression. Quantitative polymerase chain reaction (q-PCR) is a reliable and reproducible technique for the analysis of gene expression. To obtain more accurate results, it is essential to find a reference during HSCT. However, which gene is suitable during HSCT remains unclear. This study aimed to identify suitable reference genes for mRNA studies in bone marrow after HSCT. C57BL/6 mice were treated with either total body irradiation (group T) or busulfan/cyclophosphamide (BU/CY) (group B) followed by infusion of bone marrow cells. Normal mice without treatments were served as a control. All samples (group T + group B + control) were defined as group G. On days 7, 14, and 21 after transplantation, transcription levels of 7 candidate genes, ACTB, B2M, GAPDH, HMBS, HPRT, SDHA, and YWHAZ, in bone marrow cells were measured by use of real-time quantitative PCR. The expression stability of these 7 candidate reference genes were analyzed by 2 statistical software programs, GeNorm and NormFinder. Our results showed that ACTB displayed the highest expression in group G, with lowest expression of PSDHA in group T and HPRT in groups B and G. Analysis of expression stability by use of GeNorm or NormFinder demonstrated that expression of B2M in bone marrow were much more stable during HSCT, compared with other candidate genes including commonly used reference genes GAPDH and ACTB. ACTB could be used as a suitable reference gene for mRNA studies in bone marrow after HSCT. Copyright © 2016 Elsevier Inc. All rights reserved.

The LIKE system, a novel protein expression toolbox for Bacillus subtilis based on the liaI promoter

PubMed Central

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

Gene expression in spider appendages reveals reversal of exd/hth spatial specificity, altered leg gap gene dynamics, and suggests divergent distal morphogen signaling.

PubMed

Prpic, Nikola-Michael; Janssen, Ralf; Wigand, Barbara; Klingler, Martin; Damen, Wim G M

2003-12-01

Leg development in Drosophila has been studied in much detail. However, Drosophila limbs form in the larva as imaginal discs and not during embryogenesis as in most other arthropods. Here, we analyze appendage genes in the spider Cupiennius salei and the beetle Tribolium castaneum. Differences in decapentaplegic (dpp) expression suggest a different mode of distal morphogen signaling suitable for the specific geometry of growing limb buds. Also, expression of the proximal genes homothorax (hth) and extradenticle (exd) is significantly altered: in the spider, exd is restricted to the proximal leg and hth expression extends distally, while in insects, exd is expressed in the entire leg and hth is restricted to proximal parts. This reversal of spatial specificity demonstrates an evolutionary shift, which is nevertheless compatible with a conserved role of this gene pair as instructor of proximal fate. Different expression dynamics of dachshund and Distal-less point to modifications in the regulation of the leg gap gene system. We comment on the significance of this finding for attempts to homologize leg segments in different arthropod classes. Comparison of the expression profiles of H15 and optomotor-blind to the Drosophila patterns suggests modifications also in the dorsal-ventral patterning system of the legs. Together, our results suggest alterations in many components of the leg developmental system, namely proximal-distal and dorsal-ventral patterning, and leg segmentation. Thus, the leg developmental system exhibits a propensity to evolutionary change, which probably forms the basis for the impressive diversity of arthropod leg morphologies.

System for selecting relevant information for decision support.

PubMed

Kalina, Jan; Seidl, Libor; Zvára, Karel; Grünfeldová, Hana; Slovák, Dalibor; Zvárová, Jana

2013-01-01

We implemented a prototype of a decision support system called SIR which has a form of a web-based classification service for diagnostic decision support. The system has the ability to select the most relevant variables and to learn a classification rule, which is guaranteed to be suitable also for high-dimensional measurements. The classification system can be useful for clinicians in primary care to support their decision-making tasks with relevant information extracted from any available clinical study. The implemented prototype was tested on a sample of patients in a cardiological study and performs an information extraction from a high-dimensional set containing both clinical and gene expression data.

Diagnostic reasoning techniques for selective monitoring

NASA Technical Reports Server (NTRS)

Homem-De-mello, L. S.; Doyle, R. J.

1991-01-01

An architecture for using diagnostic reasoning techniques in selective monitoring is presented. Given the sensor readings and a model of the physical system, a number of assertions are generated and expressed as Boolean equations. The resulting system of Boolean equations is solved symbolically. Using a priori probabilities of component failure and Bayes' rule, revised probabilities of failure can be computed. These will indicate what components have failed or are the most likely to have failed. This approach is suitable for systems that are well understood and for which the correctness of the assertions can be guaranteed. Also, the system must be such that changes are slow enough to allow the computation.

A computational procedure for multibody systems including flexible beam dynamics

NASA Technical Reports Server (NTRS)

Downer, J. D.; Park, K. C.; Chiou, J. C.

1990-01-01

A computational procedure suitable for the solution of equations of motions for flexible multibody systems has been developed. A fully nonlinear continuum approach capable of accounting for both finite rotations and large deformations has been used to model a flexible beam component. The beam kinematics are referred directly to an inertial reference frame such that the degrees of freedom embody both the rigid and flexible deformation motions. As such, the beam inertia expression is identical to that of rigid body dynamics. The nonlinear coupling between gross body motion and elastic deformation is contained in the internal force expression. Numerical solution procedures for the integration of spatial kinematic systems can be directily applied to the generalized coordinates of both the rigid and flexible components. An accurate computation of the internal force term which is invariant to rigid motions is incorporated into the general solution procedure.

Large inserts for big data: artificial chromosomes in the genomic era.

PubMed

Tocchetti, Arianna; Donadio, Stefano; Sosio, Margherita

2018-05-01

The exponential increase in available microbial genome sequences coupled with predictive bioinformatic tools is underscoring the genetic capacity of bacteria to produce an unexpected large number of specialized bioactive compounds. Since most of the biosynthetic gene clusters (BGCs) present in microbial genomes are cryptic, i.e. not expressed under laboratory conditions, a variety of cloning systems and vectors have been devised to harbor DNA fragments large enough to carry entire BGCs and to allow their transfer in suitable heterologous hosts. This minireview provides an overview of the vectors and approaches that have been developed for cloning large BGCs, and successful examples of heterologous expression.

Overexpression of neurofilament H disrupts normal cell structure and function

NASA Technical Reports Server (NTRS)

Szebenyi, Gyorgyi; Smith, George M.; Li, Ping; Brady, Scott T.

2002-01-01

Studying exogenously expressed tagged proteins in live cells has become a standard technique for evaluating protein distribution and function. Typically, expression levels of experimentally introduced proteins are not regulated, and high levels are often preferred to facilitate detection. However, overexpression of many proteins leads to mislocalization and pathologies. Therefore, for normative studies, moderate levels of expression may be more suitable. To understand better the dynamics of intermediate filament formation, transport, and stability in a healthy, living cell, we inserted neurofilament heavy chain (NFH)-green fluorescent protein (GFP) fusion constructs in adenoviral vectors with tetracycline (tet)-regulated promoters. This system allows for turning on or off the synthesis of NFH-GFP at a selected time, for a defined period, in a dose-dependent manner. We used this inducible system for live cell imaging of changes in filament structure and cell shape, motility, and transport associated with increasing NFH-GFP expression. Cells with low to intermediate levels of NFH-GFP were structurally and functionally similar to neighboring, nonexpressing cells. In contrast, overexpression led to pathological alterations in both filament organization and cell function. Copyright 2002 Wiley-Liss, Inc.

Brain-derived neurotrophic factor and its receptors in Bergmann glia cells.

PubMed

Poblete-Naredo, Irais; Guillem, Alain M; Juárez, Claudia; Zepeda, Rossana C; Ramírez, Leticia; Caba, Mario; Hernández-Kelly, Luisa C; Aguilera, José; López-Bayghen, Esther; Ortega, Arturo

2011-12-01

Brain-derived neurotrophic factor is an abundant and widely distributed neurotrophin expressed in the Central Nervous System. It is critically involved in neuronal differentiation and survival. The expression of brain-derived neurotrophic factor and that of its catalytic active cognate receptor (TrkB) has been extensively studied in neuronal cells but their expression and function in glial cells is still controversial. Despite of this fact, brain-derived neurotrophic factor is released from astrocytes upon glutamate stimulation. A suitable model to study glia/neuronal interactions, in the context of glutamatergic synapses, is the well-characterized culture of chick cerebellar Bergmann glia cells. Using, this system, we show here that BDNF and its functional receptor are present in Bergmann glia and that BDNF stimulation is linked to the activation of the phosphatidyl-inositol 3 kinase/protein kinase C/mitogen-activated protein kinase/Activator Protein-1 signaling pathway. Accordingly, reverse transcription-polymerase chain reaction (RT-PCR) experiments predicted the expression of full-length and truncated TrkB isoforms. Our results suggest that Bergmann glia cells are able to express and respond to BDNF stimulation favoring the notion of their pivotal role in neuroprotection. Copyright © 2011 Elsevier B.V. All rights reserved.

Expression and Characterization of Glucose Oxidase from Aspergillus niger in Yarrowia lipolytica.

PubMed

Khadivi Derakshan, Fatemeh; Darvishi, Farshad; Dezfulian, Mehrouz; Madzak, Catherine

2017-08-01

Glucose oxidase (GOX) is currently used in clinical, pharmaceutical, food and chemical industries. The aim of this study was expression and characterization of Aspergillus niger glucose oxidase gene in the yeast Yarrowia lipolytica. For the first time, the GOX gene of A. niger was successfully expressed in Y. lipolytica using a mono-integrative vector containing strong hybrid promoter and secretion signal. The highest total glucose oxidase activity was 370 U/L after 7 days of cultivation. An innovative method was used to cell wall disruption in current study, and it could be recommended to use for efficiently cell wall disruption of Y. lipolytica. Optimum pH and temperature for recombinant GOX activity were 5.5 and 37 °C, respectively. A single band with a molecular weight of 80 kDa similar to the native and pure form of A. niger GOX was observed for the recombinant GOX in SDS-PAGE analysis. Y. lipolytica is a suitable and efficient eukaryotic expression system to production of recombinant GOX in compered with other yeast expression systems and could be used to production of pure form of GOX for industrial applications.

Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems.

PubMed

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.

Improved Protein Arrays for Quantitative Systems Analysis of the Dynamics of Signaling Pathway Interactions

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

Yang, Chin-Rang

Astronauts and workers in nuclear plants who repeatedly exposed to low doses of ionizing radiation (IR, <10 cGy) are likely to incur specific changes in signal transduction and gene expression in various tissues of their body. Remarkable advances in high throughput genomics and proteomics technologies enable researchers to broaden their focus from examining single gene/protein kinetics to better understanding global gene/protein expression profiling and biological pathway analyses, namely Systems Biology. An ultimate goal of systems biology is to develop dynamic mathematical models of interacting biological systems capable of simulating living systems in a computer. This Glue Grant is to complementmore » Dr. Boothman’s existing DOE grant (No. DE-FG02-06ER64186) entitled “The IGF1/IGF-1R-MAPK-Secretory Clusterin (sCLU) Pathway: Mediator of a Low Dose IR-Inducible Bystander Effect” to develop sensitive and quantitative proteomic technology that suitable for low dose radiobiology researches. An improved version of quantitative protein array platform utilizing linear Quantum dot signaling for systematically measuring protein levels and phosphorylation states for systems biology modeling is presented. The signals are amplified by a confocal laser Quantum dot scanner resulting in ~1000-fold more sensitivity than traditional Western blots and show the good linearity that is impossible for the signals of HRP-amplification. Therefore this improved protein array technology is suitable to detect weak responses of low dose radiation. Software is developed to facilitate the quantitative readout of signaling network activities. Kinetics of EGFRvIII mutant signaling was analyzed to quantify cross-talks between EGFR and other signaling pathways.« less

β-Glucuronidase is a suitable internal control gene for mRNA quantitation in pathophysiological and non-pathological livers.

PubMed

Yamaguchi, Hiromi; Matsumoto, Sawako; Ishibashi, Mariko; Hasegawa, Kiyoshi; Sugitani, Masahiko; Takayama, Tadatoshi; Esumi, Mariko

2013-10-01

The level of expression of housekeeping genes is in general considered stable, and a representative gene such as glyceraldehyde-3-phosphate dehydrogenase is commonly used as an internal control for quantitating mRNA. However, expression of housekeeping genes is not always constant under pathological conditions. To determine which genes would be most suitable as internal controls for quantitative gene expression studies in human liver diseases, we quantified 12 representative housekeeping genes in 27 non-cancerous liver tissues (normal, chronic hepatitis C with and without liver cirrhosis). We identified β-glucuronidase as the most suitable gene for studies on liver by rigorous statistical analysis of inter- and intra-group comparisons. We conclude that it is important to determine the most appropriate control gene for the particular condition to be analyzed. © 2013 Elsevier Inc. All rights reserved.

New insights into the heterogeneity and functional diversity of human mesenchymal stem cells.

PubMed

Han, Z C; Du, W J; Han, Z B; Liang, L

2017-01-01

Mesenchymal stem cells (MSCs) are being tested in several biological systems and clinical settings with the aim of exploring their therapeutic potentials for a variety of diseases. MSCs are also known to be heterogeneous populations with variable functions. In the context of this multidimensional complexity, a recurrent question is what source or population of MSCs is suitable for specific clinical indications. Here, we reported that the biological features of MSCs varied with the individual donor, the tissue source, the culture condition and the subpopulations. Placental chorionic villi (CV) derived MSCs exhibited superior activities of immunomodulation and pro-angiogenesis compared to MSCs derived from bone marrow (BM), adipose and umbilical cord (UC). We identified a subpopulation of CD106(VCAM-1)+MSCs, which are present richly in placental CV, moderately in BM, and lowly in adipose and UC. The CD106+MSCs possess significantly increased immunomodutory and pro-angiogenic activities compared to CD106-MSCs. Analysis of gene expression and cytokine secretion revealed that CD106+MSCs highly expressed several immnumodulatory and pro-angiogenic cytokines. Our data offer new insights on the identification and selection of suitable source or population of MSCs for clinical applications. Further efforts should be concentrated on standardizing methods which will ultimately allow the validation of MSC products with defined biomarkers as predictive of potency in suitable pre-clinical models and clinical settings.

Agroinfiltration: a rapid and reliable method to select suitable rose cultivars for blue flower production.

PubMed

Zeinipour, Masoume; Azadi, Pejman; Majd, Ahmad; Kermani, Maryam Jafarkhani; Irian, Saeed; Hosseini, Seyed Mohammad; Mii, Masahiro

2018-05-01

Rose cultivars with blue flower color are among the most attractive breeding targets in floriculture. However, they are difficult to produce due to the low efficiency of transformation systems, interactive effects of hosts and vectors, and lengthy processes. In this study, agroinfiltration-mediated transient expression was investigated as a tool to assess the function of flower color genes and to determine appropriate host cultivars for stable transformation in Rosa hybrida . To induce delphinidin accumulation and consequently to produce blue hue, the petals of 30 rose cultivars were infiltrated with three different expression vectors namely pBIH-35S-CcF3'5'H, pBIH-35S-Del2 and pBIH-35S-Del8, harbouring different sets of flower color genes. The results obtained showed that the ectopic expression of the genes was only detected in three cultivars with dark pink petals (i.e. 'Purple power', 'High & Mora' and 'Marina') after 6-8 days. The high performance liquid chromatography analyses confirmed delphinidin accumulation in the infiltrated petals caused by transient expression of CcF3'5'H gene. Moreover, there were significant differences in the amounts of delphinidin among the three cultivars infiltrated with the three different expression vectors. More specifically, the highest delphinidin content was detected in the cultivar 'Purple power' (4.67 µg g -1 FW), infiltrated with the pBIH-35S-Del2 vector. The expression of CcF3'5'H gene in the infiltrated petals was also confirmed by real time PCR. In conclusion and based on the findings of the present study, the agroinfiltration could be regarded as a reliable method to identify suitable rose cultivars in blue rose flower production programs.

Evaluation of Suitable Reference Genes for Normalization of qPCR Gene Expression Studies in Brinjal (Solanum melongena L.) During Fruit Developmental Stages.

PubMed

Kanakachari, Mogilicherla; Solanke, Amolkumar U; Prabhakaran, Narayanasamy; Ahmad, Israr; Dhandapani, Gurusamy; Jayabalan, Narayanasamy; Kumar, Polumetla Ananda

2016-02-01

Brinjal/eggplant/aubergine is one of the major solanaceous vegetable crops. Recent availability of genome information greatly facilitates the fundamental research on brinjal. Gene expression patterns during different stages of fruit development can provide clues towards the understanding of its biological functions. Quantitative real-time PCR (qPCR) has become one of the most widely used methods for rapid and accurate quantification of gene expression. However, its success depends on the use of a suitable reference gene for data normalization. For qPCR analysis, a single reference gene is not universally suitable for all experiments. Therefore, reference gene validation is a crucial step. Suitable reference genes for qPCR analysis of brinjal fruit development have not been investigated so far. In this study, we have selected 21 candidate reference genes from the Brinjal (Solanum melongena) Plant Gene Indices database (compbio.dfci.harvard.edu/tgi/plant.html) and studied their expression profiles by qPCR during six different fruit developmental stages (0, 5, 10, 20, 30, and 50 days post anthesis) along with leaf samples of the Pusa Purple Long (PPL) variety. To evaluate the stability of gene expression, geNorm and NormFinder analytical softwares were used. geNorm identified SAND (SAND family protein) and TBP (TATA binding protein) as the best pairs of reference genes in brinjal fruit development. The results showed that for brinjal fruit development, individual or a combination of reference genes should be selected for data normalization. NormFinder identified Expressed gene (expressed sequence) as the best single reference gene in brinjal fruit development. In this study, we have identified and validated for the first time reference genes to provide accurate transcript normalization and quantification at various fruit developmental stages of brinjal which can also be useful for gene expression studies in other Solanaceae plant species.

NATbox: a network analysis toolbox in R.

PubMed

Chavan, Shweta S; Bauer, Michael A; Scutari, Marco; Nagarajan, Radhakrishnan

2009-10-08

There has been recent interest in capturing the functional relationships (FRs) from high-throughput assays using suitable computational techniques. FRs elucidate the working of genes in concert as a system as opposed to independent entities hence may provide preliminary insights into biological pathways and signalling mechanisms. Bayesian structure learning (BSL) techniques and its extensions have been used successfully for modelling FRs from expression profiles. Such techniques are especially useful in discovering undocumented FRs, investigating non-canonical signalling mechanisms and cross-talk between pathways. The objective of the present study is to develop a graphical user interface (GUI), NATbox: Network Analysis Toolbox in the language R that houses a battery of BSL algorithms in conjunction with suitable statistical tools for modelling FRs in the form of acyclic networks from gene expression profiles and their subsequent analysis. NATbox is a menu-driven open-source GUI implemented in the R statistical language for modelling and analysis of FRs from gene expression profiles. It provides options to (i) impute missing observations in the given data (ii) model FRs and network structure from gene expression profiles using a battery of BSL algorithms and identify robust dependencies using a bootstrap procedure, (iii) present the FRs in the form of acyclic graphs for visualization and investigate its topological properties using network analysis metrics, (iv) retrieve FRs of interest from published literature. Subsequently, use these FRs as structural priors in BSL (v) enhance scalability of BSL across high-dimensional data by parallelizing the bootstrap routines. NATbox provides a menu-driven GUI for modelling and analysis of FRs from gene expression profiles. By incorporating readily available functions from existing R-packages, it minimizes redundancy and improves reproducibility, transparency and sustainability, characteristic of open-source environments. NATbox is especially suited for interdisciplinary researchers and biologists with minimal programming experience and would like to use systems biology approaches without delving into the algorithmic aspects. The GUI provides appropriate parameter recommendations for the various menu options including default parameter choices for the user. NATbox can also prove to be a useful demonstration and teaching tool in graduate and undergraduate course in systems biology. It has been tested successfully under Windows and Linux operating systems. The source code along with installation instructions and accompanying tutorial can be found at http://bioinformatics.ualr.edu/natboxWiki/index.php/Main_Page.

The combination of plant translational enhancers and terminator increase the expression of human glucocerebrosidase in Nicotiana benthamiana plants.

PubMed

Limkul, Juthamard; Misaki, Ryo; Kato, Ko; Fujiyama, Kazuhito

2015-11-01

Gaucher's disease is a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCase). It is currently treated by enzyme replacement therapy using recombinant GCase expressed in mammalian cells. Plant production systems are among the most attractive alternatives for pharmaceutical protein production due to such advantages as low-cost, high-scalability, and safety from human pathogen contamination. Because of its high biomass yield, Nicotiana benthamiana could be an economical recombinant GCase production system. In this study, a translational enhancer and suitable terminator were utilized to obtain a powerful expression system for GCase production in N. benthamiana plants. Six plasmid constructs were used. The highest activity of 44.5units/mg protein (after subtraction of endogenous glucosidase activity of the wild-type plant) was observed in transgenic plants transformed with pAt-GC-HSP combined with a 5' untranslated region of the Arabidopsis alcohol dehydrogenase gene with the Arabidopsis heat shock protein terminator. These transgenic plant lines could pave the way to a stable plant-production system for low-cost, high-yield human GCase production. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Validation of Reference Genes for Gene Expression by Quantitative Real-Time RT-PCR in Stem Segments Spanning Primary to Secondary Growth in Populus tomentosa.

PubMed

Wang, Ying; Chen, Yajuan; Ding, Liping; Zhang, Jiewei; Wei, Jianhua; Wang, Hongzhi

2016-01-01

The vertical segments of Populus stems are an ideal experimental system for analyzing the gene expression patterns involved in primary and secondary growth during wood formation. Suitable internal control genes are indispensable to quantitative real time PCR (qRT-PCR) assays of gene expression. In this study, the expression stability of eight candidate reference genes was evaluated in a series of vertical stem segments of Populus tomentosa. Analysis through software packages geNorm, NormFinder and BestKeeper showed that genes ribosomal protein (RP) and tubulin beta (TUBB) were the most unstable across the developmental stages of P. tomentosa stems, and the combination of the three reference genes, eukaryotic translation initiation factor 5A (eIF5A), Actin (ACT6) and elongation factor 1-beta (EF1-beta) can provide accurate and reliable normalization of qRT-PCR analysis for target gene expression in stem segments undergoing primary and secondary growth in P. tomentosa. These results provide crucial information for transcriptional analysis in the P. tomentosa stem, which may help to improve the quality of gene expression data in these vertical stem segments, which constitute an excellent plant system for the study of wood formation.

Expression, purification, and antibacterial activity of bovine lactoferrampin-lactoferricin in Pichia pastoris.

PubMed

Tang, Xiang-Shan; Tang, Zhi-Ru; Wang, Sheng-Ping; Feng, Ze-Meng; Zhou, Dong; Li, Tie-Jun; Yin, Yu-Long

2012-02-01

Bovine lactoferrampin (LFA) and bovine lactoferricin (LFC) are two antimicrobial peptides located in the N(1) domain of bovine lactoferrin. The bactericidal activity of the fused peptide LFA-LFC is stronger than that of either LFA or LFC. The high cost of peptide production from either native digestion or chemical synthesis limits the clinical application of antimicrobial peptides. The expression of recombinant peptides in yeast may be an effective alternative. In the current study, the expression, purification, and antibacterial activity of LFA-LFC using the Pichia pastoris expression system are reported. The linearized expression vector pPICZaA-LFA-LFC was transformed into P. pastoris KM71 by electroporation, and positive colonies harboring the target genes were screened out and used for fermentation. The recombinant LFA-LFC peptide was purified via two-step column chromatography and identified by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results indicate that P. pastoris is a suitable system for secreting LFA-LFC. The fermentation supernate and the purified LFA-LFC show high antimicrobial activities. The current study is the first to report on the expression and purification of LFA-LFC in P. pastoris and may have potential practical applications in microbial peptide production.

Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum.

PubMed

Watanabe, Masahito; Kobayashi, Mirina; Nagaya, Masaki; Matsunari, Hitomi; Nakano, Kazuaki; Maehara, Miki; Hayashida, Gota; Takayanagi, Shuko; Sakai, Rieko; Umeyama, Kazuhiro; Watanabe, Nobuyuki; Onodera, Masafumi; Nagashima, Hiroshi

2015-01-01

Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36-37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum.

Methods for Gene Transfer to the Central Nervous System

PubMed Central

Kantor, Boris; Bailey, Rachel M.; Wimberly, Keon; Kalburgi, Sahana N.; Gray, Steven J.

2015-01-01

Gene transfer is an increasingly utilized approach for research and clinical applications involving the central nervous system (CNS). Vectors for gene transfer can be as simple as an unmodified plasmid, but more commonly involve complex modifications to viruses to make them suitable gene delivery vehicles. This chapter will explain how tools for CNS gene transfer have been derived from naturally occurring viruses. The current capabilities of plasmid, retroviral, adeno-associated virus, adenovirus, and herpes simplex virus vectors for CNS gene delivery will be described. These include both focal and global CNS gene transfer strategies, with short- or long-term gene expression. As is described in this chapter, an important aspect of any vector is the cis-acting regulatory elements incorporated into the vector genome that control when, where, and how the transgene is expressed. PMID:25311922

Method To Identify Specific Inhibiutors Of Imp Dehydrogenase

DOEpatents

Collart, Frank R.; Huberman, Eliezer

2000-11-28

This invention relates to methods to identify specific inhibitors of the purine nucleotide synthesis enzyme, IMP dehydrogenase (IMPDH). IMPDH is an essential enzyme found in all free-living organisms from humans to bacteria and is an important therapeutic target. The invention allows the identification of specific inhibitors of any IMPDH enzyme which can be expressed in a functional form in a recombinant host cell. A variety of eukaryotic or prokaryotic host systems commonly used for the expression of recombinant proteins are suitable for the practice of the invention. The methods are amenable to high throughput systems for the screening of inhibitors generated by combinatorial chemistry or other methods such as antisense molecule production. Utilization of exogenous guanosine as a control component of the methods allows for the identification of inhibitors specific for IMPDH rather than other causes of decreased cell proliferation.

The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies.

PubMed

Tsukahara, T; Iwase, N; Kawakami, K; Iwasaki, M; Yamamoto, C; Ohmine, K; Uchibori, R; Teruya, T; Ido, H; Saga, Y; Urabe, M; Mizukami, H; Kume, A; Nakamura, M; Brentjens, R; Ozawa, K

2015-02-01

Engineered T-cell therapy using a CD19-specific chimeric antigen receptor (CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3(+) T-cells with stable and high-level transgene expression (~95%) produced interferon-γ upon stimulation with CD19 and specifically lysed Raji cells, a CD19(+) human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.

High-level expression of recombinant beta-galactosidases in Lactobacillus plantarum and Lactobacillus sakei using a Sakacin P-based expression system.

PubMed

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.

Evaluation of reference genes for quantitative real-time RT-PCR analysis of gene expression in Nile tilapia (Oreochromis niloticus).

PubMed

Yang, Chang Geng; Wang, Xian Li; Tian, Juan; Liu, Wei; Wu, Fan; Jiang, Ming; Wen, Hua

2013-09-15

Quantitative real-time reverse-transcriptase polymerase chain reaction (RT-qPCR) has been used frequently to study gene expression related to fish immunology. In such studies, a stable reference gene should be selected to correct the expression of the target gene. In this study, seven candidate reference genes (glyceraldehyde-3-phosphate dehydrogenase (GADPH), ubiquitin-conjugating enzyme (UBCE), 18S ribosomal RNA (18S rRNA), beta-2-microglobulin (B2M), elongation factor 1 alpha (EF1A), tubulin alpha chain-like (TUBA) and beta actin (ACTB)), were selected to analyze their stability and normalization in seven tissues (liver, spleen, kidney, brain, heart, muscle and intestine) of Nile tilapia (Oreochromis niloticus) challenged with Streptococcus agalactiae or Streptococcus iniae, respectively. The results showed that all the candidate reference genes exhibited tissue-dependent transcriptional variations. With PBS injection as a control, UBCE was the most stable and suitable single reference gene in the intestine, liver, brain, kidney, and spleen after S. iniae infection, and in the liver, kidney, and spleen after S. agalactiae infection. EF1A was the most suitable in heart and muscle after S. iniae or S. agalactiae infection. GADPH was the most suitable gene in intestine and brain after S. agalactiae infection. In normal conditions, UBCE and 18S rRNA were the most stably expressed genes across the various tissues. These results showed that for RT-qPCR analysis of tilapia, selecting two or more reference genes may be more suitable for cross-tissue analysis of gene expression. Copyright © 2013 Elsevier B.V. All rights reserved.

Selection and Validation of Reference Genes for qRT-PCR Expression Analysis of Candidate Genes Involved in Olfactory Communication in the Butterfly Bicyclus anynana

PubMed Central

Arun, Alok; Baumlé, Véronique; Amelot, Gaël; Nieberding, Caroline M.

2015-01-01

Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae), two developmental stages (pupal and adult) and two sexes (male and female), all of which were subjected to two food treatments (food stress and control feeding ad libitum). The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the expression profile of the target candidate genes. PMID:25793735

Selection and validation of reference genes for qRT-PCR expression analysis of candidate genes involved in olfactory communication in the butterfly Bicyclus anynana.

PubMed

Arun, Alok; Baumlé, Véronique; Amelot, Gaël; Nieberding, Caroline M

2015-01-01

Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae), two developmental stages (pupal and adult) and two sexes (male and female), all of which were subjected to two food treatments (food stress and control feeding ad libitum). The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the expression profile of the target candidate genes.

Validation of Suitable Reference Genes for Expression Normalization in Echinococcus spp. Larval Stages

PubMed Central

Espínola, Sergio Martin; Ferreira, Henrique Bunselmeyer; Zaha, Arnaldo

2014-01-01

In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (βTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, βACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1α and TBP were the most stable genes for both species. Interestingly, βACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the Taenia genus. PMID:25014071

Validation of suitable reference genes for expression normalization in Echinococcus spp. larval stages.

PubMed

Espínola, Sergio Martin; Ferreira, Henrique Bunselmeyer; Zaha, Arnaldo

2014-01-01

In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (βTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, βACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1α and TBP were the most stable genes for both species. Interestingly, βACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the Taenia genus.

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.

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

Effects of fourth-order dispersion in very high-speed optical time-division multiplexed transmission.

PubMed

Capmany, J; Pastor, D; Sales, S; Ortega, B

2002-06-01

We present a closed-form expression for computation of the output pulse's rms time width in an optical fiber link with up to fourth-order dispersion (FOD) by use of an optical source with arbitrary linewidth and chirp parameters. We then specialize the expression to analyze the effect of FOD on the transmission of very high-speed linear optical time-division multiplexing systems. By suitable source chirping, FOD can be compensated for to an upper link-length limit above which other techniques must be employed. Finally, a design formula to estimate the maximum attainable bit rate limited by FOD as a function of the link length is also presented.

Comparison of melibiose utilizing baker's yeast strains produced by genetic engineering and classical breeding.

PubMed

Vincent, S F; Bell, P J; Bissinger, P; Nevalainen, K M

1999-02-01

Yeast strains currently used in the baking industry cannot fully utilize the trisaccharide raffinose found in beet molasses due to the absence of melibiase (alpha-galactosidase) activity. To overcome this deficiency, the MEL1 gene encoding melibiase enzyme was introduced into baker's yeast by both classical breeding and recombinant DNA technology. Both types of yeast strains were capable of vigorous fermentation in the presence of high levels of sucrose, making them suitable for the rapidly developing Asian markets where high levels of sugar are used in bread manufacture. Melibiase expression appeared to be dosage-dependent, with relatively low expression sufficient for complete melibiose utilization in a model fermentation system.

Pressure calculation in hybrid particle-field simulations

NASA Astrophysics Data System (ADS)

Milano, Giuseppe; Kawakatsu, Toshihiro

2010-12-01

In the framework of a recently developed scheme for a hybrid particle-field simulation techniques where self-consistent field (SCF) theory and particle models (molecular dynamics) are combined [J. Chem. Phys. 130, 214106 (2009)], we developed a general formulation for the calculation of instantaneous pressure and stress tensor. The expressions have been derived from statistical mechanical definition of the pressure starting from the expression for the free energy functional in the SCF theory. An implementation of the derived formulation suitable for hybrid particle-field molecular dynamics-self-consistent field simulations is described. A series of test simulations on model systems are reported comparing the calculated pressure with those obtained from standard molecular dynamics simulations based on pair potentials.

Identification and validation of suitable reference genes for RT-qPCR analysis in mouse testis development.

PubMed

Gong, Zu-Kang; Wang, Shuang-Jie; Huang, Yong-Qi; Zhao, Rui-Qiang; Zhu, Qi-Fang; Lin, Wen-Zhen

2014-12-01

RT-qPCR is a commonly used method for evaluating gene expression; however, its accuracy and reliability are dependent upon the choice of appropriate reference gene(s), and there is limited information available on suitable reference gene(s) that can be used in mouse testis at different stages. In this study, using the RT-qPCR method, we investigated the expression variations of six reference genes representing different functional classes (Actb, Gapdh, Ppia, Tbp, Rps29, Hprt1) in mice testis during embryonic and postnatal development. The expression stabilities of putative reference genes were evaluated using five algorithms: geNorm, NormFinder, Bestkeeper, the comparative delta C(t) method and integrated tool RefFinder. Analysis of the results showed that Ppia, Gapdh and Actb were identified as the most stable genes and the geometric mean of Ppia, Gapdh and Actb constitutes an appropriate normalization factor for gene expression studies. The mRNA expression of AT1 as a test gene of interest varied depending upon which of the reference gene(s) was used as an internal control(s). This study suggested that Ppia, Gapdh and Actb are suitable reference genes among the six genes used for RT-qPCR normalization and provide crucial information for transcriptional analyses in future studies of gene expression in the developing mouse testis.

Development of an optimized tetracycline-inducible expression system to increase the accumulation of interleukin-10 in tobacco BY-2 suspension cells.

PubMed

Bortesi, Luisa; Rademacher, Thomas; Schiermeyer, Andreas; Schuster, Flora; Pezzotti, Mario; Schillberg, Stefan

2012-07-11

Plant cell suspension cultures can be used for the production of valuable pharmaceutical and industrial proteins. When the recombinant protein is secreted into the culture medium, restricting expression to a defined growth phase can improve both the quality and quantity of the recovered product by minimizing proteolytic activity. Temporal restriction is also useful for recombinant proteins whose constitutive expression affects cell growth and viability, such as viral interleukin-10 (vIL-10). We have developed a novel, tetracycline-inducible system suitable for tobacco BY-2 suspension cells which increases the yields of vIL-10. The new system is based on a binary vector that is easier to handle than conventional vectors, contains an enhanced inducible promoter and 5'-UTR to improve yields, and incorporates a constitutively-expressed visible marker gene to allow the rapid and straightforward selection of the most promising transformed clones. Stable transformation of BY-2 cells with this vector, without extensive optimization of the induction conditions, led to a 3.5 fold increase in vIL-10 levels compared to constitutive expression in the same host. We have developed an effective and straightforward molecular farming platform technology that improves both the quality and the quantity of recombinant proteins produced in plant cells, particularly those whose constitutive expression has a negative impact on plant growth and development. Although we tested the platform using vIL-10 produced in BY-2 cells, it can be applied to other host/product combinations and is also useful for basic research requiring strictly controlled transgene expression.

Gene Expression Profile Analysis is Directly Affected by the Selected Reference Gene: The Case of Leaf-Cutting Atta Sexdens

PubMed Central

Máximo, Wesley P. F.; Zanetti, Ronald; Paiva, Luciano V.

2018-01-01

Although several ant species are important targets for the development of molecular control strategies, only a few studies focus on identifying and validating reference genes for quantitative reverse transcription polymerase chain reaction (RT-qPCR) data normalization. We provide here an extensive study to identify and validate suitable reference genes for gene expression analysis in the ant Atta sexdens, a threatening agricultural pest in South America. The optimal number of reference genes varies according to each sample and the result generated by RefFinder differed about which is the most suitable reference gene. Results suggest that the RPS16, NADH and SDHB genes were the best reference genes in the sample pool according to stability values. The SNF7 gene expression pattern was stable in all evaluated sample set. In contrast, when using less stable reference genes for normalization a large variability in SNF7 gene expression was recorded. There is no universal reference gene suitable for all conditions under analysis, since these genes can also participate in different cellular functions, thus requiring a systematic validation of possible reference genes for each specific condition. The choice of reference genes on SNF7 gene normalization confirmed that unstable reference genes might drastically change the expression profile analysis of target candidate genes. PMID:29419794

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.

3D porous calcium-alginate scaffolds cell culture system improved human osteoblast cell clusters for cell therapy.

PubMed

Chen, Ching-Yun; Ke, Cherng-Jyh; Yen, Ko-Chung; Hsieh, Hui-Chen; Sun, Jui-Sheng; Lin, Feng-Huei

2015-01-01

Age-related orthopedic disorders and bone defects have become a critical public health issue, and cell-based therapy is potentially a novel solution for issues surrounding bone tissue engineering and regenerative medicine. Long-term cultures of primary bone cells exhibit phenotypic and functional degeneration; therefore, culturing cells or tissues suitable for clinical use remain a challenge. A platform consisting of human osteoblasts (hOBs), calcium-alginate (Ca-Alginate) scaffolds, and a self-made bioreactor system was established for autologous transplantation of human osteoblast cell clusters. The Ca-Alginate scaffold facilitated the growth and differentiation of human bone cell clusters, and the functionally-closed process bioreactor system supplied the soluble nutrients and osteogenic signals required to maintain the cell viability. This system preserved the proliferative ability of cells and cell viability and up-regulated bone-related gene expression and biological apatite crystals formation. The bone-like tissue generated could be extracted by removal of calcium ions via ethylenediaminetetraacetic acid (EDTA) chelation, and exhibited a size suitable for injection. The described strategy could be used in therapeutic application and opens new avenues for surgical interventions to correct skeletal defects.

Generation of protective immune response against anthrax by oral immunization with protective antigen plant-based vaccine.

PubMed

Gorantala, Jyotsna; Grover, Sonam; Rahi, Amit; Chaudhary, Prerna; Rajwanshi, Ravi; Sarin, Neera Bhalla; Bhatnagar, Rakesh

2014-04-20

In concern with frequent recurrence of anthrax in endemic areas and inadvertent use of its spores as biological weapon, the development of an effective anthrax vaccine suitable for both human and veterinary needs is highly desirable. A simple oral delivery through expression in plant system could offer promising alternative to the current methods that rely on injectable vaccines extracted from bacterial sources. In the present study, we have expressed protective antigen (PA) gene in Indian mustard by Agrobacterium-mediated transformation and in tobacco by plastid transformation. Putative transgenic lines were verified for the presence of transgene and its expression by molecular analysis. PA expressed in transgenic lines was biologically active as evidenced by macrophage lysis assay. Intraperitoneal (i.p.) and oral immunization with plant PA in murine model indicated high serum PA specific IgG and IgA antibody titers. PA specific mucosal immune response was noted in orally immunized groups. Further, antibodies indicated lethal toxin neutralizing potential in-vitro and conferred protection against in-vivo toxin challenge. Oral immunization experiments demonstrated generation of immunoprotective response in mice. Thus, our study examines the feasibility of oral PA vaccine expressed in an edible plant system against anthrax. Copyright © 2014 Elsevier B.V. All rights reserved.

Role of Renin-Angiotensin System and Oxidative Stress on Vascular Inflammation in Insulin Resistence Model

PubMed Central

Renna, N. F.; Lembo, C.; Diez, E.; Miatello, R. M.

2013-01-01

(1) This study aims to demonstrate the causal involvement of renin angiotensin system (RAS) and oxidative stress (OS) on vascular inflammation in an experimental model of metabolic syndrome (MS) achieved by fructose administration to spontaneously hypertensive rats (FFHR) during 12 weeks. (2) Chronic treatment with candesartan (C) (10 mg/kg per day for the last 6 weeks) or 4OH-Tempol (T) (10−3 mmol/L in drinking water for the last 6 weeks) reversed the increment in metabolic variables and systolic blood pressure. In addition, chronic C treatment reverted cardiovascular remodeling but not T. (3) Furthermore, chronic treatment with C was able to completely reverse the expression of NF-κB and VCAM-1, but T only reduced the expression. C reduced the expression of proatherogenic cytokines as CINC2, CINC3, VEGF, Leptin, TNF-alpha, and MCP-1 and also significantly reduced MIP-3, beta-NGF, and INF-gamma in vascular tissue in this experimental model. T was not able to substantially modify the expression of these cytokines. (4) The data suggest the involvement of RAS in the expression of inflammatory proteins at different vascular levels, allowing the creation of a microenvironment suitable for the creation, perpetuation, growth, and destabilization of vascular injury. PMID:23365721

Role of Renin-Angiotensin system and oxidative stress on vascular inflammation in insulin resistence model.

PubMed

Renna, N F; Lembo, C; Diez, E; Miatello, R M

2013-01-01

(1) This study aims to demonstrate the causal involvement of renin angiotensin system (RAS) and oxidative stress (OS) on vascular inflammation in an experimental model of metabolic syndrome (MS) achieved by fructose administration to spontaneously hypertensive rats (FFHR) during 12 weeks. (2) Chronic treatment with candesartan (C) (10 mg/kg per day for the last 6 weeks) or 4OH-Tempol (T) (10(-3) mmol/L in drinking water for the last 6 weeks) reversed the increment in metabolic variables and systolic blood pressure. In addition, chronic C treatment reverted cardiovascular remodeling but not T. (3) Furthermore, chronic treatment with C was able to completely reverse the expression of NF-κB and VCAM-1, but T only reduced the expression. C reduced the expression of proatherogenic cytokines as CINC2, CINC3, VEGF, Leptin, TNF-alpha, and MCP-1 and also significantly reduced MIP-3, beta-NGF, and INF-gamma in vascular tissue in this experimental model. T was not able to substantially modify the expression of these cytokines. (4) The data suggest the involvement of RAS in the expression of inflammatory proteins at different vascular levels, allowing the creation of a microenvironment suitable for the creation, perpetuation, growth, and destabilization of vascular injury.

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

PubMed

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

2014-12-20

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

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

PubMed

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

2014-09-30

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

Expression and characterization of codon-optimized Crimean-Congo hemorrhagic fever virus Gn glycoprotein in insect cells.

PubMed

Rahpeyma, Mehdi; Samarbaf-Zadeh, Alireza; Makvandi, Manoochehr; Ghadiri, Ata A; Dowall, Stuart D; Fotouhi, Fatemeh

2017-07-01

Crimean-Congo hemorrhagic fever virus (CCHFV) is a major cause of tick-borne viral hemorrhagic disease in the world. Despite of its importance as a deadly pathogen, there is currently no licensed vaccine against CCHF disease. The attachment glycoprotein of CCHFV (Gn) is a potentially important target for protective antiviral immune responses. To characterize the expression of recombinant CCHFV Gn in an insect-cell-based system, we developed a gene expression system expressing the full-length coding sequence under a polyhedron promoter in Sf9 cells using recombinant baculovirus. Recombinant Gn was purified by affinity chromatography, and the immunoreactivity of the protein was evaluated using sera from patients with confirmed CCHF infection. Codon-optimized Gn was successfully expressed, and the product had the expected molecular weight for CCHFV Gn glycoprotein of 37 kDa. In time course studies, the optimum expression of Gn occurred between 36 and 48 hours postinfection. The immunoreactivity of the recombinant protein in Western blot assay against human sera was positive and was similar to the results obtained with the anti-V5 tag antibody. Additionally, mice were subjected to subcutaneous injection with recombinant Gn, and the cellular and humoral immune response was monitored. The results showed that recombinant Gn protein was highly immunogenic and could elicit high titers of antigen-specific antibodies. Induction of the inflammatory cytokine interferon-gamma and the regulatory cytokine IL-10 was also detected. In conclusion, a recombinant baculovirus harboring CCHFV Gn was constructed and expressed in Sf9 host cells for the first time, and it was demonstrated that this approach is a suitable expression system for producing immunogenic CCHFV Gn protein without any biosafety concerns.

Methodology development for quantitative optimization of security enhancement in medical information systems -Case study in a PACS and a multi-institutional radiotherapy database-.

PubMed

Haneda, Kiyofumi; Umeda, Tokuo; Koyama, Tadashi; Harauchi, Hajime; Inamura, Kiyonari

2002-01-01

The target of our study is to establish the methodology for analyzing level of security requirements, for searching suitable security measures and for optimizing security distribution to every portion of medical practice. Quantitative expression must be introduced to our study as possible for the purpose of easy follow up of security procedures and easy evaluation of security outcomes or results. Results of system analysis by fault tree analysis (FTA) clarified that subdivided system elements in detail contribute to much more accurate analysis. Such subdivided composition factors very much depended on behavior of staff, interactive terminal devices, kinds of service, and routes of network. As conclusion, we found the methods to analyze levels of security requirements for each medical information systems employing FTA, basic events for each composition factor and combination of basic events. Methods for searching suitable security measures were found. Namely risk factors for each basic event, number of elements for each composition factor and candidates of security measure elements were found. Method to optimize the security measures for each medical information system was proposed. Namely optimum distribution of risk factors in terms of basic events were figured out, and comparison of them between each medical information systems became possible.

Extensive characterization of a lentiviral-derived stable cell line expressing rabbit hemorrhagic disease virus VPg protein.

PubMed

Zhu, Jie; Miao, Qiuhong; Tan, Yonggui; Guo, Huimin; Li, Chuanfeng; Chen, Zongyan; Liu, Guangqing

2016-11-01

Rabbit hemorrhagic disease virus (RHDV) is an important member of the caliciviridae family. Currently, no suitable tissue culture system is available for proliferating RHDV, which limits the study of its pathogenesis. To bypass this obstacle, we established a cell line, RK13-VPg, stably expressing the VPg gene with a lentivirus packaging system in this study. In addition, the recently constructed RHDV replicon in our laboratory provided an appropriate model for studying the pathogenesis of RHDV without in vitro RHDV propagation and culture. Using this RHDV replicon and RK13-VPg cell line, we further demonstrated that the presence of VPg protein is essential for efficient translation of an RHDV replicon. Therefore, the RK13-VPg cell line is a powerful tool for studying the replication and translation mechanisms of RHDV. Copyright © 2016 Elsevier B.V. All rights reserved.

Monitoring protein-protein interactions using split synthetic renilla luciferase protein-fragment-assisted complementation.

PubMed

Paulmurugan, R; Gambhir, S S

2003-04-01

In this study we developed an inducible synthetic renilla luciferase protein-fragment-assisted complementation-based bioluminescence assay to quantitatively measure real time protein-protein interactions in mammalian cells. We identified suitable sites to generate fragments of N and C portions of the protein that yield significant recovered activity through complementation. We validate complementation-based activation of split synthetic renilla luciferase protein driven by the interaction of two strongly interacting proteins, MyoD and Id, in five different cell lines utilizing transient transfection studies. The expression level of the system was also modulated by tumor necrosis factor alpha through NFkappaB-promoter/enhancer elements used to drive expression of the N portion of synthetic renilla luciferase reporter gene. This new system should help in studying protein-protein interactions and when used with other split reporters (e.g., split firefly luciferase) should help to monitor different components of an intracellular network.

Monitoring Protein–Protein Interactions Using Split Synthetic Renilla Luciferase Protein-Fragment-Assisted Complementation

PubMed Central

Paulmurugan, R.; Gambhir, S. S.

2014-01-01

In this study we developed an inducible synthetic renilla luciferase protein-fragment-assisted complementation-based bioluminescence assay to quantitatively measure real time protein–protein interactions in mammalian cells. We identified suitable sites to generate fragments of N and C portions of the protein that yield significant recovered activity through complementation. We validate complementation-based activation of split synthetic renilla luciferase protein driven by the interaction of two strongly interacting proteins, MyoD and Id, in five different cell lines utilizing transient transfection studies. The expression level of the system was also modulated by tumor necrosis factor α through NFκB-promoter/enhancer elements used to drive expression of the N portion of synthetic renilla luciferase reporter gene. This new system should help in studying protein–protein interactions and when used with other split reporters (e.g., split firefly luciferase) should help to monitor different components of an intracellular network. PMID:12705589

A Novel System for Simultaneous or Sequential Integration of Multiple Gene-Loading Vectors into a Defined Site of a Human Artificial Chromosome

PubMed Central

Suzuki, Teruhiko; Kazuki, Yasuhiro; Oshimura, Mitsuo; Hara, Takahiko

2014-01-01

Human artificial chromosomes (HACs) are gene-delivery vectors suitable for introducing large DNA fragments into mammalian cells. Although a HAC theoretically incorporates multiple gene expression cassettes of unlimited DNA size, its application has been limited because the conventional gene-loading system accepts only one gene-loading vector (GLV) into a HAC. We report a novel method for the simultaneous or sequential integration of multiple GLVs into a HAC vector (designated as the SIM system) via combined usage of Cre, FLP, Bxb1, and φC31 recombinase/integrase. As a proof of principle, we first attempted simultaneous integration of three GLVs encoding EGFP, Venus, and TdTomato into a gene-loading site of a HAC in CHO cells. These cells successfully expressed all three fluorescent proteins. Furthermore, microcell-mediated transfer of HACs enabled the expression of those fluorescent proteins in recipient cells. We next demonstrated that GLVs could be introduced into a HAC one-by-one via reciprocal usage of recombinase/integrase. Lastly, we introduced a fourth GLV into a HAC after simultaneous integration of three GLVs by FLP-mediated DNA recombination. The SIM system expands the applicability of HAC vectors and is useful for various biomedical studies, including cell reprogramming. PMID:25303219

A novel system for simultaneous or sequential integration of multiple gene-loading vectors into a defined site of a human artificial chromosome.

PubMed

Suzuki, Teruhiko; Kazuki, Yasuhiro; Oshimura, Mitsuo; Hara, Takahiko

2014-01-01

Human artificial chromosomes (HACs) are gene-delivery vectors suitable for introducing large DNA fragments into mammalian cells. Although a HAC theoretically incorporates multiple gene expression cassettes of unlimited DNA size, its application has been limited because the conventional gene-loading system accepts only one gene-loading vector (GLV) into a HAC. We report a novel method for the simultaneous or sequential integration of multiple GLVs into a HAC vector (designated as the SIM system) via combined usage of Cre, FLP, Bxb1, and φC31 recombinase/integrase. As a proof of principle, we first attempted simultaneous integration of three GLVs encoding EGFP, Venus, and TdTomato into a gene-loading site of a HAC in CHO cells. These cells successfully expressed all three fluorescent proteins. Furthermore, microcell-mediated transfer of HACs enabled the expression of those fluorescent proteins in recipient cells. We next demonstrated that GLVs could be introduced into a HAC one-by-one via reciprocal usage of recombinase/integrase. Lastly, we introduced a fourth GLV into a HAC after simultaneous integration of three GLVs by FLP-mediated DNA recombination. The SIM system expands the applicability of HAC vectors and is useful for various biomedical studies, including cell reprogramming.

Development of an in vitro test system measuring transcriptional downregulatory activities on IL-13.

PubMed

Choi, Jeong June; Park, Bo-Kyung; Park, Sunyoung; Yun, Chi-Young; Kim, Dong Hee; Kim, Jin Sook; Hwang, Eun Sook; Jin, Mirim

2009-03-01

Interleukin-13 (IL-13) has been proposed as a therapeutic target for bronchial asthma as it plays crucial roles in the pathogenesis of the disease. We developed an in vitro test system measuring transcriptional downregulatory activities on IL-13 as a primary screening method to select drug candidates from natural products. The promoter region of IL-13 (-2,048 to +1) was cloned into the upstream of a luciferase gene in the plasmid pGL4.14 containing the hygromycin resistance gene as a selection marker, generating pGL4.14-IL-13. The EL-4 thymoma and RBL-2H3 mast cells transiently expressing this plasmid highly produced the luciferase activities by responding to PI (PMA and ionomycin) stimulation up to 8-fold and 13-fold compared with the control, respectively, whereas cyclosporin A, a wellknown antiasthmatic agent, significantly downregulated the activities. The BF1 clone of RBL-2H3 cells constitutively expressing pGL4.14-IL-13 was established by selecting surviving cells under a constant lethal dose of hygromycin treatment. The feasibility of this system was evaluated by measuring the downregulatory activities of 354 natural products on the IL-13 promoter using the BF1 clone. An extract from Morus bombycis (named TBRC 156) significantly inhibited PI-induced luciferase activities and IL-13 mRNA expression, but not the protein expression. Fisetin (named TBRC 353) inhibited not only PI-induced luciferase activities and mRNA expression, but also the IL-13 protein secretion, whereas myricetin (named TBRC 354) could not suppress the IL-13 expression at all. Our data indicated that this in vitro test system is able to discriminate the effects on IL-13 expression, and furthermore, that it might be suitable as a simple and time-saving primary screening system to select antiasthmatic agents by measuring transcriptional activities of the IL-13 promoter.

Production of recombinant antigens and antibodies in Nicotiana benthamiana using 'magnifection' technology: GMP-compliant facilities for small- and large-scale manufacturing.

PubMed

Klimyuk, Victor; Pogue, Gregory; Herz, Stefan; Butler, John; Haydon, Hugh

2014-01-01

This review describes the adaptation of the plant virus-based transient expression system, magnICON(®) for the at-scale manufacturing of pharmaceutical proteins. The system utilizes so-called "deconstructed" viral vectors that rely on Agrobacterium-mediated systemic delivery into the plant cells for recombinant protein production. The system is also suitable for production of hetero-oligomeric proteins like immunoglobulins. By taking advantage of well established R&D tools for optimizing the expression of protein of interest using this system, product concepts can reach the manufacturing stage in highly competitive time periods. At the manufacturing stage, the system offers many remarkable features including rapid production cycles, high product yield, virtually unlimited scale-up potential, and flexibility for different manufacturing schemes. The magnICON system has been successfully adaptated to very different logistical manufacturing formats: (1) speedy production of multiple small batches of individualized pharmaceuticals proteins (e.g. antigens comprising individualized vaccines to treat NonHodgkin's Lymphoma patients) and (2) large-scale production of other pharmaceutical proteins such as therapeutic antibodies. General descriptions of the prototype GMP-compliant manufacturing processes and facilities for the product formats that are in preclinical and clinical testing are provided.

Structured representation for requirements and specifications

NASA Technical Reports Server (NTRS)

Cohen, Gerald C.; Fisher, Gene; Frincke, Deborah; Wolber, Dave

1991-01-01

This document was generated in support of NASA contract NAS1-18586, Design and Validation of Digital Flight Control Systems suitable for Fly-By-Wire Applications, Task Assignment 2. Task 2 is associated with a formal representation of requirements and specifications. In particular, this document contains results associated with the development of a Wide-Spectrum Requirements Specification Language (WSRSL) that can be used to express system requirements and specifications in both stylized and formal forms. Included with this development are prototype tools to support the specification language. In addition a preliminary requirements specification methodology based on the WSRSL has been developed. Lastly, the methodology has been applied to an Advanced Subsonic Civil Transport Flight Control System.

Design and evaluation of an autonomous, obstacle avoiding, flight control system using visual sensors

NASA Astrophysics Data System (ADS)

Crawford, Bobby Grant

In an effort to field smaller and cheaper Uninhabited Aerial Vehicles (UAVs), the Army has expressed an interest in an ability of the vehicle to autonomously detect and avoid obstacles. Current systems are not suitable for small aircraft. NASA Langley Research Center has developed a vision sensing system that uses small semiconductor cameras. The feasibility of using this sensor for the purpose of autonomous obstacle avoidance by a UAV is the focus of the research presented in this document. The vision sensor characteristics are modeled and incorporated into guidance and control algorithms designed to generate flight commands based on obstacle information received from the sensor. The system is evaluated by simulating the response to these flight commands using a six degree-of-freedom, non-linear simulation of a small, fixed wing UAV. The simulation is written using the MATLAB application and runs on a PC. Simulations were conducted to test the longitudinal and lateral capabilities of the flight control for a range of airspeeds, camera characteristics, and wind speeds. Results indicate that the control system is suitable for obstacle avoiding flight control using the simulated vision system. In addition, a method for designing and evaluating the performance of such a system has been developed that allows the user to easily change component characteristics and evaluate new systems through simulation.

Efficiency of VIGS and gene expression in a novel bipartite potexvirus vector delivery system as a function of strength of TGB1 silencing suppression.

PubMed

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.

High-yield expression in Escherichia coli, purification and application of budding yeast K2 killer protein.

PubMed

Podoliankaitė, Monika; Lukša, Juliana; Vyšniauskis, Gintautas; Sereikaitė, Jolanta; Melvydas, Vytautas; Serva, Saulius; Servienė, Elena

2014-07-01

Saccharomyces cerevisiae K2 toxin is a highly active extracellular protein, important as a biocontrol agent for biotechnological applications in the wine industry. This protein is produced at negligible levels in yeast, making difficult to isolate it in amounts sufficient for investigation and generation of analysis tools. In this work, we demonstrate the use of a bacterial system for expression of the recombinant K2 protein, suitable for generation of antibodies specific for toxin of the yeast origin. Synthesis of the full-length S. cerevisiae K2 preprotoxin in Escherichia coli was found to be toxic to the host cell, resulting in diminished growth. Such effect was abolished by the introduction of the C-terminal truncation into K2 protein, directing it into non-toxic inclusion body fraction. The obtained protein is of limited solubility thus, facilitating the purification by simple and efficient chromatography-free procedure. The protein aggregates were successfully refolded into a soluble form yielding sufficient amounts of a tag-less truncated K2 protein suitable for polyclonal antibody production. Antibodies were raised in rabbit and found to be specific for detection of both antigen and native S. cerevisiae K2 toxin.

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research.

PubMed

Weidner, Christopher; Steinfath, Matthias; Wistorf, Elisa; Oelgeschläger, Michael; Schneider, Marlon R; Schönfelder, Gilbert

2017-08-16

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

Exploratory study on the effect of discount pricing strategies for new product introduction

NASA Astrophysics Data System (ADS)

Mat Zaib, Nurul Afiqah; Bazin, Nor Erne Nazira; Mustaffa, Noorfa Haszlinna

2013-04-01

Rapid introduction of new product into the market has resulted in growing competition between retailers. Nowadays, retailers compete with one another in order to increase revenue and to maintain their position in the marketplace. This situation has forced the retailers to enhance their strategic management as well as creating competitive advantages. Generally, this situation can be observed in highly demanded product such as fashion goods and high technology electronic devices (smart phone, notebook). The consequence from the intense competition and new product introduction is difficulties in retailers pricing management. Retailers are now facing with complexity in making decisions on suitable pricing strategies and discount level for new product in association with the product life cycle. Thus, this research aims to investigate the suitable discount pricing strategies that can be integrated in every phase of product life cycle. This paper presents relationships between the discount pricing and the stages in the product life cycle in the form of conceptual diagram and mathematical expression. A system dynamic approach is used for developing the conceptual diagram and formulating the mathematical expression for the discount pricing strategies to visualize the relationship between discount pricing and product life cycle.

Reference Gene Selection for Quantitative Real-Time Reverse-Transcriptase PCR in Annual Ryegrass (Lolium multiflorum) Subjected to Various Abiotic Stresses.

PubMed

Liu, Qiuxu; Qi, Xiao; Yan, Haidong; Huang, Linkai; Nie, Gang; Zhang, Xinquan

2018-01-16

To select the most stable reference genes in annual ryegrass ( Lolium multiflorum ), we studied annual ryegrass leaf tissues exposed to various abiotic stresses by qRT-PCR and selected 11 candidate reference genes, i.e., 18S rRNA, E2, GAPDH, eIF4A, HIS3, SAMDC, TBP-1, Unigene71, Unigene77, Unigene755, and Unigene14912. We then used GeNorm, NormFinder, and BestKeeper to analyze the expression stability of these 11 genes, and used RefFinder to comprehensively rank genes according to stability. Under different stress conditions, the most suitable reference genes for studies of leaf tissues of annual ryegrass were different. The expression of the eIF4A gene was the most stable under drought stress. Under saline-alkali stress, Unigene14912 has the highest expression stability. Under acidic aluminum stress, SAMDC expression stability was highest. Under heavy metal stress, Unigene71 expression had the highest stability. According to the software analyses, Unigene14912, HIS3, and eIF4A were the most suitable for analyses of abiotic stress in tissues of annual ryegrass. GAPDH was the least suitable reference gene. In conclusion, selecting appropriate reference genes under abiotic stress not only improves the accuracy of annual ryegrass gene expression analyses, but also provides a theoretical reference for the development of reference genes in plants of the genus Lolium .

Production and crystallization of recombinant JAK proteins.

PubMed

Lucet, Isabelle S; Bamert, Rebecca

2013-01-01

JAK kinases are critical mediators in development, differentiation, and homeostasis and accordingly, have become well-validated targets for drug discovery efforts. In recent years, the integration of X-ray crystallography in kinase-focused drug discovery programs has provided a powerful rationale for chemical modification by allowing a unique glimpse of a bound inhibitor to its target. Such structural information has not only led to an improved understanding of the key drivers of potency and specificity of several JAK-specific compounds but has greatly facilitated and accelerated the design of compounds with improved pharmacokinetic properties.JAK kinases are traditionally difficult candidates to express in significant quantities, generally requiring eukaryotic expression systems, protein engineering, mutations to yield soluble, homogeneous samples suitable for crystallization studies. Here we review the key methods utilized to express, purify, and crystallize the JAK kinases and provide a detail description of the methods that we have developed to express, purify, and crystallize recombinant JAK1 and JAK2 proteins in the presence of small molecule inhibitors.

Evaluation of Reference Genes for Normalization of Gene Expression Using Quantitative RT-PCR under Aluminum, Cadmium, and Heat Stresses in Soybean.

PubMed

Gao, Mengmeng; Liu, Yaping; Ma, Xiao; Shuai, Qin; Gai, Junyi; Li, Yan

2017-01-01

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is widely used to analyze the relative gene expression level, however, the accuracy of qRT-PCR is greatly affected by the stability of reference genes, which is tissue- and environment- dependent. Therefore, choosing the most stable reference gene in a specific tissue and environment is critical to interpret gene expression patterns. Aluminum (Al), cadmium (Cd), and heat stresses are three important abiotic factors limiting soybean (Glycine max) production in southern China. To identify the suitable reference genes for normalizing the expression levels of target genes by qRT-PCR in soybean response to Al, Cd and heat stresses, we studied the expression stability of ten commonly used housekeeping genes in soybean roots and leaves under these three abiotic stresses, using five approaches, BestKeeper, Delta Ct, geNorm, NormFinder and RefFinder. We found TUA4 is the most stable reference gene in soybean root tips under Al stress. Under Cd stress, Fbox and UKN2 are the most stable reference genes in roots and leaves, respectively, while 60S is the most suitable reference gene when analyzing both roots and leaves together. For heat stress, TUA4 and UKN2 are the most stable housekeeping genes in roots and leaves, respectively, and UKN2 is the best reference gene for analysis of roots and leaves together. To validate the reference genes, we quantified the relative expression levels of six target genes that were involved in soybean response to Al, Cd or heat stresses, respectively. The expression patterns of these target genes differed between using the most and least stable reference genes, suggesting the selection of a suitable reference gene is critical for gene expression studies.

Development of quantitative security optimization approach for the picture archives and carrying system between a clinic and a rehabilitation center

NASA Astrophysics Data System (ADS)

Haneda, Kiyofumi; Kajima, Toshio; Koyama, Tadashi; Muranaka, Hiroyuki; Dojo, Hirofumi; Aratani, Yasuhiko

2002-05-01

The target of our study is to analyze the level of necessary security requirements, to search for suitable security measures and to optimize security distribution to every portion of the medical practice. Quantitative expression must be introduced to our study, if possible, to enable simplified follow-up security procedures and easy evaluation of security outcomes or results. Using fault tree analysis (FTA), system analysis showed that system elements subdivided into groups by details result in a much more accurate analysis. Such subdivided composition factors greatly depend on behavior of staff, interactive terminal devices, kinds of services provided, and network routes. Security measures were then implemented based on the analysis results. In conclusion, we identified the methods needed to determine the required level of security and proposed security measures for each medical information system, and the basic events and combinations of events that comprise the threat composition factors. Methods for identifying suitable security measures were found and implemented. Risk factors for each basic event, a number of elements for each composition factor, and potential security measures were found. Methods to optimize the security measures for each medical information system were proposed, developing the most efficient distribution of risk factors for basic events.

A genetic replacement system for selection-based engineering of essential proteins

PubMed Central

2012-01-01

Background Essential genes represent the core of biological functions required for viability. Molecular understanding of essentiality as well as design of synthetic cellular systems includes the engineering of essential proteins. An impediment to this effort is the lack of growth-based selection systems suitable for directed evolution approaches. Results We established a simple strategy for genetic replacement of an essential gene by a (library of) variant(s) during a transformation. The system was validated using three different essential genes and plasmid combinations and it reproducibly shows transformation efficiencies on the order of 107 transformants per microgram of DNA without any identifiable false positives. This allowed for reliable recovery of functional variants out of at least a 105-fold excess of non-functional variants. This outperformed selection in conventional bleach-out strains by at least two orders of magnitude, where recombination between functional and non-functional variants interfered with reliable recovery even in recA negative strains. Conclusions We propose that this selection system is extremely suitable for evaluating large libraries of engineered essential proteins resulting in the reliable isolation of functional variants in a clean strain background which can readily be used for in vivo applications as well as expression and purification for use in in vitro studies. PMID:22898007

High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

PubMed Central

Dortay, Hakan; Akula, Usha Madhuri; Westphal, Christin; Sittig, Marie; Mueller-Roeber, Bernd

2011-01-01

Protein expression in heterologous hosts for functional studies is a cumbersome effort. Here, we report a superior platform for parallel protein expression in vivo and in vitro. The platform combines highly efficient ligation-independent cloning (LIC) with instantaneous detection of expressed proteins through N- or C-terminal fusions to infrared fluorescent protein (IFP). For each open reading frame, only two PCR fragments are generated (with three PCR primers) and inserted by LIC into ten expression vectors suitable for protein expression in microbial hosts, including Escherichia coli, Kluyveromyces lactis, Pichia pastoris, the protozoon Leishmania tarentolae, and an in vitro transcription/translation system. Accumulation of IFP-fusion proteins is detected by infrared imaging of living cells or crude protein extracts directly after SDS-PAGE without additional processing. We successfully employed the LIC-IFP platform for in vivo and in vitro expression of ten plant and fungal proteins, including transcription factors and enzymes. Using the IFP reporter, we additionally established facile methods for the visualisation of protein-protein interactions and the detection of DNA-transcription factor interactions in microtiter and gel-free format. We conclude that IFP represents an excellent reporter for high-throughput protein expression and analysis, which can be easily extended to numerous other expression hosts using the setup reported here. PMID:21541323

Development of an optimized tetracycline-inducible expression system to increase the accumulation of interleukin-10 in tobacco BY-2 suspension cells

PubMed Central

2012-01-01

Background Plant cell suspension cultures can be used for the production of valuable pharmaceutical and industrial proteins. When the recombinant protein is secreted into the culture medium, restricting expression to a defined growth phase can improve both the quality and quantity of the recovered product by minimizing proteolytic activity. Temporal restriction is also useful for recombinant proteins whose constitutive expression affects cell growth and viability, such as viral interleukin-10 (vIL-10). Results We have developed a novel, tetracycline-inducible system suitable for tobacco BY-2 suspension cells which increases the yields of vIL-10. The new system is based on a binary vector that is easier to handle than conventional vectors, contains an enhanced inducible promoter and 5′-UTR to improve yields, and incorporates a constitutively-expressed visible marker gene to allow the rapid and straightforward selection of the most promising transformed clones. Stable transformation of BY-2 cells with this vector, without extensive optimization of the induction conditions, led to a 3.5 fold increase in vIL-10 levels compared to constitutive expression in the same host. Conclusions We have developed an effective and straightforward molecular farming platform technology that improves both the quality and the quantity of recombinant proteins produced in plant cells, particularly those whose constitutive expression has a negative impact on plant growth and development. Although we tested the platform using vIL-10 produced in BY-2 cells, it can be applied to other host/product combinations and is also useful for basic research requiring strictly controlled transgene expression. PMID:22784336

Gluconate as suitable potential reduction supplier in Corynebacterium glutamicum: cloning and expression of gntP and gntK in Escherichia coli.

PubMed

Porco, Antonietta; Gamero, Elida E; Mylonás, Elena; Istúriz, Tomás

2008-01-01

Corynebacterium glutamicum is widely used in the industrial production of amino acids. We have found that this bacterium grows exponentially on a mineral medium supplemented with gluconate. Gluconate permease and Gluconokinase are expressed in an inducible form and, 6-phosphogluconate dehydrogenase, although constitutively expressed, shows a 3-fold higher specific level in gluconate grown cells than those grown in fructose under similar conditions. Interestingly, these activities are lower than those detected in the strain Escherichia coli M1-8, cultivated under similar conditions. Additionally, here we also confirmed that this bacterium lacks 6-phosphogluconate dehydratase activity. Thus, gluconate must be metabolized through the pentose phosphate pathway. Genes encoding gluconate transport and its phosphorylation were cloned from C. glutamicum, and expressed in suitable E. coli mutants. Sequence analysis revealed that the amino acid sequences obtained from these genes, denoted as gntP and gntK, were similar to those found in other bacteria. Analysis of both genes by RT-PCR suggested constitutive expression, in disagreement with the inducible character of their corresponding activities. The results suggest that gluconate might be a suitable source of reduction potential for improving the efficiency in cultures engaged in amino acids production. This is the first time that gluconate specific enzymatic activities are reported in C. glutamicum.

[Targeted therapy: toward a clean and effective war against cancer].

PubMed

Castronovo, V; Waltregny, D; Detry, O; Coimbra Marques, C; De Roover, A; Honoré, P; De Pauw, E; Turtoi, A

2009-01-01

One promising avenue towards the development of more selective, better anticancer drugs consists in the targeted delivery of bioactive compounds to the tumor environment by means of binding molecules specific for tumor-associated biomarkers. Eligibility of such markers for therapeutic use implies ideally three criteria : (i) accessibility from the bloodstream, (ii) expression at sufficient level and (iii) no (or much lower) expression in normal tissues. Most current discovery strategies (such as biomarker searching into body fluids) provide no clue as to whether proteins of interest are accessible, in human tissues, to suitable high-affinity ligands, such as systemically delivered monoclonal antibodies. Innovative proteomic technologies are able to identify such accessible biomarkers and represent a key step in the clinical development of such target therapies.

Relativistic electromagnetic waves in an electron-ion plasma

NASA Technical Reports Server (NTRS)

Chian, Abraham C.-L.; Kennel, Charles F.

1987-01-01

High power laser beams can drive plasma particles to relativistic energies. An accurate description of strong waves requires the inclusion of ion dynamics in the analysis. The equations governing the propagation of relativistic electromagnetic waves in a cold electron-ion plasma can be reduced to two equations expressing conservation of energy-momentum of the system. The two conservation constants are functions of the plasma stream velocity, the wave velocity, the wave amplitude, and the electron-ion mass ratio. The dynamic parameter, expressing electron-ion momentum conversation in the laboratory frame, can be regarded as an adjustable quantity, a suitable choice of which will yield self-consistent solutions when other plasma parameters were specified. Circularly polarized electromagnetic waves and electrostatic plasma waves are used as illustrations.

Geometric algebra description of polarization mode dispersion, polarization-dependent loss, and Stokes tensor transformations.

PubMed

Soliman, George; Yevick, David; Jessop, Paul

2014-09-01

This paper demonstrates that numerous calculations involving polarization transformations can be condensed by employing suitable geometric algebra formalism. For example, to describe polarization mode dispersion and polarization-dependent loss, both the material birefringence and differential loss enter as bivectors and can be combined into a single symmetric quantity. Their frequency and distance evolution, as well as that of the Stokes vector through an optical system, can then each be expressed as a single compact expression, in contrast to the corresponding Mueller matrix formulations. The intrinsic advantage of the geometric algebra framework is further demonstrated by presenting a simplified derivation of generalized Stokes parameters that include the electric field phase. This procedure simultaneously establishes the tensor transformation properties of these parameters.

[Cordyceps sinensis enhances lymphocyte proliferation and CD markers expression in simulated microgravity environment].

PubMed

Hao, Tong; Li, Jun-Jie; Du, Zhi-Yan; Duan, Cui-Mi; Wang, Yan-Meng; Wang, Chang-Yong; Song, Jing-Ping; Wang, Lin-Jie; Li, Ying-Hui; Wang, Yan

2012-10-01

This study was aimed to explore the effect of cordyceps sinensis enhancing lymphocyte proliferation and surface CD marker expression in simulated microgravity environment. The splenic lymphocytes were separated from mice and cultured in the rotary cell culture system simulated microgravity environment. The cells were treated with different concentration of cordyceps sinensis solution (0, 6.25, 12.5, 25 and 50 µg/ml) for 24, 48 and 72 h respectively, then the cells were harvested, and analyzed for cell proliferation and the expression of cell surface markers (CD4 and CD8). The results showed that under simulated microgravity environment, the lymphocyte proliferation was inhibited. When the concentration of cordyceps sinensis was 25 or 50 µg/ml, the lymphocyte proliferation, CD4 and CD8 expressions all increased, but 50 µg/ml cordyceps sinensis could inhibit the proliferation ability with the time prolonging. It is concluded that the suitable concentration of cordyceps sinensis displayed the ability to enhance the lymphocyte proliferation and CD marker expression in simulated microgravity environment. These results may be valuable for screening drugs which can be potentially against immunosuppression under simulated microgravity.

Osmoregulated TAQ polymerase gene expression in Escherichia coli.

PubMed

Cabrera Artiles, Yeosvany; Martínez García, Duniesky; Pérez Cruz, Enrique R; Márquez Perera, Gabriel J; Feble, Manuel Luis

2002-01-01

The Thermus aquaticus DNA Polymerase I (Taq Pol I) gene was cloned into the pOSEX4 plasmid under the osmo-inducible promoter proU and subsequently expressed into the Escherichia coli MKH13 strain. The suitability of the enzyme in polymerase assays was determined in standard 35S dATP incorporation tests and by PCR. The Taq Pol I expression in this system, which is under the control of the osmotic pressure in the growth medium, was analyzed in different media and in different sodium chloride concentrations. A study of the osmolarity effects in the growth of the strain and in Taq Pol I expression shows that an increase in sodium chloride concentration limits the growth. At 0.25 M of NaCl maximum activity was observed; at higher values of osmolarity, we found an unexpected decline of activity. This is the first report of using the pOSEX vector for the expression of an heterologous protein and it is very advantageous to make a regulated, non toxic, simple and cost-effective manner of induction in a biotechnology process using just NaCl or other non-permeable osmolyte.

Rift Valley Fever Virus Structural and Nonstructural Proteins: Recombinant Protein Expression and Immunoreactivity Against Antisera from Sheep

PubMed Central

Faburay, Bonto; Wilson, William; McVey, D. Scott; Drolet, Barbara S.; Weingartl, Hana; Madden, Daniel; Young, Alan; Ma, Wenjun

2013-01-01

Abstract The Rift Valley fever virus (RVFV) encodes the structural proteins nucleoprotein (N), aminoterminal glycoprotein (Gn), carboxyterminal glycoprotein (Gc), and L protein, 78-kD, and the nonstructural proteins NSm and NSs. Using the baculovirus system, we expressed the full-length coding sequence of N, NSs, NSm, Gc, and the ectodomain of the coding sequence of the Gn glycoprotein derived from the virulent strain of RVFV ZH548. Western blot analysis using anti-His antibodies and monoclonal antibodies against Gn and N confirmed expression of the recombinant proteins, and in vitro biochemical analysis showed that the two glycoproteins, Gn and Gc, were expressed in glycosylated form. Immunoreactivity profiles of the recombinant proteins in western blot and in indirect enzyme-linked immunosorbent assay against a panel of antisera obtained from vaccinated or wild type (RVFV)-challenged sheep confirmed the results obtained with anti-His antibodies and demonstrated the suitability of the baculo-expressed antigens for diagnostic assays. In addition, these recombinant proteins could be valuable for the development of diagnostic methods that differentiate infected from vaccinated animals (DIVA). PMID:23962238

Reference genes for measuring mRNA expression.

PubMed

Dundas, Jitesh; Ling, Maurice

2012-12-01

The aim of this review is to find answers to some of the questions surrounding reference genes and their reliability for quantitative experiments. Reference genes are assumed to be at a constant expression level, over a range of conditions such as temperature. These genes, such as GADPH and beta-actin, are used extensively for gene expression studies using techniques like quantitative PCR. There have been several studies carried out on identifying reference genes. However, a lot of evidence indicates issues to the general suitability of these genes. Recent studies had shown that different factors, including the environment and methods, play an important role in changing the expression levels of the reference genes. Thus, we conclude that there is no reference gene that can deemed suitable for all the experimental conditions. In addition, we believe that every experiment will require the scientific evaluation and selection of the best candidate gene for use as a reference gene to obtain reliable scientific results.

Rapid Communication: MiR-92a as a housekeeping gene for analysis of bovine mastitis-related microRNA in milk.

PubMed

Lai, Y C; Fujikawa, T; Ando, T; Kitahara, G; Koiwa, M; Kubota, C; Miura, N

2017-06-01

Our aim was to identify a suitable microRNA housekeeping gene for real-time PCR analysis of bovine mastitis-related microRNA in milk. We identified , , and as housekeeping gene candidates on the basis of previous Solexa sequencing results. Threshold cycle (CT) values for , , and did not differ between milk from control cows and milk from mastitis-affected cows. NormFinder software identified as the most stable single housekeeping gene. We evaluated the suitability of the housekeeping gene candidates by using them to assess expression levels of the inflammation-related gene . Regardless of the housekeeping gene candidates used for normalization, relative expression levels of were significantly higher in mastitis-affected samples than in control samples. However, of all the housekeeping genes and gene combinations investigated, normalization with alone generated the difference in relative expression between mastitis-affected and control samples with the highest significance. These results suggest that is suitable for use as a housekeeping gene for analysis of bovine mastitis-related microRNA in milk.

Combining geographic information system, multicriteria evaluation techniques and fuzzy logic in siting MSW landfills

NASA Astrophysics Data System (ADS)

Gemitzi, Alexandra; Tsihrintzis, Vassilios A.; Voudrias, Evangelos; Petalas, Christos; Stravodimos, George

2007-01-01

This study presents a methodology for siting municipal solid waste landfills, coupling geographic information systems (GIS), fuzzy logic, and multicriteria evaluation techniques. Both exclusionary and non-exclusionary criteria are used. Factors, i.e., non-exclusionary criteria, are divided in two distinct groups which do not have the same level of trade off. The first group comprises factors related to the physical environment, which cannot be expressed in terms of monetary cost and, therefore, they do not easily trade off. The second group includes those factors related to human activities, i.e., socioeconomic factors, which can be expressed as financial cost, thus showing a high level of trade off. GIS are used for geographic data acquisition and processing. The analytical hierarchy process (AHP) is the multicriteria evaluation technique used, enhanced with fuzzy factor standardization. Besides assigning weights to factors through the AHP, control over the level of risk and trade off in the siting process is achieved through a second set of weights, i.e., order weights, applied to factors in each factor group, on a pixel-by-pixel basis, thus taking into account the local site characteristics. The method has been applied to Evros prefecture (NE Greece), an area of approximately 4,000 km2. The siting methodology results in two intermediate suitability maps, one related to environmental and the other to socioeconomic criteria. Combination of the two intermediate maps results in the final composite suitability map for landfill siting.

Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells

PubMed Central

Takayama, Naoya; Nishimura, Satoshi; Nakamura, Sou; Shimizu, Takafumi; Ohnishi, Ryoko; Endo, Hiroshi; Yamaguchi, Tomoyuki; Otsu, Makoto; Nishimura, Ken; Nakanishi, Mahito; Sawaguchi, Akira; Nagai, Ryozo; Takahashi, Kazutoshi; Yamanaka, Shinya; Nakauchi, Hiromitsu

2010-01-01

Human (h) induced pluripotent stem cells (iPSCs) are a potentially abundant source of blood cells, but how best to select iPSC clones suitable for this purpose from among the many clones that can be simultaneously established from an identical source is not clear. Using an in vitro culture system yielding a hematopoietic niche that concentrates hematopoietic progenitors, we show that the pattern of c-MYC reactivation after reprogramming influences platelet generation from hiPSCs. During differentiation, reduction of c-MYC expression after initial reactivation of c-MYC expression in selected hiPSC clones was associated with more efficient in vitro generation of CD41a+CD42b+ platelets. This effect was recapitulated in virus integration-free hiPSCs using a doxycycline-controlled c-MYC expression vector. In vivo imaging revealed that these CD42b+ platelets were present in thrombi after laser-induced vessel wall injury. In contrast, sustained and excessive c-MYC expression in megakaryocytes was accompanied by increased p14 (ARF) and p16 (INK4A) expression, decreased GATA1 expression, and impaired production of functional platelets. These findings suggest that the pattern of c-MYC expression, particularly its later decline, is key to producing functional platelets from selected iPSC clones. PMID:21098095

Selection of suitable reference genes from bone cells in large gradient high magnetic field based on GeNorm algorithm.

PubMed

Di, Shengmeng; Tian, Zongcheng; Qian, Airong; Gao, Xiang; Yu, Dan; Brandi, Maria Luisa; Shang, Peng

2011-12-01

Studies of animals and humans subjected to spaceflight demonstrate that weightlessness negatively affects the mass and mechanical properties of bone tissue. Bone cells could sense and respond to the gravity unloading, and genes sensitive to gravity change were considered to play a critical role in the mechanotransduction of bone cells. To evaluate the fold-change of gene expression, appropriate reference genes should be identified because there is no housekeeping gene having stable expression in all experimental conditions. Consequently, expression stability of ten candidate housekeeping genes were examined in osteoblast-like MC3T3-E1, osteocyte-like MLO-Y4, and preosteoclast-like FLG29.1 cells under different apparent gravities (μg, 1 g, and 2 g) in the high-intensity gradient magnetic field produced by a superconducting magnet. The results showed that the relative expression of these ten candidate housekeeping genes was different in different bone cells; Moreover, the most suitable reference genes of the same cells in altered gravity conditions were also different from that in strong magnetic field. It demonstrated the importance of selecting suitable reference genes in experimental set-ups. Furthermore, it provides an alternative choice to the traditionally accepted housekeeping genes used so far about studies of gravitational biology and magneto biology.

Using RNA-Seq data to select refence genes for normalizing gene expression in apple roots

USDA-ARS?s Scientific Manuscript database

Gene expression in apple roots in response to various stress conditions is a less-explored research subject. Reliable reference genes for normalizing quantitative gene expression data have not been carefully investigated. In this study, the suitability of a set of 15 apple genes were evaluated for t...

Transient Expression of Chimeric Fluorescent Reporter Proteins in Pollen Tubes to Study Protein Polar Secretion and Dynamics.

PubMed

Zhong, Guitao; Liu, Ronghe; Zhuang, Menglong; Wang, Hao

2017-01-01

Transient expression of chimeric fluorescent reporter proteins by biolistic bombardment is a quick and useful procedure for studying subcellular protein localization and dynamics in plants. It is especially beneficial in specific plant cells which are not suitable for protoplast-based and Agrobacterium-mediated protein transient expression. Polar protein secretion and vesicular trafficking play essential functions for cell polarization and tip growth. The growing pollen tube is regarded as an ideal model plant cell system to study the machinery and regulation of polar protein trafficking and targeting. A large amount of newly synthesized proteins are packed and polarly transported to the apical region to support the rapid and highly polarized tip growth. Here, we described a detailed step-by-step protocol for the transient expression of chimeric fluorescent reporter proteins in growing Arabidopsis and tobacco pollen tubes to study polar transportation logistics and mechanisms. In addition, we have optimized the Arabidopsis and tobacco in vitro pollen germination medium and the conditions to maximize the efficiency of protein expression. As a proof of concept, we have used this protocol to express actin microfilament and late endosomal fluorescent markers in Arabidopsis and tobacco pollen tubes.

Gene silencing in Escherichia coli using antisense RNAs expressed from doxycycline-inducible vectors.

PubMed

Nakashima, N; Tamura, T

2013-06-01

Here, we report on the construction of doxycycline (tetracycline analogue)-inducible vectors that express antisense RNAs in Escherichia coli. Using these vectors, the expression of genes of interest can be silenced conditionally. The expression of antisense RNAs from the vectors was more tightly regulated than the previously constructed isopropyl-β-D-galactopyranoside-inducible vectors. Furthermore, expression levels of antisense RNAs were enhanced by combining the doxycycline-inducible promoter with the T7 promoter-T7 RNA polymerase system; the T7 RNA polymerase gene, under control of the doxycycline-inducible promoter, was integrated into the lacZ locus of the genome without leaving any antibiotic marker. These vectors are useful for investigating gene functions or altering cell phenotypes for biotechnological and industrial applications. A gene silencing method using antisense RNAs in Escherichia coli is described, which facilitates the investigation of bacterial gene function. In particular, the method is suitable for comprehensive analyses or phenotypic analyses of genes essential for growth. Here, we describe expansion of vector variations for expressing antisense RNAs, allowing choice of a vector appropriate for the target genes or experimental purpose. © 2013 The Society for Applied Microbiology.

Vitellogenin and vitellogenin receptor gene expression profiles in Spodoptera exigua are related to host plant suitability.

PubMed

Zhao, Jing; Sun, Yang; Xiao, Liubin; Tan, Yongan; Jiang, Yiping; Bai, Lixin

2018-04-01

The beet armyworm Spodoptera exigua, a worldwide phytophagous pest, causes considerable economic agricultural losses. Understanding the relationship between its fecundity and the host plant is a basic and important component of early forecasting of beet armyworm outbreaks. However, little is known about the molecular mechanism by which distinct hosts affect S. exigua fecundity. In this study, key life-history parameters of S. exigua reared on distinct hosts were investigated; the host plants could be ranked as lettuce > shallot > tomato > celery in their order of suitability. Full-length S. exigua vitellogenin receptor (SeVgR) cDNA was cloned, and sex-, stage- and tissue-specific expression characteristics were assessed. Spodoptera exigua vitellogenin (SeVg) and SeVgR expression levels were markedly modulated by host nutrients (P < 0.05). SeVg and SeVgR expression levels were significantly higher in S. exigua reared on lettuce, the most preferred and most nutritive host, than in those reared on tomato and celery. Interestingly, significant linear regression correlations were found between SeVg and SeVgR expression levels and key S. exigua life-history parameters, especially life span, pupa weight, and female fecundity (P < 0.01). Host plant type and suitability could affect the expression pattern of SeVg and SeVgR, which influenced S. exigua fecundity. Vg and VgR have the potential to be used as molecular markers of S. exigua fecundity and for forecasting outbreaks of S. exigua on different hosts. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Choline supplementation alleviates fluoride-induced testicular toxicity by restoring the NGF and MEK expression in mice.

PubMed

Zhang, Jianhai; Zhang, Yufang; Liang, Chen; Wang, Nasui; Zheng, Heping; Wang, Jundong

2016-11-01

Fluoride is known to cause male reproductive toxicity, and the elucidation of its underlying mechanisms is an ongoing research focus in reproductive toxicology and epidemiology. Choline, an essential nutrient, has been extensively studied for its benefits in nervous system yet was rarely discussed for its prospective effect in male reproductive system. This study aims to explore the potential protective role of choline against NaF-induced male reproductive toxicity via MAPK pathway. The male mice were administrated by 150mg/L NaF in drinking water, 5.75g/kg choline in diet, and their combination respectively from maternal gestation to postnatal 15weeks. The results showed that fluoride exposure reduced body weight growth, lowered sperm count and survival percentages, altered testicular histology, down-regulated the mRNA expressions of NGF, Ras, Raf, and MEK genes in testes, as well as significantly decreased the expressions of both NGF and phosphor-MEK proteins in testes. Examination of data from choline-treated mice revealed that choline supplementation ameliorated these fluoride-induced changes. Taken together, our findings suggest that choline supplementation alleviates fluoride-induced testicular toxicity by restoring the NGF and phosphor-MEK expression. The suitable dosage and supplementation periods of choline await further exploration. Copyright © 2016 Elsevier Inc. All rights reserved.

Treatment strategy based on targeting P-glycoprotein on peripheral lymphocytes in patients with systemic autoimmune disease.

PubMed

Tsujimura, Shizuyo; Tanaka, Yoshiya

2012-02-01

Although corticosteroids, immunosuppressants and disease-modifying antirheumatic drugs (DMARDs) are widely used in the treatment of various systemic autoimmune diseases such as systemic lupus erythematosus (SLE), we often experience patients with systemic autoimmune diseases who are resistant to these treatments. P-glycoprotein (P-gp) of membrane transporters, a product of the multiple drug resistance (MDR)-1 gene, is known to play a pivotal role in the acquisition of drug resistance to chemotherapy in malignancy. However, the relevance of MDR-1 and P-gp to resting and activated lymphocytes, which are the major target in the treatment of systemic autoimmune diseases, remains unclear. Studies from our laboratories found surface expression of P-gp on peripheral lymphocytes in patients with SLE and a significant correlation between the expression level and disease activity. Such expression is induced not only by genotoxic stresses but also by various stimuli including cytokines, resulting in active efflux of drugs from the cytoplasm of lymphocytes, resulting in drug-resistance and high disease activity. However, the use of both P-gp antagonists (e.g., cyclosporine) and inhibition of P-gp synthesis with intensive immunosuppressive therapy successfully reduces the efflux of corticosteroids from lymphocytes in vitro, suggesting that P-gp antagonists and P-gp synthesis inhibitors could be used to overcome drug-resistance in vivo and improve outcome. In conclusion, lymphocytes activated by various stimuli in patients with highly active disease apparently acquire MDR-1-mediated multidrug resistance against corticosteroids and probably some DMARDs, which are substrates of P-gp. Inhibition/reduction of P-gp could overcome such drug resistance. The expression of P-gp on lymphocytes is a promising marker of drug resistance and a suitable target to combat drug resistance in patients with active systemic autoimmune diseases.

Identification of cis-elements conferring high levels of gene expression in non-green plastids.

PubMed

Zhang, Jiang; Ruf, Stephanie; Hasse, Claudia; Childs, Liam; Scharff, Lars B; Bock, Ralph

2012-10-01

Although our knowledge about the mechanisms of gene expression in chloroplasts has increased substantially over the past decades, next to nothing is known about the signals and factors that govern expression of the plastid genome in non-green tissues. Here we report the development of a quantitative method suitable for determining the activity of cis-acting elements for gene expression in non-green plastids. The in vivo assay is based on stable transformation of the plastid genome and the discovery that root length upon seedling growth in the presence of the plastid translational inhibitor kanamycin is directly proportional to the expression strength of the resistance gene nptII in transgenic tobacco plastids. By testing various combinations of promoters and translation initiation signals, we have used this experimental system to identify cis-elements that are highly active in non-green plastids. Surprisingly, heterologous expression elements from maize plastids were significantly more efficient in conferring high expression levels in root plastids than homologous expression elements from tobacco. Our work has established a quantitative method for characterization of gene expression in non-green plastid types, and has led to identification of cis-elements for efficient plastid transgene expression in non-green tissues, which are valuable tools for future transplastomic studies in basic and applied research. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

Evaluation of detergents for the soluble expression of alpha-helical and beta-barrel-type integral membrane proteins by a preparative scale individual cell-free expression system.

PubMed

Klammt, Christian; Schwarz, Daniel; Fendler, Klaus; Haase, Winfried; Dötsch, Volker; Bernhard, Frank

2005-12-01

Cell-free expression has become a highly promising tool for the fast and efficient production of integral membrane proteins. The proteins can be produced as precipitates that solubilize in mild detergents usually without any prior denaturation steps. Alternatively, membrane proteins can be synthesized in a soluble form by adding detergents to the cell-free system. However, the effects of a representative variety of detergents on the production, solubility and activity of a wider range of membrane proteins upon cell-free expression are currently unknown. We therefore analyzed the cell-free expression of three structurally very different membrane proteins, namely the bacterial alpha-helical multidrug transporter, EmrE, the beta-barrel nucleoside transporter, Tsx, and the porcine vasopressin receptor of the eukaryotic superfamily of G-protein coupled receptors. All three membrane proteins could be produced in amounts of several mg per one ml of reaction mixture. In general, the detergent 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] was found to be most effective for the resolubilization of membrane protein precipitates, while long chain polyoxyethylene-alkyl-ethers proved to be most suitable for the soluble expression of all three types of membrane proteins. The yield of soluble expressed membrane protein remained relatively stable above a certain threshold concentration of the detergents. We report, for the first time, the high-level cell-free expression of a beta-barrel type membrane protein in a functional form. Structural and functional variations of the analyzed membrane proteins are evident that correspond with the mode of expression and that depend on the supplied detergent.

Direct RNA detection without nucleic acid purification and PCR: Combining sandwich hybridization with signal amplification based on branched hybridization chain reaction.

PubMed

Xu, Yao; Zheng, Zhi

2016-05-15

We have developed a convenient, robust and low-cost RNA detection system suitable for high-throughput applications. This system uses a highly specific sandwich hybridization to capture target RNA directly onto solid support, followed by on-site signal amplification via 2-dimensional, branched hybridizing chain polymerization through toehold-mediated strand displacement reaction. The assay uses SYBR Green to detect targets at concentrations as low as 1 pM, without involving nucleic acid purification or any enzymatic reaction, using ordinary oligonucleotides without modification or labeling. The system was demonstrated in the detection of malaria RNA in blood and GAPDH gene expression in cell lysate. Copyright © 2015 Elsevier B.V. All rights reserved.

Limitations in transmitter-receiver distances for terrestrial microwave transmissions

NASA Astrophysics Data System (ADS)

Njau, E. C.

1990-12-01

Directional microwave repeater systems are still in significant use for radio-telephone communications as well as terrestrial TV transmissions. In these systems, the optimally maximum distance D(sub 0) between one transmitter and the next repeater-receiver partly depends on meteorological conditions since the refractive index n for microwaves is considerably influenced by these conditions. We show that, under dry atmospheric conditions, certain zonally travelling heat/temperature waves which are normally in the weather/climate system significantly vary n and hence inevitably impose specific conditions on the evaluation of D(sub 0). Finally we use some Tanzanian meteorological records to arrive at an expression for D(sub 0) that is suitable for regions whose meteorological conditions are comparable to those in Tanzania.

Functional Studies on Oligotropha carboxidovorans Molybdenum-Copper CO Dehydrogenase Produced in Escherichia coli.

PubMed

Kaufmann, Paul; Duffus, Benjamin R; Teutloff, Christian; Leimkühler, Silke

2018-04-30

The Mo/Cu-dependent CO dehydrogenase (CODH) from Oligotropha carboxidovorans is an enzyme that is able to catalyze both the oxidation of CO to CO 2 and the oxidation of H 2 to protons and electrons. Despite the close to atomic resolution structure (1.1 Å), significant uncertainties have remained with regard to the reaction mechanism of substrate oxidation at the unique Mo/Cu center, as well as the nature of intermediates formed during the catalytic cycle. So far, the investigation of the role of amino acids at the active site was hampered by the lack of a suitable expression system that allowed for detailed site-directed mutagenesis studies at the active site. Here, we report on the establishment of a functional heterologous expression system of O. carboxidovorans CODH in Escherichia coli. We characterize the purified enzyme in detail by a combination of kinetic and spectroscopic studies and show that it was purified in a form with characteristics comparable to those of the native enzyme purified from O. carboxidovorans. With this expression system in hand, we were for the first time able to generate active-site variants of this enzyme. Our work presents the basis for more detailed studies of the reaction mechanism for CO and H 2 oxidation of Mo/Cu-dependent CODHs in the future.

Tendon tissue engineering: adipose-derived stem cell and GDF-5 mediated regeneration using electrospun matrix systems.

PubMed

James, R; Kumbar, S G; Laurencin, C T; Balian, G; Chhabra, A B

2011-04-01

Tendon tissue engineering with a biomaterial scaffold that mimics the tendon extracellular matrix (ECM) and is biomechanically suitable, and when combined with readily available autologous cells, may provide successful regeneration of defects in tendon. Current repair strategies using suitable autografts and freeze-dried allografts lead to a slow repair process that is sub-optimal and fails to restore function, particularly in difficult clinical situations such as zone II flexor tendon injuries of the hand. We have investigated the effect of GDF-5 on cell proliferation and gene expression by primary rat adipose-derived stem cells (ADSCs) that were cultured on a poly(DL-lactide-co-glycolide) PLAGA fiber scaffold and compared to a PLAGA 2D film scaffold. The electrospun scaffold mimics the collagen fiber bundles present in native tendon tissue, and supports the adhesion and proliferation of multipotent ADSCs. Gene expression of scleraxis, the neotendon marker, was upregulated seven- to eightfold at 1 week with GDF-5 treatment when cultured on a 3D electrospun scaffold, and was significantly higher at 2 weeks compared to 2D films with or without GDF-5 treatment. Expression of the genes that encode the major tendon ECM protein, collagen type I, was increased by fourfold starting at 1 week on treatment with 100 ng mL(-1) GDF-5, and at all time points the expression was significantly higher compared to 2D films irrespective of GDF-5 treatment. Thus stimulation with GDF-5 can modulate primary ADSCs on a PLAGA fiber scaffold to produce a soft, collagenous musculoskeletal tissue that fulfills the need for tendon regeneration.

Tendon tissue engineering: Adipose 1 derived stem cell and GDF-5 mediated regeneration using electrospun matrix systems

PubMed Central

James, R; Kumbar, S G; Laurencin, C T; Balian, G; Chhabra, A B

2011-01-01

Tendon tissue engineering with a biomaterial scaffold that mimics the tendon extracellular matrix (ECM) and is biomechanically suitable when combined with readily available autologous cells may provide successful regeneration of defects in tendon. Current repair strategies using suitable autografts and freeze-dried allografts lead to a slow repair process that is sub-optimal and fails to restore function, particularly in difficult clinical situations such as zone II flexor tendon injuries of the hand. We have investigated the effect of GDF-5 on cell proliferation and gene expression by primary rat adipose-derived stromal cells (ADSCs) that were cultured on poly(DL-lactide-co-glycolide) PLAGA fiber scaffold and compared to PLAGA 2D film scaffold. The electrospun scaffold mimics the collagen fiber bundles present in native tendon tissue, and supports the adhesion and proliferation of multipotent ADSCs. Gene expression of scleraxis, the neotendon marker was upregulated 7 – 8 fold at 1 week with GDF-5 treatment when cultured on 3D electrospun scaffold, and was significantly higher at 2 weeks compared to 2D films with or without GDF-5 treatment. Expression of the genes that encode the major tendon ECM protein, collagen type I, was increased by 4 fold starting at 1 week on treatment with 100ng/mL GDF-5, and at all time points the expression was significantly higher compared to 2D films irrespective of GDF-5 treatment. Thus stimulation with GDF-5 can modulate primary ADSCs on PLAGA fiber scaffold to produce a soft, collagenous musculoskeletal tissue that fulfills the need for tendon regeneration. PMID:21436509

The intervening removable affinity tag (iRAT) production system facilitates Fv antibody fragment‐mediated crystallography

PubMed Central

Nomura, Yayoi; Sato, Yumi; Suno, Ryoji; Horita, Shoichiro

2016-01-01

Abstract Fv antibody fragments have been used as co‐crystallization partners in structural biology, particularly in membrane protein crystallography. However, there are inherent technical issues associated with the large‐scale production of soluble, functional Fv fragments through conventional methods in various expression systems. To circumvent these problems, we developed a new method, in which a single synthetic polyprotein consisting of a variable light (VL) domain, an intervening removable affinity tag (iRAT), and a variable heavy (VH) domain is expressed by a Gram‐positive bacterial secretion system. This method ensures stoichiometric expression of VL and VH from the monocistronic construct followed by proper folding and assembly of the two variable domains. The iRAT segment can be removed by a site‐specific protease during the purification process to yield tag‐free Fv fragments suitable for crystallization trials. In vitro refolding step is not required to obtain correctly folded Fv fragments. As a proof of concept, we tested the iRAT‐based production of multiple Fv fragments, including a crystallization chaperone for a mammalian membrane protein as well as FDA‐approved therapeutic antibodies. The resulting Fv fragments were functionally active and crystallized in complex with the target proteins. The iRAT system is a reliable, rapid and broadly applicable means of producing milligram quantities of Fv fragments for structural and biochemical studies. PMID:27595817

Mucosal and systemic immune responses elicited by recombinant Lactococcus lactis expressing a fusion protein composed of pertussis toxin and filamentous hemagglutinin from Bordetella pertussis.

PubMed

Torkashvand, Ali; Bahrami, Fariborz; Adib, Minoo; Ajdary, Soheila

2018-05-05

We constructed a food-grade expression system harboring a F1S1 fusion protein of Bordetella pertussis to be produced in Lactococcus lactis NZ3900 as a new oral vaccine model against whooping cough, caused by B. pertussis. F1S1 was composed of N-terminally truncated S1 subunit of pertussis toxin and type I immunodominant domain of filamentous hemagglutinin which are both known as protective immunogens against pertussis. The recombinant L. lactis was administered via oral or intranasal routes to BALB/c mice and the related specific systemic and mucosal immune responses were then evaluated. The results indicated significantly higher levels of specific IgA in the lung extracts and IgG in sera of mucosally-immunized mice, compared to their controls. It was revealed that higher levels of IgG2a, compared to IgG1, were produced in all mucosally-immunized mice. Moreover, immunized mice developed Th1 responses with high levels of IFN-γ production by the spleen cells. These findings provide evidence for L. lactis to be used as a suitable vehicle for expression and delivery of F1S1 fusion protein to mucosa and induction of appropriate systemic and mucosal immune responses against pertussis. Copyright © 2018 Elsevier Ltd. All rights reserved.

Adenovirus vector covalently conjugated to polyethylene glycol with a cancer-specific promoter suppresses the tumor growth through systemic administration.

PubMed

Yao, Xinglei; Yoshioka, Yasuo; Morishige, Tomohiro; Eto, Yusuke; Narimatsu, Shogo; Mizuguchi, Hiroyuki; Mukai, Yohei; Okada, Naoki; Nakagawa, Shinsaku

2010-01-01

Cancer gene therapy with adenovirus vectors (Adv) is limited to local administration because systemic administration of Adv produces a weak therapeutic effect and severe side effects. Previously, we generated a dual cancer-specific Adv system by using Adv covalently conjugated to polyethylene glycol (PEG) for transductional targeting and the telomere reverse transcriptase (TERT) promoter as a cancer-specific promoter for transcriptional targeting (PEG-Ad-TERT). We demonstrated that systemic administration of PEG-Ad-TERT showed superior antitumor effects against lung metastatic cancer with negligible side effects. Here, we investigated the therapeutic efficacy of systemic administration of PEG-Ad-TERT for the treatment of primary tumors. We first evaluated the transgene expression of PEG-Ad-TERT containing the luciferase gene (PEG-Ad-TERT/Luc) in primary tumors. Systemic administration of PEG-Ad-TERT/Luc resulted high transgene expression, similar to that observed in tumors for the conventional cytomegalovirus (CMV) promoter-driven Adv containing the luciferase gene (Ad-CMV/Luc). By comparison, transgene expression was 2500-fold lower than that of Ad-CMV/Luc in liver. We then examined the therapeutic effect of systemic administration of PEG-Ad-TERT containing the herpes simplex virus thymidine kinase (HSVtk) gene (PEG-Ad-TERT/HSVtk) for the treatment of primary tumors. We showed that PEG-Ad-TERT/HSVtk produced a notable antitumor effect against primary tumors with negligible side effects. These results demonstrated that PEG-Ad-TERT can be regarded as a prototype Adv with suitable efficacy and safety for systemic cancer gene therapy against both metastatic and primary tumors.

Use of Brevibacillus choshinensis for the production of biologically active brain-derived neurotrophic factor (BDNF).

PubMed

Angart, Phillip A; Carlson, Rebecca J; Thorwall, Sarah; Patrick Walton, S

2017-07-01

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family critical for neuronal cell survival and differentiation, with therapeutic potential for the treatment of neurological disorders and spinal cord injuries. The production of recombinant, bioactive BDNF is not practical in most traditional microbial expression systems because of the inability of the host to correctly form the characteristic cystine-knot fold of BDNF. Here, we investigated Brevibacillus choshinensis as a suitable expression host for bioactive BDNF expression, evaluating the effects of medium type (2SY and TM), temperature (25 and 30 °C), and culture time (48-120 h). Maximal BDNF bioactivity (per unit mass) was observed in cultures grown in 2SY medium at extended times (96 h at 30 °C or >72 h at 25 °C), with resulting bioactivity comparable to that of a commercially available BDNF. For cultures grown in 2SY medium at 25 °C for 72 h, the condition that led to the greatest quantity of biologically active protein in the shortest culture time, we recovered 264 μg/L of BDNF. As with other microbial expression systems, BDNF aggregates did form in all culture conditions, indicating that while we were able to recover biologically active BDNF, further optimization of the expression system could yield still greater quantities of bioactive protein. This study provides confirmation that B. choshinensis is capable of producing biologically active BDNF and that further optimization of culture conditions could prove valuable in increasing BDNF yields.

Gene transfer to brain using herpes simplex virus vectors.

PubMed

Glorioso, J C; Goins, W F; Meaney, C A; Fink, D J; DeLuca, N A

1994-01-01

Herpes simplex virus type 1 represents an ideal candidate for development as a vehicle for gene transfer to postmitotic neurons of the central nervous system. The natural biology of this virus makes it well suited for this purpose as it is capable of infecting a variety of neuronal cell types in the brain where the viral genome can persist indefinitely in a latent state. In latency, the viral lytic genes are transcriptionally silent and a unique set of latency-associated transcripts are expressed. Two impediments to using herpes simplex virus vectors must be overcome: (1) A noncytotoxic mutant virus backbone must be engineered, and (2) a suitable promoter-regulator that stably expresses foreign genes from the vector genome during latency must be constructed. Deletion of specific immediate early genes from the vector can render the virus nontoxic to neurons in culture and in vivo following stereotactic inoculation into specific regions of the brain. Because these viruses cannot replicate, they enter latency on infection of central nervous system neurons. A number of viral and cellular promoters have been tested for their ability to express genes during latency. Strong viral promoters and neurospecific promoters display transient activity. Although the promoter regions for the latency-associated transcripts are highly active in the peripheral nervous system, they show low-level but persistent activity in the brain. Experiments are in progress to exploit RNA polymerase III gene promoters or novel recombinant promoters capable of auto-inducing their own expression in order to increase gene expression during latency in brain neurons.

Selection and Evaluation of Potential Reference Genes for Gene Expression Analysis in the Brown Planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) Using Reverse-Transcription Quantitative PCR

PubMed Central

Zhu, Xun; Wan, Hu; Shakeel, Muhammad; Zhan, Sha; Jin, Byung-Rae; Li, Jianhong

2014-01-01

The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is one of the most important rice pests. Abundant genetic studies on BPH have been conducted using reverse-transcription quantitative real-time PCR (qRT-PCR). Using qRT-PCR, the expression levels of target genes are calculated on the basis of endogenous controls. These genes need to be appropriately selected by experimentally assessing whether they are stably expressed under different conditions. However, such studies on potential reference genes in N. lugens are lacking. In this paper, we presented a systematic exploration of eight candidate reference genes in N. lugens, namely, actin 1 (ACT), muscle actin (MACT), ribosomal protein S11 (RPS11), ribosomal protein S15e (RPS15), alpha 2-tubulin (TUB), elongation factor 1 delta (EF), 18S ribosomal RNA (18S), and arginine kinase (AK) and used four alternative methods (BestKeeper, geNorm, NormFinder, and the delta Ct method) to evaluate the suitability of these genes as endogenous controls. We examined their expression levels among different experimental factors (developmental stage, body part, geographic population, temperature variation, pesticide exposure, diet change, and starvation) following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guidelines. Based on the results of RefFinder, which integrates four currently available major software programs to compare and rank the tested candidate reference genes, RPS15, RPS11, and TUB were found to be the most suitable reference genes in different developmental stages, body parts, and geographic populations, respectively. RPS15 was the most suitable gene under different temperature and diet conditions, while RPS11 was the most suitable gene under different pesticide exposure and starvation conditions. This work sheds light on establishing a standardized qRT-PCR procedure in N. lugens, and serves as a starting point for screening for reference genes for expression studies of related insects. PMID:24466124

Selection and evaluation of potential reference genes for gene expression analysis in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) using reverse-transcription quantitative PCR.

PubMed

Yuan, Miao; Lu, Yanhui; Zhu, Xun; Wan, Hu; Shakeel, Muhammad; Zhan, Sha; Jin, Byung-Rae; Li, Jianhong

2014-01-01

The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is one of the most important rice pests. Abundant genetic studies on BPH have been conducted using reverse-transcription quantitative real-time PCR (qRT-PCR). Using qRT-PCR, the expression levels of target genes are calculated on the basis of endogenous controls. These genes need to be appropriately selected by experimentally assessing whether they are stably expressed under different conditions. However, such studies on potential reference genes in N. lugens are lacking. In this paper, we presented a systematic exploration of eight candidate reference genes in N. lugens, namely, actin 1 (ACT), muscle actin (MACT), ribosomal protein S11 (RPS11), ribosomal protein S15e (RPS15), alpha 2-tubulin (TUB), elongation factor 1 delta (EF), 18S ribosomal RNA (18S), and arginine kinase (AK) and used four alternative methods (BestKeeper, geNorm, NormFinder, and the delta Ct method) to evaluate the suitability of these genes as endogenous controls. We examined their expression levels among different experimental factors (developmental stage, body part, geographic population, temperature variation, pesticide exposure, diet change, and starvation) following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guidelines. Based on the results of RefFinder, which integrates four currently available major software programs to compare and rank the tested candidate reference genes, RPS15, RPS11, and TUB were found to be the most suitable reference genes in different developmental stages, body parts, and geographic populations, respectively. RPS15 was the most suitable gene under different temperature and diet conditions, while RPS11 was the most suitable gene under different pesticide exposure and starvation conditions. This work sheds light on establishing a standardized qRT-PCR procedure in N. lugens, and serves as a starting point for screening for reference genes for expression studies of related insects.

Study on efficiency of different topologies of magnetic coupled resonant wireless charging system

NASA Astrophysics Data System (ADS)

Cui, S.; Liu, Z. Z.; Hou, Y. J.; Zeng, H.; Yue, Z. K.; Liang, L. H.

2017-11-01

This paper analyses the relationship between the output power, the transmission efficiency and the frequency, load and coupling coefficient of the four kinds of magnetic coupled resonant wireless charging system topologies. Based on mutual inductance principle, four kinds of circuit models are established, and the expressions of output power and transmission efficiency of different structures are calculated. The difference between the two power characteristics and efficiency characteristics is compared by simulating the SS (series-series) and SP (series-parallel) type wireless charging systems. With the same parameters of circuit components, the SS structure is usually suitable for small load resistance. The SP structure can be applied to large load resistors, when the transmission efficiency of the system is required to keep high. If the operating frequency deviates from the system resonance frequency, the SS type system has higher transmission efficiency than the SP type system.

Closing the Gap Between Specification and Programming: VDM++ and SCALA

NASA Technical Reports Server (NTRS)

Havelund, Klaus

2011-01-01

We argue that a modern programming language such as Scala offers a level of succinctness, which makes it suitable for program and systems specification as well as for high-level programming. We illustrate this by comparing the language with the Vdm++ specification language. The comparison also identifies areas where Scala perhaps could be improved, inspired by Vdm++. We furthermore illustrate Scala's potential as a specification language by augmenting it with a combination of parameterized state machines and temporal logic, defined as a library, thereby forming an expressive but simple runtime verification framework.

Reference gene stability of a synanthropic fly, Chrysomya megacephala.

PubMed

Wang, Xiaoyun; Xiong, Mei; Wang, Jialu; Lei, Chaoliang; Zhu, Fen

2015-10-29

Stable reference genes are essential for accurate normalization in gene expression studies with reverse transcription quantitative polymerase chain reaction (qPCR). A synanthropic fly, Chrysomya megacephala, is a well known medical vector and forensic indicator. Unfortunately, previous studies did not look at the stability of reference genes used in C. megacephala. In this study, the expression level of Actin, ribosomal protein L8 (Rpl8), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1α (EF1), α-tubulin (α-TUB), β-tubulin (β-TUB), TATA binding box (TBP), 18S rRNA (18S) and ribosomal protein S7 (Rps7) were evaluated for their stability using online software RefFinder, which combines the normal software of the ΔCt method, BestKeeper, Normfinder, and geNorm. Moreover the number of suitable reference gene pairs was also suggested by Excel-based geNorm. The expression levels of these reference genes were evaluated under different experimental conditions with special perspectives of forensic applications: developmental stages (eggs, first, second and third instar larvae, pupae and adults); food sources of larvae (pork, fish and chicken); feeding larvae with drugs (untreated control, Estazolam and Marvelon); feeding larvae with heavy metals (untreated control, cadmium and zinc); tissues of adults (head, thorax, abdomen, legs and wings). According to RefFinder, EF1 was the most suitable reference gene of developmental stages, food and tissues; 18S and GAPDH were the most suitable reference genes for drugs and heavy metals, respectively, which could be widely used for quantification of target gene expression with qPCR in C. megacephala. Suitable reference gene pairs were also suggested by geNorm. This fundamental but vital work should facilitate the gene studies of related biological processes and deepen the understanding in physiology, toxicology, and especially medical and forensic entomology of C. megacephala.

Identification of Reference Genes for Normalizing Quantitative Real-Time PCR in Urechis unicinctus

NASA Astrophysics Data System (ADS)

Bai, Yajiao; Zhou, Di; Wei, Maokai; Xie, Yueyang; Gao, Beibei; Qin, Zhenkui; Zhang, Zhifeng

2018-06-01

The reverse transcription quantitative real-time PCR (RT-qPCR) has become one of the most important techniques of studying gene expression. A set of valid reference genes are essential for the accurate normalization of data. In this study, five candidate genes were analyzed with geNorm, NormFinder, BestKeeper and ΔCt methods to identify the genes stably expressed in echiuran Urechis unicinctus, an important commercial marine benthic worm, under abiotic (sulfide stress) and normal (adult tissues, embryos and larvae at different development stages) conditions. The comprehensive results indicated that the expression of TBP was the most stable at sulfide stress and in developmental process, while the expression of EF- 1- α was the most stable at sulfide stress and in various tissues. TBP and EF- 1- α were recommended as a suitable reference gene combination to accurately normalize the expression of target genes at sulfide stress; and EF- 1- α, TBP and TUB were considered as a potential reference gene combination for normalizing the expression of target genes in different tissues. No suitable gene combination was obtained among these five candidate genes for normalizing the expression of target genes for developmental process of U. unicinctus. Our results provided a valuable support for quantifying gene expression using RT-qPCR in U. unicinctus.

The N Domain of Human Angiotensin-I-converting Enzyme

PubMed Central

Anthony, Colin S.; Corradi, Hazel R.; Schwager, Sylva L. U.; Redelinghuys, Pierre; Georgiadis, Dimitris; Dive, Vincent; Acharya, K. Ravi; Sturrock, Edward D.

2010-01-01

Angiotensin-I-converting enzyme (ACE) plays a critical role in the regulation of blood pressure through its central role in the renin-angiotensin and kallikrein-kinin systems. ACE contains two domains, the N and C domains, both of which are heavily glycosylated. Structural studies of ACE have been fraught with severe difficulties because of surface glycosylation of the protein. In order to investigate the role of glycosylation in the N domain and to create suitable forms for crystallization, we have investigated the importance of the 10 potential N-linked glycan sites using enzymatic deglycosylation, limited proteolysis, and mass spectrometry. A number of glycosylation mutants were generated via site-directed mutagenesis, expressed in CHO cells, and analyzed for enzymatic activity and thermal stability. At least eight of 10 of the potential glycan sites are glycosylated; three C-terminal sites were sufficient for expression of active N domain, whereas two N-terminal sites are important for its thermal stability. The minimally glycosylated Ndom389 construct was highly suitable for crystallization studies. The structure in the presence of an N domain-selective phosphinic inhibitor RXP407 was determined to 2.0 Å resolution. The Ndom389 structure revealed a hinge region that may contribute to the breathing motion proposed for substrate binding. PMID:20826823

Development of a Fluorescent Bodipy Probe for Visualization of the Serotonin 5-HT1A Receptor in Native Cells of the Immune System.

PubMed

Hernández-Torres, Gloria; Enríquez-Palacios, Ernesto; Mecha, Miriam; Feliú, Ana; Rueda-Zubiaurre, Ainoa; Angelina, Alba; Martín-Cruz, Leticia; Martín-Fontecha, Mar; Palomares, Oscar; Guaza, Carmen; Peña-Cabrera, Eduardo; López-Rodríguez, María L; Ortega-Gutiérrez, Silvia

2018-05-14

Serotonin (5-HT) modulates key aspects of the immune system. However, its precise function and the receptors involved in the observed effects have remained elusive. Among the different serotonin receptors, 5-HT 1A plays an important role in the immune system given its presence in cells involved in both the innate and adaptive immune responses, but its actual levels of expression under different conditions have not been comprehensively studied due to the lack of suitable tools. To further clarify the role of 5-HT 1A receptor in the immune system, we have developed a fluorescent small molecule probe that enables the direct study of the receptor levels in native cells. This probe allows direct profiling of the receptor expression in immune cells using flow cytometry. Our results show that important subsets of immune cells including human monocytes and dendritic cells express functional 5-HT 1A and that its activation is associated with anti-inflammatory signaling. Furthermore, application of the probe to the experimental autoimmune encephalomyelitis model of multiple sclerosis demonstrates its potential to detect the specific overexpression of the 5-HT 1A receptor in CD4+ T cells. Accordingly, the probe reported herein represents a useful tool whose use can be extended to study the levels of 5-HT 1A receptor in ex vivo samples of different immune system conditions.

An Efficient PEG/CaCl₂-Mediated Transformation Approach for the Medicinal Fungus Wolfiporia cocos.

PubMed

Sun, Qiao; Wei, Wei; Zhao, Juan; Song, Jia; Peng, Fang; Zhang, Shaopeng; Zheng, Yonglian; Chen, Ping; Zhu, Wenjun

2015-09-01

Sclerotia of Wolfiporia cocos are of medicinal and culinary value. The genes and molecular mechanisms involved in W. cocos sclerotial formation are poorly investigated because of the lack of a suitable and reproducible transformation system for W. cocos. In this study, a PEG/ CaCl₂-mediated genetic transformation system for W. cocos was developed. The promoter Pgpd from Ganoderma lucidum effectively drove expression of the hygromycin B phosphotransferase gene in W. cocos, and approximately 30 transformants were obtained per 10 μg DNA when the protoplast suspension density was 10(6) protoplasts/ml. However, no transformants were obtained under the regulation of the PtrpC promoter from Aspergillus nidulans.

Overview of expression of hepatitis B surface antigen in transgenic plants.

PubMed

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.

Development of Bacteriocinogenic Strains of Saccharomyces cerevisiae Heterologously Expressing and Secreting the Leaderless Enterocin L50 Peptides L50A and L50B from Enterococcus faecium L50▿

PubMed Central

Basanta, Antonio; Herranz, Carmen; Gutiérrez, Jorge; Criado, Raquel; Hernández, Pablo E.; Cintas, Luis M.

2009-01-01

A segregationally stable expression and secretion vector for Saccharomyces cerevisiae, named pYABD01, was constructed by cloning the yeast gene region encoding the mating pheromone α-factor 1 secretion signal (MFα1s) into the S. cerevisiae high-copy-number expression vector pYES2. The structural genes of the two leaderless peptides of enterocin L50 (EntL50A and EntL50B) from Enterococcus faecium L50 were cloned, separately (entL50A or entL50B) and together (entL50AB), into pYABD01 under the control of the galactose-inducible promoter PGAL1. The generation of recombinant S. cerevisiae strains heterologously expressing and secreting biologically active EntL50A and EntL50B demonstrates the suitability of the MFα1s-containing vector pYABD01 to direct processing and secretion of these antimicrobial peptides through the S. cerevisiae Sec system. PMID:19218405

Transient gene expression in epidermal cells of plant leaves by biolistic DNA delivery.

PubMed

Ueki, Shoko; Magori, Shimpei; Lacroix, Benoît; Citovsky, Vitaly

2013-01-01

Transient gene expression is a useful approach for studying the functions of gene products. In the case of plants, Agrobacterium infiltration is a method of choice for transient introduction of genes for many species. However, this technique does not work efficiently in some species, such as Arabidopsis thaliana. Moreover, the infection of Agrobacterium is known to induce dynamic changes in gene expression patterns in the host plants, possibly affecting the function and localization of the proteins to be tested. These problems can be circumvented by biolistic delivery of the genes of interest. Here, we present an optimized protocol for biolistic delivery of plasmid DNA into epidermal cells of plant leaves, which can be easily performed using the Bio-Rad Helios gene gun system. This protocol allows efficient and reproducible transient expression of diverse genes in Arabidopsis, Nicotiana benthamiana and N. tabacum, and is suitable for studies of the biological function and subcellular localization of the gene products directly in planta. The protocol also can be easily adapted to other species by optimizing the delivery gas pressure.

Development of a keratinocyte-based screening model for antipsoriatic drugs using green fluorescent protein under the control of an endogenous promoter.

PubMed

Pol, Arno; van Ruissen, Fred; Schalkwijk, Joost

2002-08-01

Inflamed epidermis (psoriasis, wound healing, ultraviolet-irradiated skin) harbors keratinocytes that are hyperproliferative and display an abnormal differentiation program. A distinct feature of this so-called regenerative maturation pathway is the expression of proteins such as the cytokeratins CK6, CK16, and CK17 and the antiinflammatory protein SKALP/elafin. These proteins are absent in normal skin but highly induced in lesional psoriatic skin. Expression of these genes can be used as a surrogate marker for psoriasis in drug-screening procedures of large compound libraries. The aim of this study was to develop a keratinocyte cell line that contained a reporter gene under the control of a psoriasis-associated endogenous promoter and demonstrate its use in an assay suitable for screening. We generated a stably transfected keratinocyte cell line that expresses enhanced green fluorescent protein (EGFP), under the control of a 0.8-kb fragment derived from the promoter of the SKALP/elafin gene, which confers high levels of tissue-specific expression at the mRNA level. Induction of the SKALP promoter by tumor necrosis factor-alpha resulted in increased expression levels of the secreted SKALP-EGFP fusion protein as assessed by direct readout of fluorescence and fluorescence polarization in 96-well cell culture plates. The fold stimulation of the reporter gene was comparable to that of the endogenous SKALP gene as assessed by enzyme-linked immunosorbent assay. Although the dynamic range of the screening system is limited, the small standard deviation yields a Z factor of 0.49. This indicates that the assay is suitable as a high-throughput screen, and provides proof of the concept that a secreted EGFP fusion protein under the control of a physiologically relevant endogenous promoter can be used as a fluorescence-based high-throughput screen for differentiation-modifying or antiinflammatory compounds that act via the keratinocyte.

Selection of suitable reference genes for gene expression studies in Staphylococcus capitis during growth under erythromycin stress.

PubMed

Cui, Bintao; Smooker, Peter M; Rouch, Duncan A; Deighton, Margaret A

2016-08-01

Accurate and reproducible measurement of gene transcription requires appropriate reference genes, which are stably expressed under different experimental conditions to provide normalization. Staphylococcus capitis is a human pathogen that produces biofilm under stress, such as imposed by antimicrobial agents. In this study, a set of five commonly used staphylococcal reference genes (gyrB, sodA, recA, tuf and rpoB) were systematically evaluated in two clinical isolates of Staphylococcus capitis (S. capitis subspecies urealyticus and capitis, respectively) under erythromycin stress in mid-log and stationary phases. Two public software programs (geNorm and NormFinder) and two manual calculation methods, reference residue normalization (RRN) and relative quantitative (RQ), were applied. The potential reference genes selected by the four algorithms were further validated by comparing the expression of a well-studied biofilm gene (icaA) with phenotypic biofilm formation in S. capitis under four different experimental conditions. The four methods differed considerably in their ability to predict the most suitable reference gene or gene combination for comparing icaA expression under different conditions. Under the conditions used here, the RQ method provided better selection of reference genes than the other three algorithms; however, this finding needs to be confirmed with a larger number of isolates. This study reinforces the need to assess the stability of reference genes for analysis of target gene expression under different conditions and the use of more than one algorithm in such studies. Although this work was conducted using a specific human pathogen, it emphasizes the importance of selecting suitable reference genes for accurate normalization of gene expression more generally.

In-depth analysis of internal control genes for quantitative real-time PCR in Brassica oleracea var. botrytis.

PubMed

Sheng, X G; Zhao, Z Q; Yu, H F; Wang, J S; Zheng, C F; Gu, H H

2016-07-15

Quantitative reverse-transcription PCR (qRT-PCR) is a versatile technique for the analysis of gene expression. The selection of stable reference genes is essential for the application of this technique. Cauliflower (Brassica oleracea L. var. botrytis) is a commonly consumed vegetable that is rich in vitamin, calcium, and iron. Thus far, to our knowledge, there have been no reports on the validation of suitable reference genes for the data normalization of qRT-PCR in cauliflower. In the present study, we analyzed 12 candidate housekeeping genes in cauliflower subjected to different abiotic stresses, hormone treatment conditions, and accessions. geNorm and NormFinder algorithms were used to assess the expression stability of these genes. ACT2 and TIP41 were selected as suitable reference genes across all experimental samples in this study. When different accessions were compared, ACT2 and UNK3 were found to be the most suitable reference genes. In the hormone and abiotic stress treatments, ACT2, TIP41, and UNK2 were the most stably expressed. Our study also provided guidelines for selecting the best reference genes under various experimental conditions.

Selection of suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using quantitative real-time polymerase chain reaction.

PubMed

Zornhagen, K W; Kristensen, A T; Hansen, A E; Oxboel, J; Kjaer, A

2015-12-01

Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is a sensitive technique for quantifying gene expression. Stably expressed reference genes are necessary for normalization of RT-qPCR data. Only a few articles have been published on reference genes in canine tumours. The objective of this study was to demonstrate how to identify suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using RT-qPCR. Primer pairs for 17 potential reference genes were designed and tested in archival tumour biopsies from six dogs. The geNorm algorithm was used to analyse the most suitable reference genes. Eight potential reference genes were excluded from this final analysis because of their dissociation curves. β-Glucuronidase (GUSB) and proteasome subunit, beta type, 6 (PSMB6) were most stably expressed with an M value of 0.154 and a CV of 0.053 describing their average stability. We suggest that choice of reference genes should be based on specific testing in every new experimental set-up. © 2014 John Wiley & Sons Ltd.

Serum- and Growth-Factor-Free Three-Dimensional Culture System Supports Cartilage Tissue Formation by Promoting Collagen Synthesis via Sox9–Col2a1 Interaction

PubMed Central

Ahmed, Nazish; Iu, Jonathan; Brown, Chelsea E.; Taylor, Drew Wesley

2014-01-01

Objective: One of the factors preventing clinical application of regenerative medicine to degenerative cartilage diseases is a suitable source of cells. Chondrocytes, the only cell type of cartilage, grown in vitro under culture conditions to expand cell numbers lose their phenotype along with the ability to generate hyaline cartilaginous tissue. In this study we determine that a serum- and growth-factor-free three-dimensional (3D) culture system restores the ability of the passaged chondrocytes to form cartilage tissue in vitro, a process that involves sox9. Methods: Bovine articular chondrocytes were passaged twice to allow for cell number expansion (P2) and cultured at high density on 3D collagen-type-II-coated membranes in high glucose content media supplemented with insulin and dexamethasone (SF3D). The cells were characterized after monolayer expansion and following 3D culture by flow cytometry, gene expression, and histology. The early changes in signaling transduction pathways during redifferentiation were characterized. Results: The P2 cells showed a progenitor-like antigen profile of 99% CD44+ and 40% CD105+ and a gene expression profile suggestive of interzone cells. P2 in SF3D expressed chondrogenic genes and accumulated extracellular matrix. Downregulating insulin receptor (IR) with HNMPA-(AM3) or the PI-3/AKT kinase pathway (activated by insulin treatment) with Wortmannin inhibited collagen synthesis. HNMPA-(AM3) reduced expression of Col2, Col11, and IR genes as well as Sox6 and -9. Co-immunoprecipitation and chromatin immunoprecipitation analyses of HNMPA-(AM3)-treated cells showed binding of the coactivators Sox6 and Med12 with Sox9 but reduced Sox9–Col2a1 binding. Conclusions: We describe a novel culture method that allows for increase in the number of chondrocytes and promotes hyaline-like cartilage tissue formation in part by insulin-mediated Sox9–Col2a1 binding. The suitability of the tissue generated via this approach for use in joint repair needs to be examined in vivo. PMID:24606204

Expression and characterization of a recombinant single-domain monoclonal antibody against MUC1 mucin in tobacco plants.

PubMed

Rajabi-Memari, H; Jalali-Javaran, M; Rasaee, M J; Rahbarizadeh, F; Forouzandeh-Moghadam, M; Esmaili, A

2006-08-01

A promising alternative to conventional antibodies is the single-domain antibody fragment of the Camelidae (V(HH)), which (because of features such as small length, high expression, solubility, and stability) is preferred to other antibody derivatives. In this report, a recombinant single-domain antibody (V(HH)) against MUC1 mucin in the tobacco plant, which may be considered as a suitable and economical alternative expression system, was produced. This antibody was expressed under the control of a strong constitutive promoter, CaMV35S, and NOS terminator. A plant high-expression sequence (Kozak sequence) was linked at the 5' end for overexpression of the V(HH) gene. The constructed cassette (pBIV(HH)) was transferred to agrobacterium, and the VHH gene was inserted into the plant genome by agrobacterium-mediated transformation. Transgenic lines were selected on kanamycin (100 mg/L) and maintained in soil, and subsequent generations were obtained. The presence and expression of the transgene was confirmed in the transformants by polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and Western blot. Tobacco transgenic lines leave expressed V(HH) at levels varying from 1.12% to 1.63% of the total soluble protein. This report examines the transformation and expression of recombinant single-domain antibody (V(HH)) against antigen-associated tumor in tobacco plants.

A Conserved Odorant Receptor Tuned to Floral Volatiles in Three Heliothinae Species

PubMed Central

Cao, Song; Liu, Yang; Guo, Mengbo; Wang, Guirong

2016-01-01

Odorant receptors (ORs) play an important role in insects to monitor and adapt to the external environment, such as host plant location, oviposition-site selection, mate recognition and natural enemy avoidance. In our study, we identified and characterized OR12 from three closely-related species, Helicoverpa armigera, Helicoverpa assulta, Heliothis virescens, sharing between 90 and 98% of their amino acids. The tissue expression pattern analysis in H. armigera showed that HarmOR12 was strongly expressed both in male and female antennae, but not in other tissues. Functional analysis performed in the heterologous Xenopus expression system showed that all three OR12 were tuned to six structurally related plant volatiles. Electroantennogram recordings from male and female antennae of H. armigera closely matched the data of in vitro functional studies. Our results revealed that OR12 has a conserved role in Heliothinae moths and might represent a suitable target for the control of these crop pests. PMID:27163122

Validation of the β-amy1 transcription profiling assay and selection of reference genes suited for a RT-qPCR assay in developing barley caryopsis.

PubMed

Ovesná, Jaroslava; Kučera, Ladislav; Vaculová, Kateřina; Štrymplová, Kamila; Svobodová, Ilona; Milella, Luigi

2012-01-01

Reverse transcription coupled with real-time quantitative PCR (RT-qPCR) is a frequently used method for gene expression profiling. Reference genes (RGs) are commonly employed to normalize gene expression data. A limited information exist on the gene expression and profiling in developing barley caryopsis. Expression stability was assessed by measuring the cycle threshold (Ct) range and applying both the GeNorm (pair-wise comparison of geometric means) and Normfinder (model-based approach) principles for the calculation. Here, we have identified a set of four RGs suitable for studying gene expression in the developing barley caryopsis. These encode the proteins GAPDH, HSP90, HSP70 and ubiquitin. We found a correlation between the frequency of occurrence of a transcript in silico and its suitability as an RG. This set of RGs was tested by comparing the normalized level of β-amylase (β-amy1) transcript with directly measured quantities of the BMY1 gene product in the developing barley caryopsis. This panel of genes could be used for other gene expression studies, as well as to optimize β-amy1 analysis for study of the impact of β-amy1 expression upon barley end-use quality.

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

Aoyagi, Yasuyuki; Department of Genome Research and Clinical Application, Graduate School of Medicine, Chiba University, Chiba; Kuroda, Masayuki, E-mail: kurodam@faculty.chiba-u.jp

Adipose tissue is expected to provide a source of cells for protein replacement therapies via auto-transplantation. However, the conditioning of the environment surrounding the transplanted adipocytes for their long-term survival and protein secretion properties has not been established. We have recently developed a preparation procedure for preadipocytes, ceiling culture-derived proliferative adipocytes (ccdPAs), as a therapeutic gene vehicle suitable for stable gene product secretion. We herein report the results of our evaluation of using fibrin glue as a scaffold for the transplanted ccdPAs for the expression of a transduced gene in a three-dimensional culture system. The ccdPAs secreted the functional proteinmore » translated from an exogenously transduced gene, as well as physiological adipocyte proteins, and the long viability of ccdPAs (up to 84 days) was dependent on the fibrinogen concentrations. The ccdPAs spontaneously accumulated lipid droplets, and their expression levels of the transduced exogenous gene with its product were maintained for at least 56 days. The fibrinogen concentration modified the adipogenic differentiation of ccdPAs and their exogenous gene expression levels, and the levels of exogenously transduced gene expression at the different fibrinogen concentrations were dependent on the extent of adipogenic differentiation in the gel. These results indicate that fibrin glue helps to maintain the high adipogenic potential of cultured adipocytes after passaging in a 3D culture system, and suggests that once they are successfully implanted at the transplantation site, the cells exhibit increased expression of the transduced gene with adipogenic differentiation.« less

Expression and in vitro functional analyses of recombinant Gam1 protein

PubMed Central

Avila, Gustavo A.; Ramirez, Daniel H.; Hildenbrand, Zacariah L.; Jacquez, Pedro; Chiocca, Susanna; Sun, Jianjun; Rosas-Acosta, German; Xiao, Chuan

2014-01-01

Gam1, an early gene product of an avian adenovirus, is essential for viral replication. Gam1 is the first viral protein found to globally inhibit cellular SUMOylation, a critical posttranslational modification that alters the function and cellular localization of proteins. The interaction details at the interface between Gam1 and its cellular targets remain unclear due to the lack of structural information. Although Gam1 has been previously characterized, the purity of the protein was not suitable for structural investigations. In the present study, the gene of Gam1 was cloned and expressed in various bacterial expression systems to obtain pure and soluble recombinant Gam1 protein for in vitro functional and structural studies. While Gam1 was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that both low temperature induction and the chaperone function of TF play critical roles in increasing Gam1 solubility. Soluble Gam1 was purified to homogeneity through sequential chromatography techniques. Monomeric Gam1 was obtained via size exclusion chromatography and analyzed by dynamic light scattering. The SUMOylation inhibitory function of the purified Gam1 was confirmed in an in vitro assay. These results have built the foundation for further structural investigations that will broaden our understanding of Gam1’s roles in viral replication. PMID:25450237

Expression and in vitro functional analyses of recombinant Gam1 protein.

PubMed

Avila, Gustavo A; Ramirez, Daniel H; Hildenbrand, Zacariah L; Jacquez, Pedro; Chiocca, Susanna; Sun, Jianjun; Rosas-Acosta, German; Xiao, Chuan

2015-01-01

Gam1, an early gene product of an avian adenovirus, is essential for viral replication. Gam1 is the first viral protein found to globally inhibit cellular SUMOylation, a critical posttranslational modification that alters the function and cellular localization of proteins. The interaction details at the interface between Gam1 and its cellular targets remain unclear due to the lack of structural information. Although Gam1 has been previously characterized, the purity of the protein was not suitable for structural investigations. In the present study, the gene of Gam1 was cloned and expressed in various bacterial expression systems to obtain pure and soluble recombinant Gam1 protein for in vitro functional and structural studies. While Gam1 was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that both low temperature induction and the chaperone function of TF play critical roles in increasing Gam1 solubility. Soluble Gam1 was purified to homogeneity through sequential chromatography techniques. Monomeric Gam1 was obtained via size exclusion chromatography and analyzed by dynamic light scattering. The SUMOylation inhibitory function of the purified Gam1 was confirmed in an in vitro assay. These results have built the foundation for further structural investigations that will broaden our understanding of Gam1's roles in viral replication. Copyright © 2014 Elsevier Inc. All rights reserved.

A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine.

PubMed

Chen, Yang; Guo, Wanzhu; Xu, Zhiwen; Yan, Qigui; Luo, Yan; Shi, Qian; Chen, Dishi; Zhu, Ling; Wang, Xiaoyu

2011-06-16

Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene. A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA) analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein. Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28) following virulent PPV challenge compared with the control (7 of 31). Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts. In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection.

Energy-momentum tensors in linearized Einstein's theory and massive gravity: The question of uniqueness

NASA Astrophysics Data System (ADS)

Bičák, Jiří; Schmidt, Josef

2016-01-01

The question of the uniqueness of energy-momentum tensors in the linearized general relativity and in the linear massive gravity is analyzed without using variational techniques. We start from a natural ansatz for the form of the tensor (for example, that it is a linear combination of the terms quadratic in the first derivatives), and require it to be conserved as a consequence of field equations. In the case of the linear gravity in a general gauge we find a four-parametric system of conserved second-rank tensors which contains a unique symmetric tensor. This turns out to be the linearized Landau-Lifshitz pseudotensor employed often in full general relativity. We elucidate the relation of the four-parametric system to the expression proposed recently by Butcher et al. "on physical grounds" in harmonic gauge, and we show that the results coincide in the case of high-frequency waves in vacuum after a suitable averaging. In the massive gravity we show how one can arrive at the expression which coincides with the "generalized linear symmetric Landau-Lifshitz" tensor. However, there exists another uniquely given simpler symmetric tensor which can be obtained by adding the divergence of a suitable superpotential to the canonical energy-momentum tensor following from the Fierz-Pauli action. In contrast to the symmetric tensor derived by the Belinfante procedure which involves the second derivatives of the field variables, this expression contains only the field and its first derivatives. It is simpler than the generalized Landau-Lifshitz tensor but both yield the same total quantities since they differ by the divergence of a superpotential. We also discuss the role of the gauge conditions in the proofs of the uniqueness. In the Appendix, the symbolic tensor manipulation software cadabra is briefly described. It is very effective in obtaining various results which would otherwise require lengthy calculations.

Gaussian discriminating strength

NASA Astrophysics Data System (ADS)

Rigovacca, L.; Farace, A.; De Pasquale, A.; Giovannetti, V.

2015-10-01

We present a quantifier of nonclassical correlations for bipartite, multimode Gaussian states. It is derived from the Discriminating Strength measure, introduced for finite dimensional systems in Farace et al., [New J. Phys. 16, 073010 (2014), 10.1088/1367-2630/16/7/073010]. As the latter the new measure exploits the quantum Chernoff bound to gauge the susceptibility of the composite system with respect to local perturbations induced by unitary gates extracted from a suitable set of allowed transformations (the latter being identified by posing some general requirements). Closed expressions are provided for the case of two-mode Gaussian states obtained by squeezing or by linearly mixing via a beam splitter a factorized two-mode thermal state. For these density matrices, we study how nonclassical correlations are related with the entanglement present in the system and with its total photon number.

Permanent, lowered HLA class I expression using lentivirus vectors with shRNA constructs: Averting cytotoxicity by alloreactive T lymphocytes.

PubMed

Haga, K; Lemp, N A; Logg, C R; Nagashima, J; Faure-Kumar, E; Gomez, G G; Kruse, C A; Mendez, R; Stripecke, R; Kasahara, N; Kasahara, N A; Cicciarelli, J C

2006-12-01

Transplantation of many tissues requires histocompatibility matching of human leukocyte antigens (HLA) to prevent graft rejection, to reduce the level of immunosuppression needed to maintain graft survival, and to minimize the risk of graft-versus-host disease, particularly in the case of bone marrow transplantation. However, recent advances in fields of gene delivery and genetic regulation technologies have opened the possibility of engineering grafts that display reduced levels of HLA expression. Suppression of HLA expression could help to overcome the limitations imposed by extensive HLA polymorphisms that restrict the availability of suitable donors, necessitate the maintenance of large donor registries, and complicate the logistics of procuring and delivering matched tissues and organs to the recipient. Accordingly, we investigated whether knockdown of HLA by RNA interference (RNAi), a ubiquitous regulatory system that can efficiently and selectively inhibit the expression of specific gene products, would enable allogeneic cells to evade immune recognition. For efficient and stable delivery of short hairpin-type RNAi constructs (shRNA), we employed lentivirus-based gene transfer vectors, which provide a delivery system that can achieve integration into genomic DNA, thereby permanently modifying transduced graft cells. Our results show that lentivirus-mediated delivery of shRNA targeting pan-Class I and allele-specific HLA can achieve efficient and dose-dependent reduction in surface expression of HLA in human cells, associated with enhanced resistance to alloreactive T lymphocyte-mediated cytotoxicity, while avoiding MHC-non-restricted killing. We hypothesize that RNAi-induced silencing of HLA expression has the potential to create histocompatibility-enhanced, and, eventually, perhaps "universally" compatible cellular grafts.

Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions

PubMed Central

Barsalobres-Cavallari, Carla F; Severino, Fábio E; Maluf, Mirian P; Maia, Ivan G

2009-01-01

Background Quantitative data from gene expression experiments are often normalized by transcription levels of reference or housekeeping genes. An inherent assumption for their use is that the expression of these genes is highly uniform in living organisms during various phases of development, in different cell types and under diverse environmental conditions. To date, the validation of reference genes in plants has received very little attention and suitable reference genes have not been defined for a great number of crop species including Coffea arabica. The aim of the research reported herein was to compare the relative expression of a set of potential reference genes across different types of tissue/organ samples of coffee. We also validated the expression profiles of the selected reference genes at various stages of development and under a specific biotic stress. Results The expression levels of five frequently used housekeeping genes (reference genes), namely alcohol dehydrogenase (adh), 14-3-3, polyubiquitin (poly), β-actin (actin) and glyceraldehyde-3-phosphate dehydrogenase (gapdh) was assessed by quantitative real-time RT-PCR over a set of five tissue/organ samples (root, stem, leaf, flower, and fruits) of Coffea arabica plants. In addition to these commonly used internal controls, three other genes encoding a cysteine proteinase (cys), a caffeine synthase (ccs) and the 60S ribosomal protein L7 (rpl7) were also tested. Their stability and suitability as reference genes were validated by geNorm, NormFinder and BestKeeper programs. The obtained results revealed significantly variable expression levels of all reference genes analyzed, with the exception of gapdh, which showed no significant changes in expression among the investigated experimental conditions. Conclusion Our data suggests that the expression of housekeeping genes is not completely stable in coffee. Based on our results, gapdh, followed by 14-3-3 and rpl7 were found to be homogeneously expressed and are therefore adequate for normalization purposes, showing equivalent transcript levels in different tissue/organ samples. Gapdh is therefore the recommended reference gene for measuring gene expression in Coffea arabica. Its use will enable more accurate and reliable normalization of tissue/organ-specific gene expression studies in this important cherry crop plant. PMID:19126214

In vivo production of non-glycosylated recombinant proteins in Nicotiana benthamiana plants by co-expression with Endo-β-N-acetylglucosaminidase H (Endo H) of Streptomyces plicatus

PubMed Central

Cicek, Kader; Gulec, Burcu; Ungor, Rifat; Hasanova, Gulnara

2017-01-01

A plant transient expression system, with eukaryotic post-translational modification machinery, offers superior efficiency, scalability, safety, and lower cost over other expression systems. However, due to aberrant N-glycosylation, this expression system may not be a suitable expression platform for proteins not carrying N-linked glycans in the native hosts. Therefore, it is crucial to develop a strategy to produce target proteins in a non-glycosylated form while preserving their native sequence, conformation and biological activity. Previously, we developed a strategy for enzymatic deglycosylation of proteins in planta by co-expressing bacterial peptide-N-glycosidase F (PNGase F). Though PNGase F removes oligosaccharides from glycosylated proteins, in so doing it causes an amino acid change due to the deamidation of asparagine to aspartate in the N-X-S/T site. Endo-β-N-acetylglucosaminidase (EC3.2.1.96, Endo H), another deglycosylating enzyme, catalyzes cleavage between two N-Acetyl-D-glucosamine residues of the chitobiose core of N-linked glycans, leaving a single N-Acetyl-D-glucosamine residue without the concomitant deamidation of asparagine. In this study, a method for in vivo deglycosylation of recombinant proteins in plants by transient co-expression with bacterial Endo H is described for the first time. Endo H was fully active in vivo. and successfully cleaved N-linked glycans from glycoproteins were tested. In addition, unlike the glycosylated form, in vivo Endo H deglycosylated Pfs48/45 was recognized by conformational specific Pfs48/45 monoclonal antibody, in a manner similar to its PNGase F deglycosylated counterpart. Furthermore, the deglycosylated PA83 molecule produced by Endo H showed better stability than a PNGase F deglycosylated counterpart. Thus, an Endo H in vivo deglycosylation approach provides another opportunity to develop vaccine antigens, therapeutic proteins, antibodies, and industrial enzymes. PMID:28827815

Highly efficient mesophyll protoplast isolation and PEG-mediated transient gene expression for rapid and large-scale gene characterization in cassava (Manihot esculenta Crantz).

PubMed

Wu, Jun-Zheng; Liu, Qin; Geng, Xiao-Shan; Li, Kai-Mian; Luo, Li-Juan; Liu, Jin-Ping

2017-03-14

Cassava (Manihot esculenta Crantz) is a major crop extensively cultivated in the tropics as both an important source of calories and a promising source for biofuel production. Although stable gene expression have been used for transgenic breeding and gene function study, a quick, easy and large-scale transformation platform has been in urgent need for gene functional characterization, especially after the cassava full genome was sequenced. Fully expanded leaves from in vitro plantlets of Manihot esculenta were used to optimize the concentrations of cellulase R-10 and macerozyme R-10 for obtaining protoplasts with the highest yield and viability. Then, the optimum conditions (PEG4000 concentration and transfection time) were determined for cassava protoplast transient gene expression. In addition, the reliability of the established protocol was confirmed for subcellular protein localization. In this work we optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and PEG-mediated transient gene expression in cassava. The suitable enzyme digestion system was established with the combination of 1.6% cellulase R-10 and 0.8% macerozyme R-10 for 16 h of digestion in the dark at 25 °C, resulting in the high yield (4.4 × 10 7 protoplasts/g FW) and vitality (92.6%) of mesophyll protoplasts. The maximum transfection efficiency (70.8%) was obtained with the incubation of the protoplasts/vector DNA mixture with 25% PEG4000 for 10 min. We validated the applicability of the system for studying the subcellular localization of MeSTP7 (an H + /monosaccharide cotransporter) with our transient expression protocol and a heterologous Arabidopsis transient gene expression system. We optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and transient gene expression in cassava, which will facilitate large-scale characterization of genes and pathways in cassava.

Secretory production of a beta-mannanase and a chitosanase using a Lactobacillus plantarum expression system.

PubMed

Sak-Ubol, Suttipong; Namvijitr, Peenida; Pechsrichuang, Phornsiri; Haltrich, Dietmar; Nguyen, Thu-Ha; Mathiesen, Geir; Eijsink, Vincent G H; Yamabhai, Montarop

2016-05-12

Heterologous production of hydrolytic enzymes is important for green and white biotechnology since these enzymes serve as efficient biocatalysts for the conversion of a wide variety of raw materials into value-added products. Lactic acid bacteria are interesting cell factories for the expression of hydrolytic enzymes as many of them are generally recognized as safe and require only a simple cultivation process. We are studying a potentially food-grade expression system for secretion of hydrolytic enzymes into the culture medium, since this enables easy harvesting and purification, while allowing direct use of the enzymes in food applications. We studied overexpression of a chitosanase (CsnA) and a β-mannanase (ManB), from Bacillus licheniformis and Bacillus subtilis, respectively, in Lactobacillus plantarum, using the pSIP system for inducible expression. The enzymes were over-expressed in three forms: without a signal peptide, with their natural signal peptide and with the well-known OmpA signal peptide from Escherichia coli. The total production levels and secretion efficiencies of CsnA and ManB were highest when using the native signal peptides, and both were reduced considerably when using the OmpA signal. At 20 h after induction with 12.5 ng/mL of inducing peptide in MRS media containing 20 g/L glucose, the yields and secretion efficiencies of the proteins with their native signal peptides were 50 kU/L and 84% for ManB, and 79 kU/L and 56% for CsnA, respectively. In addition, to avoid using antibiotics, the erythromycin resistance gene was replaced on the expression plasmid with the alanine racemase (alr) gene, which led to comparable levels of protein production and secretion efficiency in a suitable, alr-deficient L. plantarum host. ManB and CsnA were efficiently produced and secreted in L. plantarum using pSIP-based expression vectors containing either an erythromycin resistance or the alr gene as selection marker.

Enzyme efficiency: An open reaction system perspective

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

Banerjee, Kinshuk, E-mail: kb36@rice.edu; Bhattacharyya, Kamal, E-mail: pchemkb@gmail.com

2015-12-21

A measure of enzyme efficiency is proposed for an open reaction network that, in suitable form, applies to closed systems as well. The idea originates from the description of classical enzyme kinetics in terms of cycles. We derive analytical expressions for the efficiency measure by treating the network not only deterministically but also stochastically. The latter accounts for any significant amount of noise that can be present in biological systems and hence reveals its impact on efficiency. Numerical verification of the results is also performed. It is found that the deterministic equation overestimates the efficiency, the more so for verymore » small system sizes. Roles of various kinetics parameters and system sizes on the efficiency are thoroughly explored and compared with the standard definition k{sub 2}/K{sub M}. Study of substrate fluctuation also indicates an interesting efficiency-accuracy balance.« less

STINGRAY: system for integrated genomic resources and analysis.

PubMed

Wagner, Glauber; Jardim, Rodrigo; Tschoeke, Diogo A; Loureiro, Daniel R; Ocaña, Kary A C S; Ribeiro, Antonio C B; Emmel, Vanessa E; Probst, Christian M; Pitaluga, André N; Grisard, Edmundo C; Cavalcanti, Maria C; Campos, Maria L M; Mattoso, Marta; Dávila, Alberto M R

2014-03-07

The STINGRAY system has been conceived to ease the tasks of integrating, analyzing, annotating and presenting genomic and expression data from Sanger and Next Generation Sequencing (NGS) platforms. STINGRAY includes: (a) a complete and integrated workflow (more than 20 bioinformatics tools) ranging from functional annotation to phylogeny; (b) a MySQL database schema, suitable for data integration and user access control; and (c) a user-friendly graphical web-based interface that makes the system intuitive, facilitating the tasks of data analysis and annotation. STINGRAY showed to be an easy to use and complete system for analyzing sequencing data. While both Sanger and NGS platforms are supported, the system could be faster using Sanger data, since the large NGS datasets could potentially slow down the MySQL database usage. STINGRAY is available at http://stingray.biowebdb.org and the open source code at http://sourceforge.net/projects/stingray-biowebdb/.

STINGRAY: system for integrated genomic resources and analysis

PubMed Central

2014-01-01

Background The STINGRAY system has been conceived to ease the tasks of integrating, analyzing, annotating and presenting genomic and expression data from Sanger and Next Generation Sequencing (NGS) platforms. Findings STINGRAY includes: (a) a complete and integrated workflow (more than 20 bioinformatics tools) ranging from functional annotation to phylogeny; (b) a MySQL database schema, suitable for data integration and user access control; and (c) a user-friendly graphical web-based interface that makes the system intuitive, facilitating the tasks of data analysis and annotation. Conclusion STINGRAY showed to be an easy to use and complete system for analyzing sequencing data. While both Sanger and NGS platforms are supported, the system could be faster using Sanger data, since the large NGS datasets could potentially slow down the MySQL database usage. STINGRAY is available at http://stingray.biowebdb.org and the open source code at http://sourceforge.net/projects/stingray-biowebdb/. PMID:24606808

Efficient siRNA delivery system using carboxilated single-wall carbon nanotubes in cancer treatment.

PubMed

Neagoe, Ioana Berindan; Braicu, Cornelia; Matea, Cristian; Bele, Constantin; Florin, Graur; Gabriel, Katona; Veronica, Chedea; Irimie, Alexandru

2012-08-01

Several functionalized carbon nanotubes have been designed and tested for the purpose of nucleic acid delivery. In this study, the capacity of SWNTC-COOH for siRNA deliverey were investigated delivery in parallel with an efficient commercial system. Hep2G cells were reverse-transfected with 50 nM siRNA (p53 siRNA, TNF-alphasiRNA, VEGFsiRNA) using the siPORT NeoFX (Ambion) transfection agent in paralel with SWNTC-COOH, functionalised with siRNA. The highest level of gene inhibition was observed in the cases treated with p53 siRNA gene; in the case of transfection with siPort, the NeoFX value was 33.8%, while in the case of SWNTC-COOH as delivery system for p53 siRNA was 37.5%. The gene silencing capacity for VEGF was 53.7%, respectively for TNF-alpha 56.7% for siPORT NeoFX delivery systems versus 47.7% (VEGF) and 46.5% (TNF-alpha) for SWNTC-COOH delivery system. SWNTC-COOH we have been showed to have to be an efficient carrier system. The results from the inhibition of gene expresion for both transfection systems were confirmed at protein level. Overall, the lowest mRNA expression was confirmed at protein level, especially in the case of p53 siRNA and TNF-alpha siRNA transfection. Less efficient reduction protein expressions were observed in the case of VEGF siRNA, for both transfection systems at 24 h; only at 48 h, there was a statistically significant reduction of VEGF protein expression. SWCNT-COOH determined an efficient delivery of siRNA. SWNTC-COOH, combined with suitable tumor markers like p53 siRNA, TNFalpha siRNA or VEGF siRNA can be used for the efficient delivery of siRNA.

Expression, purification, and characterization of the Necator americanus aspartic protease-1 (Na-APR-1 (M74)) antigen, a component of the bivalent human hookworm vaccine.

PubMed

Seid, Christopher A; Curti, Elena; Jones, R Mark; Hudspeth, Elissa; Rezende, Wanderson; Pollet, Jeroen; Center, Lori; Versteeg, Leroy; Pritchard, Sonya; Musiychuk, Konstantin; Yusibov, Vidadi; Hotez, Peter J; Bottazzi, Maria Elena

2015-01-01

Over 400 million people living in the world's poorest developing nations are infected with hookworms, mostly of the genus Necator americanus. A bivalent human hookworm vaccine composed of the Necator americanus Glutathione S-Transferase-1 (Na-GST-1) and the Necator americanus Aspartic Protease-1 (Na-APR-1 (M74)) is currently under development by the Sabin Vaccine Institute Product Development Partnership (Sabin PDP). Both monovalent vaccines are currently in Phase 1 trials. Both Na-GST-1 and Na-APR-1 antigens are expressed as recombinant proteins. While Na-GST-1 was found to express with high yields in Pichia pastoris, the level of expression of Na-APR-1 in this host was too low to be suitable for a manufacturing process. When the tobacco plant Nicotiana benthamiana was evaluated as an expression system, acceptable levels of solubility, yield, and stability were attained. Observed expression levels of Na-APR-1 (M74) using this system are ∼300 mg/kg. Here we describe the achievements and obstacles encountered during process development as well as characterization and stability of the purified Na-APR-1 (M74) protein and formulated vaccine. The expression, purification and analysis of purified Na-APR-1 (M74) protein obtained from representative 5 kg reproducibility runs performed to qualify the Na-APR-1 (M74) production process is also presented. This process has been successfully transferred to a pilot plant and a 50 kg scale manufacturing campaign under current Good Manufacturing Practice (cGMP) has been performed. The 50 kg run has provided a sufficient amount of protein to support the ongoing hookworm vaccine development program of the Sabin PDP.

A stepwise approach for the reproducible optimization of PAMO expression in Escherichia coli for whole-cell biocatalysis

PubMed Central

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

Properties of a recombinant bovine tissue factor expressed by Silkworm pupae and its performance as an Owren-type prothrombin time reagent for warfarin monitoring.

PubMed

Okuda, Masahiro; Taniguchi, Tomokuni; Takamiya, Osamu

2012-09-01

Tissue factor (TF), or thromboplastin, is a glycoprotein that triggers the extrinsic coagulation pathway. In blood coagulation testing, TF has been used as a natural source for determining Quick prothrombin time (PT) or the Owren PT (OBT). Currently, natural sources are being replaced with recombinant proteins because of their uniform characteristics and the possibility of stable mass production of PT reagents. Because bovine spongiform encephalopathy (BSE)-infected cows are widespread in Japan, we prepared a recombinant bovine TF (rbTF) with a baculovirus expression system using silkworms. To overcome the limitations of natural TF, especially in bovine brain, we expressed a full-length rbTF protein in Silkworm pupae with a baculovirus expression system. Baculovirus inactivation and the presence of DNA fragments in the rbTF fraction were confirmed using Reed-Muench and polymerase chain reaction methods after inactivation with a detergent. The rbTF fraction prepared by an immobilized anti-Silkworm pupae fluid protein Sepharose 4B column was identified as a visible band on western blots with a polyclonal antibody against human TF with cross-reactivity with TFs. The inhibition of the polyclonal antibody against human TF by the clotting assay for PT was identified, and amidolytic biological activity through activated factor VII on S-2288 substrate was observed. In conclusion, the rbTF expressed by the baculovirus system using Silkworm pupae was uniformly specific for bovine TF. The OBT reagent incorporated by this rbTF was similar to those of commercial reagents. It also showed a suitable International Sensitivity Index and reproducibility precision, thereby allowing for diagnostic use. Copyright © 2012 Elsevier Ltd. All rights reserved.

Determination of Highly Sensitive Biological Cell Model Systems to Screen BPA-Related Health Hazards Using Pathway Studio.

PubMed

Ryu, Do-Yeal; Rahman, Md Saidur; Pang, Myung-Geol

2017-09-06

Bisphenol-A (BPA) is a ubiquitous endocrine-disrupting chemical. Recently, many issues have arisen surrounding the disease pathogenesis of BPA. Therefore, several studies have been conducted to investigate the proteomic biomarkers of BPA that are associated with disease processes. However, studies on identifying highly sensitive biological cell model systems in determining BPA health risk are lacking. Here, we determined suitable cell model systems and potential biomarkers for predicting BPA-mediated disease using the bioinformatics tool Pathway Studio. We compiled known BPA-mediated diseases in humans, which were categorized into five major types. Subsequently, we investigated the differentially expressed proteins following BPA exposure in several cell types, and analyzed the efficacy of altered proteins to investigate their associations with BPA-mediated diseases. Our results demonstrated that colon cancer cells (SW480), mammary gland, and Sertoli cells were highly sensitive biological model systems, because of the efficacy of predicting the majority of BPA-mediated diseases. We selected glucose-6-phosphate dehydrogenase (G6PD), cytochrome b-c1 complex subunit 1 (UQCRC1), and voltage-dependent anion-selective channel protein 2 (VDAC2) as highly sensitive biomarkers to predict BPA-mediated diseases. Furthermore, we summarized proteomic studies in spermatozoa following BPA exposure, which have recently been considered as another suitable cell type for predicting BPA-mediated diseases.

Determination of Highly Sensitive Biological Cell Model Systems to Screen BPA-Related Health Hazards Using Pathway Studio

PubMed Central

Ryu, Do-Yeal

2017-01-01

Bisphenol-A (BPA) is a ubiquitous endocrine-disrupting chemical. Recently, many issues have arisen surrounding the disease pathogenesis of BPA. Therefore, several studies have been conducted to investigate the proteomic biomarkers of BPA that are associated with disease processes. However, studies on identifying highly sensitive biological cell model systems in determining BPA health risk are lacking. Here, we determined suitable cell model systems and potential biomarkers for predicting BPA-mediated disease using the bioinformatics tool Pathway Studio. We compiled known BPA-mediated diseases in humans, which were categorized into five major types. Subsequently, we investigated the differentially expressed proteins following BPA exposure in several cell types, and analyzed the efficacy of altered proteins to investigate their associations with BPA-mediated diseases. Our results demonstrated that colon cancer cells (SW480), mammary gland, and Sertoli cells were highly sensitive biological model systems, because of the efficacy of predicting the majority of BPA-mediated diseases. We selected glucose-6-phosphate dehydrogenase (G6PD), cytochrome b-c1 complex subunit 1 (UQCRC1), and voltage-dependent anion-selective channel protein 2 (VDAC2) as highly sensitive biomarkers to predict BPA-mediated diseases. Furthermore, we summarized proteomic studies in spermatozoa following BPA exposure, which have recently been considered as another suitable cell type for predicting BPA-mediated diseases. PMID:28878155

Jacobian-free approximate solvers for hyperbolic systems: Application to relativistic magnetohydrodynamics

NASA Astrophysics Data System (ADS)

Castro, Manuel J.; Gallardo, José M.; Marquina, Antonio

2017-10-01

We present recent advances in PVM (Polynomial Viscosity Matrix) methods based on internal approximations to the absolute value function, and compare them with Chebyshev-based PVM solvers. These solvers only require a bound on the maximum wave speed, so no spectral decomposition is needed. Another important feature of the proposed methods is that they are suitable to be written in Jacobian-free form, in which only evaluations of the physical flux are used. This is particularly interesting when considering systems for which the Jacobians involve complex expressions, e.g., the relativistic magnetohydrodynamics (RMHD) equations. On the other hand, the proposed Jacobian-free solvers have also been extended to the case of approximate DOT (Dumbser-Osher-Toro) methods, which can be regarded as simple and efficient approximations to the classical Osher-Solomon method, sharing most of it interesting features and being applicable to general hyperbolic systems. To test the properties of our schemes a number of numerical experiments involving the RMHD equations are presented, both in one and two dimensions. The obtained results are in good agreement with those found in the literature and show that our schemes are robust and accurate, running stable under a satisfactory time step restriction. It is worth emphasizing that, although this work focuses on RMHD, the proposed schemes are suitable to be applied to general hyperbolic systems.

10 CFR 963.16 - Postclosure suitability evaluation method.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 10 Energy 4 2014-01-01 2014-01-01 false Postclosure suitability evaluation method. 963.16 Section... Determination, Methods, and Criteria § 963.16 Postclosure suitability evaluation method. (a) DOE will evaluate postclosure suitability using the total system performance assessment method. DOE will conduct a total system...

10 CFR 963.16 - Postclosure suitability evaluation method.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 10 Energy 4 2011-01-01 2011-01-01 false Postclosure suitability evaluation method. 963.16 Section... Determination, Methods, and Criteria § 963.16 Postclosure suitability evaluation method. (a) DOE will evaluate postclosure suitability using the total system performance assessment method. DOE will conduct a total system...

10 CFR 963.16 - Postclosure suitability evaluation method.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 10 Energy 4 2012-01-01 2012-01-01 false Postclosure suitability evaluation method. 963.16 Section... Determination, Methods, and Criteria § 963.16 Postclosure suitability evaluation method. (a) DOE will evaluate postclosure suitability using the total system performance assessment method. DOE will conduct a total system...

Multiscale Embedded Gene Co-expression Network Analysis

PubMed Central

Song, Won-Min; Zhang, Bin

2015-01-01

Gene co-expression network analysis has been shown effective in identifying functional co-expressed gene modules associated with complex human diseases. However, existing techniques to construct co-expression networks require some critical prior information such as predefined number of clusters, numerical thresholds for defining co-expression/interaction, or do not naturally reproduce the hallmarks of complex systems such as the scale-free degree distribution of small-worldness. Previously, a graph filtering technique called Planar Maximally Filtered Graph (PMFG) has been applied to many real-world data sets such as financial stock prices and gene expression to extract meaningful and relevant interactions. However, PMFG is not suitable for large-scale genomic data due to several drawbacks, such as the high computation complexity O(|V|3), the presence of false-positives due to the maximal planarity constraint, and the inadequacy of the clustering framework. Here, we developed a new co-expression network analysis framework called Multiscale Embedded Gene Co-expression Network Analysis (MEGENA) by: i) introducing quality control of co-expression similarities, ii) parallelizing embedded network construction, and iii) developing a novel clustering technique to identify multi-scale clustering structures in Planar Filtered Networks (PFNs). We applied MEGENA to a series of simulated data and the gene expression data in breast carcinoma and lung adenocarcinoma from The Cancer Genome Atlas (TCGA). MEGENA showed improved performance over well-established clustering methods and co-expression network construction approaches. MEGENA revealed not only meaningful multi-scale organizations of co-expressed gene clusters but also novel targets in breast carcinoma and lung adenocarcinoma. PMID:26618778

Multiscale Embedded Gene Co-expression Network Analysis.

PubMed

Song, Won-Min; Zhang, Bin

2015-11-01

Gene co-expression network analysis has been shown effective in identifying functional co-expressed gene modules associated with complex human diseases. However, existing techniques to construct co-expression networks require some critical prior information such as predefined number of clusters, numerical thresholds for defining co-expression/interaction, or do not naturally reproduce the hallmarks of complex systems such as the scale-free degree distribution of small-worldness. Previously, a graph filtering technique called Planar Maximally Filtered Graph (PMFG) has been applied to many real-world data sets such as financial stock prices and gene expression to extract meaningful and relevant interactions. However, PMFG is not suitable for large-scale genomic data due to several drawbacks, such as the high computation complexity O(|V|3), the presence of false-positives due to the maximal planarity constraint, and the inadequacy of the clustering framework. Here, we developed a new co-expression network analysis framework called Multiscale Embedded Gene Co-expression Network Analysis (MEGENA) by: i) introducing quality control of co-expression similarities, ii) parallelizing embedded network construction, and iii) developing a novel clustering technique to identify multi-scale clustering structures in Planar Filtered Networks (PFNs). We applied MEGENA to a series of simulated data and the gene expression data in breast carcinoma and lung adenocarcinoma from The Cancer Genome Atlas (TCGA). MEGENA showed improved performance over well-established clustering methods and co-expression network construction approaches. MEGENA revealed not only meaningful multi-scale organizations of co-expressed gene clusters but also novel targets in breast carcinoma and lung adenocarcinoma.

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

PubMed Central

Minaba, Masaomi

2014-01-01

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

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

PubMed

Buntru, Matthias; Vogel, Simon; Stoff, Katrin; Spiegel, Holger; Schillberg, Stefan

2015-05-01

Cell-free protein synthesis is a powerful method for the high-throughput production of recombinant proteins, especially proteins that are difficult to express in living cells. Here we describe a coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates (BYLs). Using a combination of fractional factorial designs and response surface models, we developed a cap-independent system that produces more than 250 μg/mL of functional enhanced yellow fluorescent protein (eYFP) and about 270 μg/mL of firefly luciferase using plasmid templates, and up to 180 μg/mL eYFP using linear templates (PCR products) in 18 h batch reactions. The BYL contains actively-translocating microsomal vesicles derived from the endoplasmic reticulum, promoting the formation of disulfide bonds, glycosylation and the cotranslational integration of membrane proteins. This was demonstrated by expressing a functional full-size antibody (∼ 150 μg/mL), the model enzyme glucose oxidase (GOx) (∼ 7.3 U/mL), and a transmembrane growth factor (∼ 25 μg/mL). Subsequent in vitro treatment of GOx with peptide-N-glycosidase F confirmed the presence of N-glycans. Our results show that the BYL can be used as a high-throughput expression and screening platform that is particularly suitable for complex and cytotoxic proteins. © 2014 Wiley Periodicals, Inc.

Polycistronic gene expression in Aspergillus niger.

PubMed

Schuetze, Tabea; Meyer, Vera

2017-09-25

Genome mining approaches predict dozens of biosynthetic gene clusters in each of the filamentous fungal genomes sequenced so far. However, the majority of these gene clusters still remain cryptic because they are not expressed in their natural host. Simultaneous expression of all genes belonging to a biosynthetic pathway in a heterologous host is one approach to activate biosynthetic gene clusters and to screen the metabolites produced for bioactivities. Polycistronic expression of all pathway genes under control of a single and tunable promoter would be the method of choice, as this does not only simplify cloning procedures, but also offers control on timing and strength of expression. However, polycistronic gene expression is a feature not commonly found in eukaryotic host systems, such as Aspergillus niger. In this study, we tested the suitability of the viral P2A peptide for co-expression of three genes in A. niger. Two genes descend from Fusarium oxysporum and are essential to produce the secondary metabolite enniatin (esyn1, ekivR). The third gene (luc) encodes the reporter luciferase which was included to study position effects. Expression of the polycistronic gene cassette was put under control of the Tet-On system to ensure tunable gene expression in A. niger. In total, three polycistronic expression cassettes which differed in the position of luc were constructed and targeted to the pyrG locus in A. niger. This allowed direct comparison of the luciferase activity based on the position of the luciferase gene. Doxycycline-mediated induction of the Tet-On expression cassettes resulted in the production of one long polycistronic mRNA as proven by Northern analyses, and ensured comparable production of enniatin in all three strains. Notably, gene position within the polycistronic expression cassette matters, as, luciferase activity was lowest at position one and had a comparable activity at positions two and three. The P2A peptide can be used to express at least three genes polycistronically in A. niger. This approach can now be applied to heterologously express entire secondary metabolite gene clusters polycistronically or to co-express any genes of interest in equimolar amounts.

Significant expression of thyroid transcription factor-1 in pulmonary squamous cell carcinoma detected by SPT24 monoclonal antibody and CSA-II system.

PubMed

Kashima, Kenji; Hashimoto, Hisashi; Nishida, Haruto; Arakane, Motoki; Yada, Naomi; Daa, Tsutomu; Yokoyama, Shigeo

2014-01-01

In contrast to the usefulness of thyroid transcription factor-1 (TTF-1) in distinguishing primary adenocarcinoma of the lung from metastatic lesions, TTF-1 expression in pulmonary squamous cell carcinoma is reported to be at low level and not a suitable immunohistochemical marker. We hypothesized that the highly sensitive detection system, CSA-II, can visualize even faint expression of TTF-1 in pulmonary squamous cell carcinoma. In this study, 2 commercially available clones of TTF-1 monoclonal antibody, 8G7G3/1 and SPT24, were used for staining 38 cases of pulmonary squamous cell carcinoma, in combination with the CSA-II and the conventional detection system, EnVision. The combined use of the 8G7G3/1 clone with EnVision and CSA-II showed a positive reaction in only 1 and 4 cases, respectively. The use of SPT24 clone showed positive staining in 5 cases with EnVision and in 20 of 38 cases (52.6%) with the CSA-II. Interestingly, positive staining by the SPT24-CSA-II technique of samples from tissue blocks preserved for <2 years was 73.6% compared with only 31.5% in those preserved for >2 years. In addition, a 6-month preservation of the cut sections resulted in stain fading and decreased positivity (50%), compared with freshly cut sections. We conclude that the use of the SPT24 monoclonal antibody with the CSA-II system can detect even weak expression of TTF-1 in pulmonary squamous cell carcinoma. This staining technique can potentially allow the discrimination of primary squamous cell carcinoma of the lung from metastatic lesions, especially in freshly prepared paraffin sections.

Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes.

PubMed

Ramadan, Abdelaziz; Nemoto, Keiichirou; Seki, Motoaki; Shinozaki, Kazuo; Takeda, Hiroyuki; Takahashi, Hirotaka; Sawasaki, Tatsuya

2015-11-10

Protein ubiquitination is a ubiquitous mechanism in eukaryotes. In Arabidopsis, ubiquitin modification is mainly mediated by two ubiquitin activating enzymes (E1s), 37 ubiquitin conjugating enzymes (E2s), and more than 1300 predicted ubiquitin ligase enzymes (E3s), of which ~470 are RING-type E3s. A large proportion of the RING E3's gene products have yet to be characterised in vitro, likely because of the laborious work involved in large-scale cDNA cloning and protein expression, purification, and characterisation. In addition, several E2s, which might be necessary for the activity of certain E3 ligases, cannot be expressed by Escherichia coli or cultured insect cells and, therefore, remain uncharacterised. Using the RIKEN Arabidopsis full-length cDNA library (RAFL) with the 'split-primer' PCR method and a wheat germ cell-free system, we established protein libraries of Arabidopsis E2 and RING E3 enzymes. We expressed 35 Arabidopsis E2s including six enzymes that have not been previously expressed, and 204 RING proteins, most of which had not been functionally characterised. Thioester assays using dithiothreitol (DTT) showed DTT-sensitive ubiquitin thioester formation for all E2s expressed. In expression assays of RING proteins, 31 proteins showed high molecular smears, which are probably the result of their functional activity. The activities of another 27 RING proteins were evaluated with AtUBC10 and/or a group of different E2s. All the 27 RING E3s tested showed ubiquitin ligase activity, including 17 RING E3s. Their activities are reported for the first time. The wheat germ cell-free system used in our study, which is a eukaryotic expression system and more closely resembles the endogenous expression of plant proteins, is very suitable for expressing Arabidopsis E2s and RING E3s in their functional form. In addition, the protein libraries described here can be used for further understanding E2-E3 specificities and as platforms for protein-protein interaction screening.

A Cost-effective and Reliable Method to Predict Mechanical Stress in Single-use and Standard Pumps

PubMed Central

Dittler, Ina; Dornfeld, Wolfgang; Schöb, Reto; Cocke, Jared; Rojahn, Jürgen; Kraume, Matthias; Eibl, Dieter

2015-01-01

Pumps are mainly used when transferring sterile culture broths in biopharmaceutical and biotechnological production processes. However, during the pumping process shear forces occur which can lead to qualitative and/or quantitative product loss. To calculate the mechanical stress with limited experimental expense, an oil-water emulsion system was used, whose suitability was demonstrated for drop size detections in bioreactors1. As drop breakup of the oil-water emulsion system is a function of mechanical stress, drop sizes need to be counted over the experimental time of shear stress investigations. In previous studies, the inline endoscopy has been shown to be an accurate and reliable measurement technique for drop size detections in liquid/liquid dispersions. The aim of this protocol is to show the suitability of the inline endoscopy technique for drop size measurements in pumping processes. In order to express the drop size, the Sauter mean diameter d32 was used as the representative diameter of drops in the oil-water emulsion. The results showed low variation in the Sauter mean diameters, which were quantified by standard deviations of below 15%, indicating the reliability of the measurement technique. PMID:26274765

A low temperature furnace for solution crystal growth on the International Space Station

NASA Astrophysics Data System (ADS)

Baç, Nurcan; Harpster, Joseph; Maston, Robert A.; Sacco, Albert

2000-01-01

The Zeolite Crystal Growth Furnace Unit (ZCG-FU) is the first module in an integrated payload designed for low temperature crystal growth in solutions on the International Space Station (ISS). This payload is scheduled to fly on the ISS flight 7A.1 in an EXPRESS rack. Its name originated from early shuttle flight experiments limited to the growth of zeolite crystals but has since grown to include other materials of significant commercial interest using the solution method of crystal growth. Zeolites, ferroelectrics, piezeoelectrics and silver halides are some of the materials considered. The ZCG-FU experiment consists of a furnace unit and its electronic control system, and mechanically complex, crystal growth autoclaves suitable for use with a particular furnace and solution. The ZCG facility is being designed to grow into four independent furnaces controlled by IZECS (Improved Zeolite Electronic Control System). IZECS provides monitoring of critical parameters, data logging, safety monitoring, air-to-ground control and operator interfacing. It is suitable for controlling the four furnaces either individually or all at one time. It also contains the power management solid-state drivers and switches for the ZCG-FU furnace. The furnace contains 19 tubes operating at three different temperature zones. .

Landau-Zener extension of the Tavis-Cummings model: Structure of the solution

DOE PAGES

Sun, Chen; Sinitsyn, Nikolai A.

2016-09-07

We explore the recently discovered solution of the driven Tavis-Cummings model (DTCM). It describes interaction of an arbitrary number of two-level systems with a bosonic mode that has linearly time-dependent frequency. We derive compact and tractable expressions for transition probabilities in terms of the well-known special functions. In this form, our formulas are suitable for fast numerical calculations and analytical approximations. As an application, we obtain the semiclassical limit of the exact solution and compare it to prior approximations. Furthermore, we also reveal connection between DTCM and q-deformed binomial statistics.

Semiclassical description of resonance-assisted tunneling in one-dimensional integrable models

NASA Astrophysics Data System (ADS)

Le Deunff, Jérémy; Mouchet, Amaury; Schlagheck, Peter

2013-10-01

Resonance-assisted tunneling is investigated within the framework of one-dimensional integrable systems. We present a systematic recipe, based on Hamiltonian normal forms, to construct one-dimensional integrable models that exhibit resonance island chain structures with accurately controlled sizes and positions of the islands. Using complex classical trajectories that evolve along suitably defined paths in the complex time domain, we construct a semiclassical theory of the resonance-assisted tunneling process. This semiclassical approach yields a compact analytical expression for tunnelling-induced level splittings which is found to be in very good agreement with the exact splittings obtained through numerical diagonalization.

Molecular profiles to biology and pathways: a systems biology approach.

PubMed

Van Laere, Steven; Dirix, Luc; Vermeulen, Peter

2016-06-16

Interpreting molecular profiles in a biological context requires specialized analysis strategies. Initially, lists of relevant genes were screened to identify enriched concepts associated with pathways or specific molecular processes. However, the shortcoming of interpreting gene lists by using predefined sets of genes has resulted in the development of novel methods that heavily rely on network-based concepts. These algorithms have the advantage that they allow a more holistic view of the signaling properties of the condition under study as well as that they are suitable for integrating different data types like gene expression, gene mutation, and even histological parameters.

RPL13A and EEF1A1 Are Suitable Reference Genes for qPCR during Adipocyte Differentiation of Vascular Stromal Cells from Patients with Different BMI and HOMA-IR.

PubMed

Gentile, Adriana-Mariel; Lhamyani, Said; Coín-Aragüez, Leticia; Oliva-Olivera, Wilfredo; Zayed, Hatem; Vega-Rioja, Antonio; Monteseirin, Javier; Romero-Zerbo, Silvana-Yanina; Tinahones, Francisco-José; Bermúdez-Silva, Francisco-Javier; El Bekay, Rajaa

2016-01-01

Real-time or quantitative PCR (qPCR) is a useful technique that requires reliable reference genes for data normalization in gene expression analysis. Adipogenesis is among the biological processes suitable for this technique. The selection of adequate reference genes is essential for qPCR gene expression analysis of human Vascular Stromal Cells (hVSCs) during their differentiation into adipocytes. To the best of our knowledge, there are no studies validating reference genes for the analyses of visceral and subcutaneous adipose tissue hVSCs from subjects with different Body Mass Index (BMI) and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index. The present study was undertaken to analyze this question. We first analyzed the stability of expression of five potential reference genes: CYC, GAPDH, RPL13A, EEF1A1, and 18S ribosomal RNA, during in vitro adipogenic differentiation, in samples from these types of patients. The expression of RPL13A and EEF1A1 was not affected by differentiation, thus being these genes the most stable candidates, while CYC, GAPDH, and 18S were not suitable for this sort of analysis. This work highlights that RPL13A and EEF1A1 are good candidates as reference genes for qPCR analysis of hVSCs differentiation into adipocytes from subjects with different BMI and HOMA-IR.

RPL13A and EEF1A1 Are Suitable Reference Genes for qPCR during Adipocyte Differentiation of Vascular Stromal Cells from Patients with Different BMI and HOMA-IR

PubMed Central

Gentile, Adriana-Mariel; Lhamyani, Said; Coín-Aragüez, Leticia; Oliva-Olivera, Wilfredo; Zayed, Hatem; Vega-Rioja, Antonio; Monteseirin, Javier; Romero-Zerbo, Silvana-Yanina; Tinahones, Francisco-José; Bermúdez-Silva, Francisco-Javier; El Bekay, Rajaa

2016-01-01

Real-time or quantitative PCR (qPCR) is a useful technique that requires reliable reference genes for data normalization in gene expression analysis. Adipogenesis is among the biological processes suitable for this technique. The selection of adequate reference genes is essential for qPCR gene expression analysis of human Vascular Stromal Cells (hVSCs) during their differentiation into adipocytes. To the best of our knowledge, there are no studies validating reference genes for the analyses of visceral and subcutaneous adipose tissue hVSCs from subjects with different Body Mass Index (BMI) and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index. The present study was undertaken to analyze this question. We first analyzed the stability of expression of five potential reference genes: CYC, GAPDH, RPL13A, EEF1A1, and 18S ribosomal RNA, during in vitro adipogenic differentiation, in samples from these types of patients. The expression of RPL13A and EEF1A1 was not affected by differentiation, thus being these genes the most stable candidates, while CYC, GAPDH, and 18S were not suitable for this sort of analysis. This work highlights that RPL13A and EEF1A1 are good candidates as reference genes for qPCR analysis of hVSCs differentiation into adipocytes from subjects with different BMI and HOMA-IR. PMID:27304673

Cell line with endogenous EGFRvIII expression is a suitable model for research and drug development purposes.

PubMed

Stec, Wojciech J; Rosiak, Kamila; Siejka, Paulina; Peciak, Joanna; Popeda, Marta; Banaszczyk, Mateusz; Pawlowska, Roza; Treda, Cezary; Hulas-Bigoszewska, Krystyna; Piaskowski, Sylwester; Stoczynska-Fidelus, Ewelina; Rieske, Piotr

2016-05-31

Glioblastoma is the most common and malignant brain tumor, characterized by high cellular heterogeneity. About 50% of glioblastomas are positive for EGFR amplification, half of which express accompanying EGFR mutation, encoding truncated and constitutively active receptor termed EGFRvIII. Currently, no cell models suitable for development of EGFRvIII-targeting drugs exist, while the available ones lack the intratumoral heterogeneity or extrachromosomal nature of EGFRvIII.The reports regarding the biology of EGFRvIII expressed in the stable cell lines are often contradictory in observations and conclusions. In the present study, we use DK-MG cell line carrying endogenous non-modified EGFRvIII amplicons and derive a sub-line that is near depleted of amplicons, whilst remaining identical on the chromosomal level. By direct comparison of the two lines, we demonstrate positive effects of EGFRvIII on cell invasiveness and populational growth as a result of elevated cell survival but not proliferation rate. Investigation of the PI3K/Akt indicated no differences between the lines, whilst NFκB pathway was over-active in the line strongly expressing EGFRvIII, finding further supported by the effects of NFκB pathway specific inhibitors. Taken together, these results confirm the important role of EGFRvIII in intrinsic and extrinsic regulation of tumor behavior. Moreover, the proposed models are stable, making them suitable for research purposes as well as drug development process utilizing high throughput approach.

Cell line with endogenous EGFRvIII expression is a suitable model for research and drug development purposes

PubMed Central

Stec, Wojciech J.; Rosiak, Kamila; Siejka, Paulina; Peciak, Joanna; Popeda, Marta; Banaszczyk, Mateusz; Pawlowska, Roza; Treda, Cezary; Hulas-Bigoszewska, Krystyna; Piaskowski, Sylwester; Stoczynska-Fidelus, Ewelina; Rieske, Piotr

2016-01-01

Glioblastoma is the most common and malignant brain tumor, characterized by high cellular heterogeneity. About 50% of glioblastomas are positive for EGFR amplification, half of which express accompanying EGFR mutation, encoding truncated and constitutively active receptor termed EGFRvIII. Currently, no cell models suitable for development of EGFRvIII-targeting drugs exist, while the available ones lack the intratumoral heterogeneity or extrachromosomal nature of EGFRvIII. The reports regarding the biology of EGFRvIII expressed in the stable cell lines are often contradictory in observations and conclusions. In the present study, we use DK-MG cell line carrying endogenous non-modified EGFRvIII amplicons and derive a sub-line that is near depleted of amplicons, whilst remaining identical on the chromosomal level. By direct comparison of the two lines, we demonstrate positive effects of EGFRvIII on cell invasiveness and populational growth as a result of elevated cell survival but not proliferation rate. Investigation of the PI3K/Akt indicated no differences between the lines, whilst NFκB pathway was over-active in the line strongly expressing EGFRvIII, finding further supported by the effects of NFκB pathway specific inhibitors. Taken together, these results confirm the important role of EGFRvIII in intrinsic and extrinsic regulation of tumor behavior. Moreover, the proposed models are stable, making them suitable for research purposes as well as drug development process utilizing high throughput approach. PMID:27004406

Array-based comparative genomic hybridization-guided identification of reference genes for normalization of real-time quantitative polymerase chain reaction assay data for lymphomas, histiocytic sarcomas, and osteosarcomas of dogs.

PubMed

Tsai, Pei-Chien; Breen, Matthew

2012-09-01

To identify suitable reference genes for normalization of real-time quantitative PCR (RT-qPCR) assay data for common tumors of dogs. Malignant lymph node (n = 8), appendicular osteosarcoma (9), and histiocytic sarcoma (12) samples and control samples of various nonneoplastic canine tissues. Array-based comparative genomic hybridization (aCGH) data were used to guide selection of 9 candidate reference genes. Expression stability of candidate reference genes and 4 commonly used reference genes was determined for tumor samples with RT-qPCR assays and 3 software programs. LOC611555 was the candidate reference gene with the highest expression stability among the 3 tumor types. Of the commonly used reference genes, expression stability of HPRT was high in histiocytic sarcoma samples, and expression stability of Ubi and RPL32 was high in osteosarcoma samples. Some of the candidate reference genes had higher expression stability than did the commonly used reference genes. Data for constitutively expressed genes with high expression stability are required for normalization of RT-qPCR assay results. Without such data, accurate quantification of gene expression in tumor tissue samples is difficult. Results of the present study indicated LOC611555 may be a useful RT-qPCR assay reference gene for multiple tissue types. Some commonly used reference genes may be suitable for normalization of gene expression data for tumors of dogs, such as lymphomas, osteosarcomas, or histiocytic sarcomas.

Identification of Suitable Reference Genes for Investigating Gene Expression in Anterior Cruciate Ligament Injury by Using Reverse Transcription-Quantitative PCR.

PubMed

Leal, Mariana Ferreira; Astur, Diego Costa; Debieux, Pedro; Arliani, Gustavo Gonçalves; Silveira Franciozi, Carlos Eduardo; Loyola, Leonor Casilla; Andreoli, Carlos Vicente; Smith, Marília Cardoso; Pochini, Alberto de Castro; Ejnisman, Benno; Cohen, Moises

2015-01-01

The anterior cruciate ligament (ACL) is one of the most frequently injured structures during high-impact sporting activities. Gene expression analysis may be a useful tool for understanding ACL tears and healing failure. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) has emerged as an effective method for such studies. However, this technique requires the use of suitable reference genes for data normalization. Here, we evaluated the suitability of six reference genes (18S, ACTB, B2M, GAPDH, HPRT1, and TBP) by using ACL samples of 39 individuals with ACL tears (20 with isolated ACL tears and 19 with ACL tear and combined meniscal injury) and of 13 controls. The stability of the candidate reference genes was determined by using the NormFinder, geNorm, BestKeeper DataAssist, and RefFinder software packages and the comparative ΔCt method. ACTB was the best single reference gene and ACTB+TBP was the best gene pair. The GenEx software showed that the accumulated standard deviation is reduced when a larger number of reference genes is used for gene expression normalization. However, the use of a single reference gene may not be suitable. To identify the optimal combination of reference genes, we evaluated the expression of FN1 and PLOD1. We observed that at least 3 reference genes should be used. ACTB+HPRT1+18S is the best trio for the analyses involving isolated ACL tears and controls. Conversely, ACTB+TBP+18S is the best trio for the analyses involving (1) injured ACL tears and controls, and (2) ACL tears of patients with meniscal tears and controls. Therefore, if the gene expression study aims to compare non-injured ACL, isolated ACL tears and ACL tears from patients with meniscal tear as three independent groups ACTB+TBP+18S+HPRT1 should be used. In conclusion, 3 or more genes should be used as reference genes for analysis of ACL samples of individuals with and without ACL tears.

Identification of reference genes and validation for gene expression studies in diverse axolotl (Ambystoma mexicanum) tissues.

PubMed

Guelke, Eileen; Bucan, Vesna; Liebsch, Christina; Lazaridis, Andrea; Radtke, Christine; Vogt, Peter M; Reimers, Kerstin

2015-04-10

For the precise quantitative RT-PCR normalization a set of valid reference genes is obligatory. Moreover have to be taken into concern the experimental conditions as they bias the regulation of reference genes. Up till now, no reference targets have been described for the axolotl (Ambystoma mexicanum). In a search in the public database SalSite for genetic information of the axolotl we identified fourteen presumptive reference genes, eleven of which were further tested for their gene expression stability. This study characterizes the expressional patterns of 11 putative endogenous control genes during axolotl limb regeneration and in an axolotl tissue panel. All 11 reference genes showed variable expression. Strikingly, ACTB was to be found most stable expressed in all comparative tissue groups, so we reason it to be suitable for all different kinds of axolotl tissue-type investigations. Moreover do we suggest GAPDH and RPLP0 as suitable for certain axolotl tissue analysis. When it comes to axolotl limb regeneration, a validated pair of reference genes is ODC and RPLP0. With these findings, new insights into axolotl gene expression profiling might be gained. Copyright © 2015 Elsevier B.V. All rights reserved.

MicroRNA expression in melanocytic nevi: the usefulness of formalin-fixed, paraffin-embedded material for miRNA microarray profiling.

PubMed

Glud, Martin; Klausen, Mikkel; Gniadecki, Robert; Rossing, Maria; Hastrup, Nina; Nielsen, Finn C; Drzewiecki, Krzysztof T

2009-05-01

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate cellular differentiation, proliferation, and apoptosis. MiRNAs are expressed in a developmentally regulated and tissue-specific manner. Aberrant expression may contribute to pathological processes such as cancer, and miRNA may therefore serve as biomarkers that may be useful in a clinical environment for diagnosis of various diseases. Most miRNA profiling studies have used fresh tissue samples. However, in some types of cancer, including malignant melanoma, fresh material is difficult to obtain from primary tumors, and most surgical specimens are formalin fixed and paraffin embedded (FFPE). To explore whether FFPE material would be suitable for miRNA profiling in melanocytic lesions, we compared miRNA expression patterns in FFPE versus fresh frozen samples, obtained from 15 human melanocytic nevi. Out of microarray data, we identified 84 miRNAs that were expressed in both types of samples and represented an miRNA profile of melanocytic nevi. Our results showed a high correlation in miRNA expression (Spearman r-value of 0.80) between paired FFPE and fresh frozen material. The data were further validated by quantitative RT-PCR. In conclusion, FFPE specimens of melanocytic lesions are suitable as a source for miRNA microarray profiling.

Recent advances in live cell imaging of hepatoma cells

PubMed Central

2014-01-01

Live cell imaging enables the study of dynamic processes of living cells in real time by use of suitable reporter proteins and the staining of specific cellular structures and/or organelles. With the availability of advanced optical devices and improved cell culture protocols it has become a rapidly growing research methodology. The success of this technique relies mainly on the selection of suitable reporter proteins, construction of recombinant plasmids possessing cell type specific promoters as well as reliable methods of gene transfer. This review aims to provide an overview of the recent developments in the field of marker proteins (bioluminescence and fluorescent) and methodologies (fluorescent resonance energy transfer, fluorescent recovery after photobleaching and proximity ligation assay) employed as to achieve an improved imaging of biological processes in hepatoma cells. Moreover, different expression systems of marker proteins and the modes of gene transfer are discussed with emphasis on the study of lipid droplet formation in hepatocytes as an example. PMID:25005127

Application of the SeDeM Diagram and a new mathematical equation in the design of direct compression tablet formulation.

PubMed

Suñé-Negre, Josep M; Pérez-Lozano, Pilar; Miñarro, Montserrat; Roig, Manel; Fuster, Roser; Hernández, Carmen; Ruhí, Ramon; García-Montoya, Encarna; Ticó, Josep R

2008-08-01

Application of the new SeDeM Method is proposed for the study of the galenic properties of excipients in terms of the applicability of direct-compression technology. Through experimental studies of the parameters of the SeDeM Method and their subsequent mathematical treatment and graphical expression (SeDeM Diagram), six different DC diluents were analysed to determine whether they were suitable for direct compression (DC). Based on the properties of these diluents, a mathematical equation was established to identify the best DC diluent and the optimum amount to be used when defining a suitable formula for direct compression, depending on the SeDeM properties of the active pharmaceutical ingredient (API) to be used. The results obtained confirm that the SeDeM Method is an appropriate system, effective tool for determining a viable formulation for tablets prepared by direct compression, and can thus be used as the basis for the relevant pharmaceutical development.

Selection of novel reference genes for use in the human central nervous system: a BrainNet Europe Study.

PubMed

Durrenberger, Pascal F; Fernando, Francisca S; Magliozzi, Roberta; Kashefi, Samira N; Bonnert, Timothy P; Ferrer, Isidro; Seilhean, Danielle; Nait-Oumesmar, Brahim; Schmitt, Andrea; Gebicke-Haerter, Peter J; Falkai, Peter; Grünblatt, Edna; Palkovits, Miklos; Parchi, Piero; Capellari, Sabina; Arzberger, Thomas; Kretzschmar, Hans; Roncaroli, Federico; Dexter, David T; Reynolds, Richard

2012-12-01

The use of an appropriate reference gene to ensure accurate normalisation is crucial for the correct quantification of gene expression using qPCR assays and RNA arrays. The main criterion for a gene to qualify as a reference gene is a stable expression across various cell types and experimental settings. Several reference genes are commonly in use but more and more evidence reveals variations in their expression due to the presence of on-going neuropathological disease processes, raising doubts concerning their use. We conducted an analysis of genome-wide changes of gene expression in the human central nervous system (CNS) covering several neurological disorders and regions, including the spinal cord, and were able to identify a number of novel stable reference genes. We tested the stability of expression of eight novel (ATP5E, AARS, GAPVD1, CSNK2B, XPNPEP1, OSBP, NAT5 and DCTN2) and four more commonly used (BECN1, GAPDH, QARS and TUBB) reference genes in a smaller cohort using RT-qPCR. The most stable genes out of the 12 reference genes were tested as normaliser to validate increased levels of a target gene in CNS disease. We found that in human post-mortem tissue the novel reference genes, XPNPEP1 and AARS, were efficient in replicating microarray target gene expression levels and that XPNPEP1 was more efficient as a normaliser than BECN1, which has been shown to change in expression as a consequence of neuronal cell loss. We provide herein one more suitable novel reference gene, XPNPEP1, with no current neuroinflammatory or neurodegenerative associations that can be used for gene quantitative gene expression studies with human CNS post-mortem tissue and also suggest a list of potential other candidates. These data also emphasise the importance of organ/tissue-specific stably expressed genes as reference genes for RNA studies.

Versatile plasmid-based expression systems for Gram-negative bacteria--General essentials exemplified with the bacterium Ralstonia eutropha H16.

PubMed

Gruber, Steffen; Schwab, Helmut; Koefinger, Petra

2015-12-25

The Gram-negative bacterium Escherichia coli is currently the most efficient and widely used prokaryotic host for recombinant protein and metabolite production. However, due to some limitations and to various interesting features of other Gram-negative bacteria efficient vector systems applicable to a broad range are desired. Basic building blocks for plasmid-based vectors include besides the need for a suitable selection marker in the first line a proper replication and maintenance system. In addition to these basic requirements, further elements are needed for Gram-negative bacteria beyond E. coli, such as Pseudomonas pudita, Ralstonia eutropha, Burkholderia glumae or Acinetobacter sp.. Established building blocks have to be adapted and new building blocks providing the desired functions need to be identified and exploited. This minireview addresses so far described and used genetic elements for broad host range replication, efficient plasmid maintenance, and conjugative plasmid transfer as well as expression elements and protein secretion signals. The industrially important bacterium R. eutropha H16 was chosen as a model organism to provide specific data on the effectivity and utility of building blocks based on such genetic elements. Copyright © 2015 Elsevier B.V. All rights reserved.

Power Spectrum of a Noisy System Close to a Heteroclinic Orbit

NASA Astrophysics Data System (ADS)

Giner-Baldó, Jordi; Thomas, Peter J.; Lindner, Benjamin

2017-07-01

We consider a two-dimensional dynamical system that possesses a heteroclinic orbit connecting four saddle points. This system is not able to show self-sustained oscillations on its own. If endowed with white Gaussian noise it displays stochastic oscillations, the frequency and quality factor of which are controlled by the noise intensity. This stochastic oscillation of a nonlinear system with noise is conveniently characterized by the power spectrum of suitable observables. In this paper we explore different analytical and semianalytical ways to compute such power spectra. Besides a number of explicit expressions for the power spectrum, we find scaling relations for the frequency, spectral width, and quality factor of the stochastic heteroclinic oscillator in the limit of weak noise. In particular, the quality factor shows a slow logarithmic increase with decreasing noise of the form Q˜ [ln (1/D)]^2. Our results are compared to numerical simulations of the respective Langevin equations.

MALDI-TOF mass spectrometry for quantitative gene expression analysis of acid responses in Staphylococcus aureus.

PubMed

Rode, Tone Mari; Berget, Ingunn; Langsrud, Solveig; Møretrø, Trond; Holck, Askild

2009-07-01

Microorganisms are constantly exposed to new and altered growth conditions, and respond by changing gene expression patterns. Several methods for studying gene expression exist. During the last decade, the analysis of microarrays has been one of the most common approaches applied for large scale gene expression studies. A relatively new method for gene expression analysis is MassARRAY, which combines real competitive-PCR and MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry. In contrast to microarray methods, MassARRAY technology is suitable for analysing a larger number of samples, though for a smaller set of genes. In this study we compare the results from MassARRAY with microarrays on gene expression responses of Staphylococcus aureus exposed to acid stress at pH 4.5. RNA isolated from the same stress experiments was analysed using both the MassARRAY and the microarray methods. The MassARRAY and microarray methods showed good correlation. Both MassARRAY and microarray estimated somewhat lower fold changes compared with quantitative real-time PCR (qRT-PCR). The results confirmed the up-regulation of the urease genes in acidic environments, and also indicated the importance of metal ion regulation. This study shows that the MassARRAY technology is suitable for gene expression analysis in prokaryotes, and has advantages when a set of genes is being analysed for an organism exposed to many different environmental conditions.

A unified classifier for robust face recognition based on combining multiple subspace algorithms

NASA Astrophysics Data System (ADS)

Ijaz Bajwa, Usama; Ahmad Taj, Imtiaz; Waqas Anwar, Muhammad

2012-10-01

Face recognition being the fastest growing biometric technology has expanded manifold in the last few years. Various new algorithms and commercial systems have been proposed and developed. However, none of the proposed or developed algorithm is a complete solution because it may work very well on one set of images with say illumination changes but may not work properly on another set of image variations like expression variations. This study is motivated by the fact that any single classifier cannot claim to show generally better performance against all facial image variations. To overcome this shortcoming and achieve generality, combining several classifiers using various strategies has been studied extensively also incorporating the question of suitability of any classifier for this task. The study is based on the outcome of a comprehensive comparative analysis conducted on a combination of six subspace extraction algorithms and four distance metrics on three facial databases. The analysis leads to the selection of the most suitable classifiers which performs better on one task or the other. These classifiers are then combined together onto an ensemble classifier by two different strategies of weighted sum and re-ranking. The results of the ensemble classifier show that these strategies can be effectively used to construct a single classifier that can successfully handle varying facial image conditions of illumination, aging and facial expressions.

Potential Role of Circulating MiR-21 in the Diagnosis and Prognosis of Digestive System Cancer: A Systematic Review and Meta-Analysis.

PubMed

Yin, Chengqiang; Zhou, Xiaoying; Dang, Yini; Yan, Jin; Zhang, Guoxin

2015-12-01

Recent evidences indicate that circulating microRNAs (miRNAs) exhibit aberrant expression in the plasma of patients suffering from cancer compared to normal individuals, suggesting that it may be a useful noninvasion diagnostic method. MiR-21 plays crucial roles in carcinogenesis and can be served as a biomarker for the detection of various cancers. Therefore, the aim of this meta-analysis is to assess the potential role of miR-21 for digestive system cancer. By searching the PubMed, Embase, and Web of Science for publications concerning the diagnostic value of miR-21 for digestive system cancer, total of 23 publications were included in this meta-analysis. Receiver operating characteristic curves (ROC) were used to check the overall test performance. For prognostic meta-analysis, pooled hazard ratios (HRs) of circulating miR-21 for survival were calculated. Totally 23 eligible publications were included in this meta-analysis (15 articles for diagnosis and 8 articles for prognosis). For diagnostic meta-analysis, the summary estimates revealed that the pooled sensitivity and specificity were 0.76 (95% CI = 0.70-0.82) and 0.84 (95% CI = 0.78-0.89). Besides, the area under the summary ROC curve (AUC) is 0.87. For prognostic meta-analysis, the pooled HR of higher miR-21 expression in circulation was 1.94 (95% CI = 0.99-3.82, P = 0.055), which indicated higher miR-21 expression could be likely to predict poorer survival in digestive system cancer. The subgroup analysis implied the higher expression of miR-21 was correlated with worse overall survival in the Asian population in digestive system cancer (HR = 2.41, 95% CI = 1.21-4.77, P = 0.012). The current evidence suggests circulating miR-21 may be suitable to be a diagnostic and prognostic biomarker for digestive system cancer in the Asians.

Transcriptome-wide identification of reference genes for expression analysis of soybean responses to drought stress along the day

USDA-ARS?s Scientific Manuscript database

The soybean transcriptome displays strong variation along the day in optimal growth conditions and also in response to adverse circumstances, like drought stress. However, no study conducted to date has presented suitable reference genes, with stable expression along the day, for relative gene expre...

On the Denesting of Nested Square Roots

ERIC Educational Resources Information Center

Gkioulekas, Eleftherios

2017-01-01

We present the basic theory of denesting nested square roots, from an elementary point of view, suitable for lower level coursework. Necessary and sufficient conditions are given for direct denesting, where the nested expression is rewritten as a sum of square roots of rational numbers, and for indirect denesting, where the nested expression is…

Cloning and expression of soluble truncated variants of Borrelia OspA, OspB and Vmp7

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

Dunn, J.J.; Barbour, A.G.

1996-11-05

A method is provided for preparing soluble recombinant variations of Borrelia lipoproteins such as Borrelia burgdorferi outer surface protein A (OspA) and outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The method includes synthesizing a set of oligonucleotide primers, amplifying the template DNA utilizing the PCR, purifying the amplification products, cloning the amplification products into a suitable expression vector, transforming a suitable host utilizing the cloned expression vector, cultivating the transformed host for protein production and subsequently isolating and purifying the resulting protein. Also provided are soluble, recombinant variations of Borrelia burgdorferi outer surface proteinmore » A (OspA), outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The expression vectors harboring DNA encoding the recombinant variations, pET9-OspA, pET9-OspB and pET9-Vmp7, as well as the E. coli host BL21(DE3)/pLysS transformed with each of these vectors, are also disclosed. 38 figs.« less

Cloning and expression of soluble truncated variants of Borrelia OspA, OspB and Vmp7

DOEpatents

Dunn, John J.; Barbour, Alan G.

1996-11-05

A method is provided herein for preparing soluble recombinant variations of Borrelia lipoproteins such as Borrelia burgdorferi outer surface protein A (OspA) and outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The method includes synthesizing a set of oligonucleotide primers, amplifying the template DNA utilizing the PCR, purifying the amplification products, cloning the amplification products into a suitable expression vector, transforming a suitable host utilizing the cloned expression vector, cultivating the transformed host for protein production and subsequently isolating and purifying the resulting protein. Also provided are soluble, recombinant variations of Borrelia burgdorferi outer surface protein A (OspA), outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The expression vectors harboring DNA encoding the recombinant variations, pET9-OspA, pET9-OspB and pET9-Vmp7, as well as the E. coli host BL21(DE3)/pLysS transformed with each of these vectors, are also disclosed.

Cloning and expression of soluble truncated variants of Borrelia OspA, OspB and Vmp7

DOEpatents

Dunn, J.J.; Barbour, A.G.

1996-11-05

A method is provided for preparing soluble recombinant variations of Borrelia lipoproteins such as Borrelia burgdorferi outer surface protein A (OspA) and outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The method includes synthesizing a set of oligonucleotide primers, amplifying the template DNA utilizing the PCR, purifying the amplification products, cloning the amplification products into a suitable expression vector, transforming a suitable host utilizing the cloned expression vector, cultivating the transformed host for protein production and subsequently isolating and purifying the resulting protein. Also provided are soluble, recombinant variations of Borrelia burgdorferi outer surface protein A (OspA), outer surface protein B (OspB), and B. hermsii variable major protein 7 (Vmp7). The expression vectors harboring DNA encoding the recombinant variations, pET9-OspA, pET9-OspB and pET9-Vmp7, as well as the E. coli host BL21(DE3)/pLysS transformed with each of these vectors, are also disclosed. 38 figs.

Selection of reference genes for expression analysis of Kumamoto and Portuguese oysters and their hybrid

NASA Astrophysics Data System (ADS)

Yan, Lulu; Su, Jiaqi; Wang, Zhaoping; Yan, Xiwu; Yu, Ruihai

2017-12-01

Quantitative real-time polymerase chain reaction (qRT-PCR) is a rapid and reliable technique which has been widely used to quantifying gene transcripts (expression analysis). It is also employed for studying heterosis, hybridization breeding and hybrid tolerability of oysters, an ecologically and economically important taxonomic group. For these studies, selection of a suitable set of housekeeping genes as references is crucial for correct interpretation of qRT-PCR data. To identify suitable reference genes for oysters during low temperature and low salinity stresses, we analyzed twelve genes from the gill tissue of Crassostrea sikamea (SS), Crassostrea angulata (AA) and their hybrid (SA), which included three ribosomal genes, 28S ribosomal protein S5 ( RPS5), ribosomal protein L35 ( RPL35), and 60S ribosomal protein L29 ( RPL29); three structural genes, tubulin gamma ( TUBγ), annexin A6 and A7 ( AA6 and AA7); three metabolic pathway genes, ornithine decarboxylase ( OD), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH) and glutathione S-transferase P1 ( GSP); two transcription factors, elongation factor 1 alpha and beta ( EF1α and EF1β); and one protein synthesis gene (ubiquitin ( UBQ). Primers specific for these genes were successfully developed for the three groups of oysters. Three different algorithms, geNorm, NormFinder and BestKeeper, were used to evaluate the expression stability of these candidate genes. BestKeeper program was found to be the most reliable. Based on our analysis, we found that the expression of RPL35 and EF1α was stable under low salinity stress, and the expression of OD, GAPDH and EF1α was stable under low temperature stress in hybrid (SA) oyster; the expression of RPS5 and GAPDH was stable under low salinity stress, and the expression of RPS5, UBQ, GAPDH was stable under low temperature stress in SS oyster; the expression of RPS5, GAPDH, EF1β and AA7 was stable under low salinity stress, and the expression of RPL35, EF1α, GAPDH and EF1β was stable under low temperature stress in AA oyster. Furthermore, to evaluate their suitability, the reference genes were used to quantify six target genes. In conclusion, we have successfully developed primers appropriate for the expression analysis in SS, SA and AA.

A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector

PubMed Central

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

A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector.

PubMed

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.

Harnessing the complexity of gene expression data from cancer: from single gene to structural pathway methods

PubMed Central

2012-01-01

High-dimensional gene expression data provide a rich source of information because they capture the expression level of genes in dynamic states that reflect the biological functioning of a cell. For this reason, such data are suitable to reveal systems related properties inside a cell, e.g., in order to elucidate molecular mechanisms of complex diseases like breast or prostate cancer. However, this is not only strongly dependent on the sample size and the correlation structure of a data set, but also on the statistical hypotheses tested. Many different approaches have been developed over the years to analyze gene expression data to (I) identify changes in single genes, (II) identify changes in gene sets or pathways, and (III) identify changes in the correlation structure in pathways. In this paper, we review statistical methods for all three types of approaches, including subtypes, in the context of cancer data and provide links to software implementations and tools and address also the general problem of multiple hypotheses testing. Further, we provide recommendations for the selection of such analysis methods. Reviewers This article was reviewed by Arcady Mushegian, Byung-Soo Kim and Joel Bader. PMID:23227854

Semantic word impressions expressed by hue.

PubMed

Shinomori, Keizo; Komatsu, Honami

2018-04-01

We investigated the possibility of whether impressions of semantic words showing complex concepts could be stably expressed by hues. Using a paired comparison method, we asked ten subjects to select from a pair of hues the one that more suitably matched a word impression. We employed nine Japanese semantic words and used twelve hues from vivid tones in the practical color coordinate system. As examples of the results, for the word "vigorous" the most frequently selected color was yellow and the least selected was blue to purple; for "tranquil" the most selected was yellow to green and the least selected was red. Principal component analysis of the selection data indicated that the cumulative contribution rate of the first two components was 94.6%, and in the two-dimensional space of the components, all hues were distributed as a hue-circle shape. In addition, comparison with additional data of color impressions measured by a semantic differential method suggested that most semantic word impressions can be stably expressed by hue, but the impression of some words, such as "magnificent" cannot. These results suggest that semantic word impression can be expressed reasonably well by color, and that hues are treated as impressions from the hue circle, not from color categories.

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

PubMed

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

2016-04-01

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

CNS Schwann cells display oligodendrocyte precursor-like potassium channel activation and antigenic expression in vitro.

PubMed

Kegler, Kristel; Imbschweiler, Ilka; Ulrich, Reiner; Kovermann, Peter; Fahlke, Christoph; Deschl, Ulrich; Kalkuhl, Arno; Baumgärnter, Wolfgang; Wewetzer, Konstantin

2014-06-01

Central nervous system (CNS) injury triggers production of myelinating Schwann cells from endogenous oligodendrocyte precursors (OLPs). These CNS Schwann cells may be attractive candidates for novel therapeutic strategies aiming to promote endogenous CNS repair. However, CNS Schwann cells have been so far mainly characterized in situ regarding morphology and marker expression, and it has remained enigmatic whether they display functional properties distinct from peripheral nervous system (PNS) Schwann cells. Potassium channels (K+) have been implicated in progenitor and glial cell proliferation after injury and may, therefore, represent a suitable pharmacological target. In the present study, we focused on the function and expression of voltage-gated K+ channels Kv(1-12) and accessory β-subunits in purified adult canine CNS and PNS Schwann cell cultures using electrophysiology and microarray analysis and characterized their antigenic phenotype. We show here that K+ channels differed significantly in both cell types. While CNS Schwann cells displayed prominent K D-mediated K+ currents, PNS Schwann cells elicited K(D-) and K(A-type) K+ currents. Inhibition of K+ currents by TEA and Ba2+ was more effective in CNS Schwann cells. These functional differences were not paralleled by differential mRNA expression of Kv(1-12) and accessory β-subunits. However, O4/A2B5 and GFAP expressions were significantly higher and lower, respectively, in CNS than in PNS Schwann cells. Taken together, this is the first evidence that CNS Schwann cells display specific properties not shared by their peripheral counterpart. Both Kv currents and increased O4/A2B5 expression were reminiscent of OLPs suggesting that CNS Schwann cells retain OLP features during maturation.

A new approach to gene therapy using Sleeping Beauty to genetically modify clinical-grade T cells to target CD19

PubMed Central

Singh, Harjeet; Huls, Helen; Cooper, Laurence JN

2014-01-01

Summary The advent of efficient approaches to the genetic modification of T cells has provided investigators with clinically appealing approaches to improve the potency of tumor-specific clinical grade T cells. For example, gene therapy has been successfully used to enforce expression of chimeric antigen receptors (CAR) that provide T cells with ability to directly recognize tumor-associated antigens without the need for presentation by human leukocyte antigen. Gene transfer of CARs can be undertaken using viral-based and non-viral approaches. We have advanced DNA vectors derived from the Sleeping Beauty (SB) system to avoid the expense and manufacturing difficulty associated with transducing T cells with recombinant viral vectors. After electroporation, the transposon/transposase system improves the efficiency of integration of plasmids used to express CAR and other transgenes in T cells. The SB system combined with artificial antigen-presenting cells (aAPC) can selectively propagate and thus retrieve CAR+ T cells suitable for human application. This review describes the translation of the SB system and aAPC for use in clinical trials and highlights how a nimble and cost-effective approach to developing genetically modified T cells can be used to implement clinical trials infusing next-generation T cells with improved therapeutic potential. PMID:24329797

Potential Roles of Dental Pulp Stem Cells in Neural Regeneration and Repair

PubMed Central

Luo, Lihua; Wang, Xiaoyan; Key, Brian; Lee, Bae Hoon

2018-01-01

This review summarizes current advances in dental pulp stem cells (DPSCs) and their potential applications in the nervous diseases. Injured adult mammalian nervous system has a limited regenerative capacity due to an insufficient pool of precursor cells in both central and peripheral nervous systems. Nerve growth is also constrained by inhibitory factors (associated with central myelin) and barrier tissues (glial scarring). Stem cells, possessing the capacity of self-renewal and multicellular differentiation, promise new therapeutic strategies for overcoming these impediments to neural regeneration. Dental pulp stem cells (DPSCs) derive from a cranial neural crest lineage, retain a remarkable potential for neuronal differentiation, and additionally express multiple factors that are suitable for neuronal and axonal regeneration. DPSCs can also express immunomodulatory factors that stimulate formation of blood vessels and enhance regeneration and repair of injured nerve. These unique properties together with their ready accessibility make DPSCs an attractive cell source for tissue engineering in injured and diseased nervous systems. In this review, we interrogate the neuronal differentiation potential as well as the neuroprotective, neurotrophic, angiogenic, and immunomodulatory properties of DPSCs and its application in the injured nervous system. Taken together, DPSCs are an ideal stem cell resource for therapeutic approaches to neural repair and regeneration in nerve diseases. PMID:29853908

Engineering zucchini yellow mosaic potyvirus as a non-pathogenic vector for expression of heterologous proteins in cucurbits.

PubMed

Arazi, T; Slutsky, S G; Shiboleth, Y M; Wang, Y; Rubinstein, M; Barak, S; Yang, J; Gal-On, A

2001-04-27

Plant virus vectors provide an attractive biotechnological tool for the transient expression of foreign genes in whole plants. As yet there has been no use of recombinant viruses for the improvement of commercial crops. This is mainly because the viruses used to create vectors usually cause significant yield loss and can be transmitted in the field. A novel attenuated zucchini yellow mosaic potyvirus (AG) was used for the development of an environmentally safe non-pathogenic virus vector. The suitability of AG as an expression vector in plants was tested by analysis of two infectious viral constructs, each containing a distinct gene insertion site. Introduction of a foreign viral coat protein gene into AG genome between the P1 and HC-Pro genes, resulted in no expression in planta. In contrast, the same gene was stably expressed when inserted between NIb and CP genes, suggesting that this site is more suitable for a gene vector. Virus-mediated expression of reporter genes was observed in squash and cucumber leaves, stems, roots and edible fruit. Furthermore, AG stably expressed human interferon-alpha 2, an important human anti-viral drug, without affecting plant development and yield. Interferon biological activity was measured in cucumber and squash fruit. Together, these data corroborate a biotechnological utility of AG as a non-pathogenic vector for the expression of a foreign gene, as a benefit trait, in cucurbits and their edible fruit.

Choline supplementation alleviates fluoride-induced testicular toxicity by restoring the NGF and MEK expression in mice

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

Zhang, Jianhai

Fluoride is known to cause male reproductive toxicity, and the elucidation of its underlying mechanisms is an ongoing research focus in reproductive toxicology and epidemiology. Choline, an essential nutrient, has been extensively studied for its benefits in nervous system yet was rarely discussed for its prospective effect in male reproductive system. This study aims to explore the potential protective role of choline against NaF-induced male reproductive toxicity via MAPK pathway. The male mice were administrated by 150 mg/L NaF in drinking water, 5.75 g/kg choline in diet, and their combination respectively from maternal gestation to postnatal 15 weeks. The resultsmore » showed that fluoride exposure reduced body weight growth, lowered sperm count and survival percentages, altered testicular histology, down-regulated the mRNA expressions of NGF, Ras, Raf, and MEK genes in testes, as well as significantly decreased the expressions of both NGF and phosphor-MEK proteins in testes. Examination of data from choline-treated mice revealed that choline supplementation ameliorated these fluoride-induced changes. Taken together, our findings suggest that choline supplementation alleviates fluoride-induced testicular toxicity by restoring the NGF and phosphor-MEK expression. The suitable dosage and supplementation periods of choline await further exploration. - Highlights: • Fluoride exposure altered the growth and development, sperm count and sperm survival percentages, testicular histology • Fluoride exposure decreased NGF, Ras, and Mek mRNA and NGF and p-MEK protein expressions in testis of mice. • Choline supplementation diminishes fluoride-induced testicular toxicity.« less

A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine

PubMed Central

2011-01-01

Background Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene. Methods A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA) analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein. Results Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28) following virulent PPV challenge compared with the control (7 of 31). Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts. Conclusions In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection. PMID:21679423

Information processing of motion in facial expression and the geometry of dynamical systems

NASA Astrophysics Data System (ADS)

Assadi, Amir H.; Eghbalnia, Hamid; McMenamin, Brenton W.

2005-01-01

An interesting problem in analysis of video data concerns design of algorithms that detect perceptually significant features in an unsupervised manner, for instance methods of machine learning for automatic classification of human expression. A geometric formulation of this genre of problems could be modeled with help of perceptual psychology. In this article, we outline one approach for a special case where video segments are to be classified according to expression of emotion or other similar facial motions. The encoding of realistic facial motions that convey expression of emotions for a particular person P forms a parameter space XP whose study reveals the "objective geometry" for the problem of unsupervised feature detection from video. The geometric features and discrete representation of the space XP are independent of subjective evaluations by observers. While the "subjective geometry" of XP varies from observer to observer, levels of sensitivity and variation in perception of facial expressions appear to share a certain level of universality among members of similar cultures. Therefore, statistical geometry of invariants of XP for a sample of population could provide effective algorithms for extraction of such features. In cases where frequency of events is sufficiently large in the sample data, a suitable framework could be provided to facilitate the information-theoretic organization and study of statistical invariants of such features. This article provides a general approach to encode motion in terms of a particular genre of dynamical systems and the geometry of their flow. An example is provided to illustrate the general theory.

Production of halophilic proteins using Haloferax volcanii H1895 in a stirred-tank bioreactor.

PubMed

Strillinger, Eva; Grötzinger, Stefan Wolfgang; Allers, Thorsten; Eppinger, Jörg; Weuster-Botz, Dirk

2016-02-01

The success of biotechnological processes is based on the availability of efficient and highly specific biocatalysts, which can satisfy industrial demands. Extreme and remote environments like the deep brine pools of the Red Sea represent highly interesting habitats for the discovery of novel halophilic and thermophilic enzymes. Haloferax volcanii constitutes a suitable expression system for halophilic enzymes obtained from such brine pools. We developed a batch process for the cultivation of H. volcanii H1895 in controlled stirred-tank bioreactors utilising knockouts of components of the flagella assembly system. The standard medium Hv-YPC was supplemented to reach a higher cell density. Without protein expression, cell dry weight reaches 10 g L(-1). Two halophilic alcohol dehydrogenases were expressed under the control of the tryptophanase promoter p.tna with 16.8 and 3.2 mg gCDW (-1), respectively, at a maximum cell dry weight of 6.5 g L(-1). Protein expression was induced by the addition of L-tryptophan. Investigation of various expression strategies leads to an optimised two-step induction protocol introducing 6 mM L-tryptophan at an OD650 of 0.4 followed by incubation for 16 h and a second induction step with 3 mM L-tryptophan followed by a final incubation time of 4 h. Compared with the uncontrolled shaker-flask cultivations used until date, dry cell mass concentrations were improved by a factor of more than 5 and cell-specific enzyme activities showed an up to 28-fold increased yield of the heterologous proteins.

Unexpected role of the IMD pathway in Drosophila gut defense against Staphylococcus aureus.

PubMed

Hori, Aki; Kurata, Shoichiro; Kuraishi, Takayuki

2018-01-01

In this study, fruit fly of the genus Drosophila is utilized as a suitable model animal to investigate the molecular mechanisms of innate immunity. To combat orally transmitted pathogenic Gram-negative bacteria, the Drosophila gut is armed with the peritrophic matrix, which is a physical barrier composed of chitin and glycoproteins: the Duox system that produces reactive oxygen species (ROS), which in turn sterilize infected microbes, and the IMD pathway that regulates the expression of antimicrobial peptides (AMPs), which in turn control ROS-resistant pathogens. However, little is known about the defense mechanisms against Gram-positive bacteria in the fly gut. Here, we show that the peritrophic matrix protects Drosophila against Gram-positive bacteria S. aureus. We also define the few roles of ROS in response to the infection and show that the IMD pathway is required for the clearance of ingested microbes, possibly independently from AMP expression. These findings provide a new aspect of the gut defense system of Drosophila, and helps to elucidate the processes of gut-microbe symbiosis and pathogenesis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Membrane Protein Production in E. coli Lysates in Presence of Preassembled Nanodiscs.

PubMed

Rues, Ralf-Bernhardt; Gräwe, Alexander; Henrich, Erik; Bernhard, Frank

2017-01-01

Cell-free expression allows to synthesize membrane proteins in completely new formats that can relatively easily be customized for particular applications. Amphiphilic superstructures such as micelles, lipomicelles, or nanodiscs can be provided as nano-devices for the solubilization of membrane proteins. Defined empty bilayers in the form of nanodiscs offer native like environments for membrane proteins, supporting functional folding, proper oligomeric assembly as well as stability. Even very difficult and detergent-sensitive membrane proteins can be addressed by the combination of nanodisc technology with efficient cell-free expression systems as the direct co-translational insertion of nascent membrane proteins into supplied preassembled nanodiscs is possible. This chapter provides updated protocols for the synthesis of membrane proteins in presence of preassembled nanodiscs suitable for emerging applications such as screening of lipid effects on membrane protein function and the modulation of oligomeric complex formation.

Heterologous laccase production and its role in industrial applications

PubMed Central

Pezzella, Cinzia; Giardina, Paola; Faraco, Vincenza; Sannia, Giovanni

2010-01-01

Laccases are blue multicopper oxidases, catalyzing the oxidation of an array of aromatic substrates concomitantly with the reduction of molecular oxygen to water. These enzymes are implicated in a variety of biological activities. Most of the laccases studied thus far are of fungal origin. The large range of substrates oxidized by laccases has raised interest in using them within different industrial fields, such as pulp delignification, textile dye bleaching and bioremediation. Laccases secreted from native sources are usually not suitable for large-scale purposes, mainly due to low production yields and high cost of preparation/purification procedures. Heterologous expression may provide higher enzyme yields and may permit to produce laccases with desired properties (such as different substrate specificities, or improved stabilities) for industrial applications. This review surveys researches on heterologous laccase expression focusing on the pivotal role played by recombinant systems towards the development of robust tools for greening modern industry. PMID:21327057

An Organotypic 3D Assay for Primary Human Mammary Epithelial Cells that Recapitulates Branching Morphogenesis.

PubMed

Linnemann, Jelena R; Meixner, Lisa K; Miura, Haruko; Scheel, Christina H

2017-01-01

We have developed a three-dimensional organotypic culture system for primary human mammary epithelial cells (HMECs) in which the cells are cultured in free floating collagen type I gels. In this assay, luminal cells predominantly form multicellular spheres, while basal/myoepithelial cells form complex branched structures resembling terminal ductal lobular units (TDLUs), the functional units of the human mammary gland in situ. The TDLU-like organoids can be cultured for at least 3 weeks and can then be passaged multiple times. Subsequently, collagen gels can be stained with carmine or by immunofluorescence to allow for the analysis of morphology, protein expression and polarization, and to facilitate quantification of structures. In addition, structures can be isolated for gene expression analysis. In summary, this technique is suitable for studying branching morphogenesis, regeneration, and differentiation of HMECs as well as their dependence on the physical environment.

Cattaneo-Christov double-diffusion theory for three-dimensional flow of viscoelastic nanofluid with the effect of heat generation/absorption

NASA Astrophysics Data System (ADS)

Hayat, Tasawar; Qayyum, Sajid; Shehzad, Sabir Ali; Alsaedi, Ahmed

2018-03-01

The present research article focuses on three-dimensional flow of viscoelastic(second grade) nanofluid in the presence of Cattaneo-Christov double-diffusion theory. Flow caused is due to stretching sheet. Characteristics of heat transfer are interpreted by considering the heat generation/absorption. Nanofluid theory comprises of Brownian motion and thermophoresis. Cattaneo-Christov double-diffusion theory is introduced in the energy and concentration expressions. Such diffusions are developed as a part of formulating the thermal and solutal relaxation times framework. Suitable variables are implemented for the conversion of partial differential systems into a sets of ordinary differential equations. The transformed expressions have been explored through homotopic algorithm. Behavior of sundry variables on the velocities, temperature and concentration are scrutinized graphically. Numerical values of skin friction coefficients are also calculated and examined. Here thermal field enhances for heat generation parameter while reverse situation is noticed for heat absorption parameter.

Cells for tissue engineering of cardiac valves.

PubMed

Jana, Soumen; Tranquillo, Robert T; Lerman, Amir

2016-10-01

Heart valve tissue engineering is a promising alternative to prostheses for the replacement of diseased or damaged heart valves, because tissue-engineered valves have the ability to remodel, regenerate and grow. To engineer heart valves, cells are harvested, seeded onto or into a three-dimensional (3D) matrix platform to generate a tissue-engineered construct in vitro, and then implanted into a patient's body. Successful engineering of heart valves requires a thorough understanding of the different types of cells that can be used to obtain the essential phenotypes that are expressed in native heart valves. This article reviews different cell types that have been used in heart valve engineering, cell sources for harvesting, phenotypic expression in constructs and suitability in heart valve tissue engineering. Natural and synthetic biomaterials that have been applied as scaffold systems or cell-delivery platforms are discussed with each cell type. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

A Single-Expression Formula for Inverting Strain-Life and Stress-Strain Relationships

NASA Technical Reports Server (NTRS)

Manson, S. S.; Muralidharan, U.

1981-01-01

Starting with the basic fatigue lift formula, an inversion formula is derived. The inversion formula is valid over the entire life range of engineering interest for all materials examined. Conformity between the two equations is extremely close, suitable for all engineering problems. The approach used to invert the life relation is also suitable for the inversion of other formulas involving the sum of two power-law terms.

Suitability of a Saccharomyces cerevisiae-based assay to assess the toxicity of pyrimethanil sprayed soils via surface runoff: comparison with standard aquatic and soil toxicity assays.

PubMed

Gil, Fátima N; Moreira-Santos, Matilde; Chelinho, Sónia; Pereira, Carla; Feliciano, Joana R; Leitão, Jorge H; Sousa, José P; Ribeiro, Rui; Viegas, Cristina A

2015-02-01

The present study is aimed at evaluating whether a gene expression assay with the microbial eukaryotic model Saccharomyces cerevisiae could be used as a suitable warning tool for the rapid preliminary screening of potential toxic effects on organisms due to scenarios of soil and water contamination with pyrimethanil. The assay consisted of measuring changes in the expression of the selected pyrimethanil-responsive genes ARG3 and ARG5,6 in a standardized yeast population. Evaluation was held by assessing the toxicity of surface runoff, a major route of pesticide exposure in aquatic systems due to non-point-source pollution, which was simulated with a pyrimethanil formulation at a semifield scale mimicking worst-case scenarios of soil contamination (e.g. accident or improper disposal). Yeast cells 2-h exposure to the runoff samples led to a significant 2-fold increase in the expression of both indicator genes. These results were compared with those from assays with organisms relevant for the aquatic and soil compartments, namely the nematode Caenorhabditis elegans (reproduction), the freshwater cladoceran Daphnia magna (survival and reproduction), the benthic midge Chironomus riparius (growth), and the soil invertebrates Folsomia candida and Enchytraeus crypticus (survival and reproduction). Under the experimental conditions used to simulate accidental discharges into soil, runoff waters were highly toxic to the standard test organisms, except for C. elegans. Overall, results point out the usefulness of the yeast assay to provide a rapid preview of the toxicity level in preliminary screenings of environmental samples in situations of inadvertent high pesticide contamination. Advantages and limitations of this novel method are discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification and Validation of Reference Genes for RT-qPCR Analysis in Non-Heading Chinese Cabbage Flowers

PubMed Central

Wang, Cheng; Cui, Hong-Mi; Huang, Tian-Hong; Liu, Tong-Kun; Hou, Xi-Lin; Li, Ying

2016-01-01

Non-heading Chinese cabbage (Brassica rapa ssp. chinensis Makino) is an important vegetable member of Brassica rapa crops. It exhibits a typical sporophytic self-incompatibility (SI) system and is an ideal model plant to explore the mechanism of SI. Gene expression research are frequently used to unravel the complex genetic mechanism and in such studies appropriate reference selection is vital. Validation of reference genes have neither been conducted in Brassica rapa flowers nor in SI trait. In this study, 13 candidate reference genes were selected and examined systematically in 96 non-heading Chinese cabbage flower samples that represent four strategic groups in compatible and self-incompatible lines of non-heading Chinese cabbage. Two RT-qPCR analysis software, geNorm and NormFinder, were used to evaluate the expression stability of these genes systematically. Results revealed that best-ranked references genes should be selected according to specific sample subsets. DNAJ, UKN1, and PP2A were identified as the most stable reference genes among all samples. Moreover, our research further revealed that the widely used reference genes, CYP and ACP, were the least suitable reference genes in most non-heading Chinese cabbage flower sample sets. To further validate the suitability of the reference genes identified in this study, the expression level of SRK and Exo70A1 genes which play important roles in regulating interaction between pollen and stigma were studied. Our study presented the first systematic study of reference gene(s) selection for SI study and provided guidelines to obtain more accurate RT-qPCR results in non-heading Chinese cabbage. PMID:27375663

Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro.

PubMed

Cong, Shan; Cao, Guifang; Liu, Dongjun

2014-12-01

To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1-5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic fibroblast feeder layers of mouse and bovine embryonic fibroblast at different ratios and sources including mouse fibroblast cell lines. The bovine embryonic stem cell-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as OCT4, stage-specific embryonic antigen 1 and 4, alkaline phosphatase, SOX2, and NANOG mRNA levels. When feeder layers and cell growth factors were removed, the bovine embryonic stem cell-like cells formed embryoid bodies in a suspension culture. Furthermore, we compared the expression of the pluripotent markers during bovine embryonic stem cell-like cell in culture on mixed embryonic fibroblast feeder layers, including mouse fibroblast cell lines feeder layers and mouse embryonic fibroblast feeder layers by real-time quantitative polymerase chain reaction. Results suggested that mixed embryonic fibroblast and sources including mouse fibroblast cell lines feeder layers were more suitable for long-term culture and growth of bovine embryonic stem cell-like cells than mouse embryonic fibroblast feeder layers. The findings may provide useful experimental data for the establishment of an appropriate culture system for bovine embryonic stem cell lines.

Understanding the dynamics of superparamagnetic particles under the influence of high field gradient arrays

NASA Astrophysics Data System (ADS)

Barnsley, Lester C.; Carugo, Dario; Aron, Miles; Stride, Eleanor

2017-03-01

The aim of this study was to characterize the behaviour of superparamagnetic particles in magnetic drug targeting (MDT) schemes. A 3-dimensional mathematical model was developed, based on the analytical derivation of the trajectory of a magnetized particle suspended inside a fluid channel carrying laminar flow and in the vicinity of an external source of magnetic force. Semi-analytical expressions to quantify the proportion of captured particles, and their relative accumulation (concentration) as a function of distance along the wall of the channel were also derived. These were expressed in terms of a non-dimensional ratio of the relevant physical and physiological parameters corresponding to a given MDT protocol. The ability of the analytical model to assess magnetic targeting schemes was tested against numerical simulations of particle trajectories. The semi-analytical expressions were found to provide good first-order approximations for the performance of MDT systems in which the magnetic force is relatively constant over a large spatial range. The numerical model was then used to test the suitability of a range of different designs of permanent magnet assemblies for MDT. The results indicated that magnetic arrays that emit a strong magnetic force that varies rapidly over a confined spatial range are the most suitable for concentrating magnetic particles in a localized region. By comparison, commonly used magnet geometries such as button magnets and linear Halbach arrays result in distributions of accumulated particles that are less efficient for delivery. The trajectories predicted by the numerical model were verified experimentally by acoustically focusing magnetic microbeads flowing in a glass capillary channel, and optically tracking their path past a high field gradient Halbach array.

Identifying optimal reference genes for the normalization of microRNA expression in cucumber under viral stress

PubMed Central

Liang, Chaoqiong; Hao, Jianjun; Meng, Yan; Luo, Laixin; Li, Jianqiang

2018-01-01

Cucumber green mottle mosaic virus (CGMMV) is an economically important pathogen and causes significant reduction of both yield and quality of cucumber (Cucumis sativus). Currently, there were no satisfied strategies for controlling the disease. A better understanding of microRNA (miRNA) expression related to the regulation of plant-virus interactions and virus resistance would be of great assistance when developing control strategies for CGMMV. However, accurate expression analysis is highly dependent on robust and reliable reference gene used as an internal control for normalization of miRNA expression. Most commonly used reference genes involved in CGMMV-infected cucumber are not universally expressed depending on tissue types and stages of plant development. It is therefore crucial to identify suitable reference genes in investigating the role of miRNA expression. In this study, seven reference genes, including Actin, Tubulin, EF-1α, 18S rRNA, Ubiquitin, GAPDH and Cyclophilin, were evaluated for the most accurate results in analyses using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Gene expression was assayed on cucumber leaves, stems and roots that were collected at different days post inoculation with CGMMV. The expression data were analyzed using algorithms including delta-Ct, geNorm, NormFinder, and BestKeeper as well as the comparative tool RefFinder. The reference genes were subsequently validated using miR159. The results showed that EF-1α and GAPDH were the most reliable reference genes for normalizing miRNA expression in leaf, root and stem samples, while Ubiquitin and EF-1α were the most suitable combination overall. PMID:29543906

An image-processing strategy to extract important information suitable for a low-size stimulus pattern in a retinal prosthesis.

PubMed

Chen, Yili; Fu, Jixiang; Chu, Dawei; Li, Rongmao; Xie, Yaoqin

2017-11-27

A retinal prosthesis is designed to help the blind to obtain some sight. It consists of an external part and an internal part. The external part is made up of a camera, an image processor and an RF transmitter. The internal part is made up of an RF receiver, implant chip and microelectrode. Currently, the number of microelectrodes is in the hundreds, and we do not know the mechanism for using an electrode to stimulate the optic nerve. A simple hypothesis is that the pixels in an image correspond to the electrode. The images captured by the camera should be processed by suitable strategies to correspond to stimulation from the electrode. Thus, it is a question of how to obtain the important information from the image captured in the picture. Here, we use the region of interest (ROI), a useful algorithm for extracting the ROI, to retain the important information, and to remove the redundant information. This paper explains the details of the principles and functions of the ROI. Because we are investigating a real-time system, we need a fast processing ROI as a useful algorithm to extract the ROI. Thus, we simplified the ROI algorithm and used it in an outside image-processing digital signal processing (DSP) system of the retinal prosthesis. The results show that our image-processing strategies are suitable for a real-time retinal prosthesis and can eliminate redundant information and provide useful information for expression in a low-size image.

Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II

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

Tykvart, J.; Sacha, P.; Barinka, C.

2012-02-07

Affinity purification is a useful approach for purification of recombinant proteins. Eukaryotic expression systems have become more frequently used at the expense of prokaryotic systems since they afford recombinant eukaryotic proteins with post-translational modifications similar or identical to the native ones. Here, we present a one-step affinity purification set-up suitable for the purification of secreted proteins. The set-up is based on the interaction between biotin and mutated streptavidin. Drosophila Schneider 2 cells are chosen as the expression host, and a biotin acceptor peptide is used as an affinity tag. This tag is biotinylated by Escherichia coli biotin-protein ligase in vivo.more » We determined that localization of the ligase within the ER led to the most effective in vivo biotinylation of the secreted proteins. We optimized a protocol for large-scale expression and purification of AviTEV-tagged recombinant human glutamate carboxypeptidase II (Avi-GCPII) with milligram yields per liter of culture. We also determined the 3D structure of Avi-GCPII by X-ray crystallography and compared the enzymatic characteristics of the protein to those of its non-tagged variant. These experiments confirmed that AviTEV tag does not affect the biophysical properties of its fused partner. Purification approach, developed here, provides not only a sufficient amount of highly homogenous protein but also specifically and effectively biotinylates a target protein and thus enables its subsequent visualization or immobilization.« less

Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions.

PubMed

Chen, Lei; Zhong, Hai-ying; Kuang, Jian-fei; Li, Jian-guo; Lu, Wang-jin; Chen, Jian-ye

2011-08-01

Reverse transcription quantitative real-time PCR (RT-qPCR) is a sensitive technique for quantifying gene expression, but its success depends on the stability of the reference gene(s) used for data normalization. Only a few studies on validation of reference genes have been conducted in fruit trees and none in banana yet. In the present work, 20 candidate reference genes were selected, and their expression stability in 144 banana samples were evaluated and analyzed using two algorithms, geNorm and NormFinder. The samples consisted of eight sample sets collected under different experimental conditions, including various tissues, developmental stages, postharvest ripening, stresses (chilling, high temperature, and pathogen), and hormone treatments. Our results showed that different suitable reference gene(s) or combination of reference genes for normalization should be selected depending on the experimental conditions. The RPS2 and UBQ2 genes were validated as the most suitable reference genes across all tested samples. More importantly, our data further showed that the widely used reference genes, ACT and GAPDH, were not the most suitable reference genes in many banana sample sets. In addition, the expression of MaEBF1, a gene of interest that plays an important role in regulating fruit ripening, under different experimental conditions was used to further confirm the validated reference genes. Taken together, our results provide guidelines for reference gene(s) selection under different experimental conditions and a foundation for more accurate and widespread use of RT-qPCR in banana.

Suitable for Whom? The Case of System of Rice Intensification in Tanzania

ERIC Educational Resources Information Center

Tumusiime, Emmanuel

2017-01-01

Purpose: This study examines the suitability of the system of rice intensification (SRI) for diverse small-scale farmers in Tanzania by exploring if poor and non-poor farmers adopt the system to a similar extent. Originality: The suitability of low-external input technologies such as SRI for diverse African farmers is a contentious issue. Existing…

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Federal Register 2010, 2011, 2012, 2013, 2014

2010-09-17

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Mapping gene expression patterns during myeloid differentiation using the EML hematopoietic progenitor cell line.

PubMed

Du, Yang; Campbell, Janee L; Nalbant, Demet; Youn, Hyewon; Bass, Ann C Hughes; Cobos, Everardo; Tsai, Schickwann; Keller, Jonathan R; Williams, Simon C

2002-07-01

The detailed examination of the molecular events that control the early stages of myeloid differentiation has been hampered by the relative scarcity of hematopoietic stem cells and the lack of suitable cell line models. In this study, we examined the expression of several myeloid and nonmyeloid genes in the murine EML hematopoietic stem cell line. Expression patterns for 19 different genes were examined by Northern blotting and RT-PCR in RNA samples from EML, a variety of other immortalized cell lines, and purified murine hematopoietic stem cells. Representational difference analysis (RDA) was performed to identify differentially expressed genes in EML. Expression patterns of genes encoding transcription factors (four members of the C/EBP family, GATA-1, GATA-2, PU.1, CBFbeta, SCL, and c-myb) in EML were examined and were consistent with the proposed functions of these proteins in hematopoietic differentiation. Expression levels of three markers of terminal myeloid differentiation (neutrophil elastase, proteinase 3, and Mac-1) were highest in EML cells at the later stages of differentiation. In a search for genes that were differentially expressed in EML cells during myeloid differentiation, six cDNAs were isolated. These included three known genes (lysozyme, histidine decarboxylase, and tryptophan hydroxylase) and three novel genes. Expression patterns of known genes in differentiating EML cells accurately reflected their expected expression patterns based on previous studies. The identification of three novel genes, two of which encode proteins that may act as regulators of hematopoietic differentiation, suggests that EML is a useful model system for the molecular analysis of hematopoietic differentiation.

Development of expert system for biobased polymer material selection: food packaging application.

PubMed

Sanyang, M L; Sapuan, S M

2015-10-01

Biobased food packaging materials are gaining more attention owing to their intrinsic biodegradable nature and renewability. Selection of suitable biobased polymers for food packaging applications could be a tedious task with potential mistakes in choosing the best materials. In this paper, an expert system was developed using Exsys Corvid software to select suitable biobased polymer materials for packaging fruits, dry food and dairy products. If - Then rule based system was utilized to accomplish the material selection process whereas a score system was formulated to facilitate the ranking of selected materials. The expert system selected materials that satisfied all constraints and selection results were presented in suitability sequence depending on their scores. The expert system selected polylactic acid (PLA) as the most suitable material.

Suitable pitch difference to realize anti-loosening performance for various bolts-nuts diameter

NASA Astrophysics Data System (ADS)

Kubo, S.; Tateishi, K.; Noda, N.-A.; Sano, Y.; Takase, Y.; Honda, K.

2018-06-01

In bolt-nut connection, the anti-loosening performance and high fatigue strength are always required with low cost to ensure the connected structure’s safety. In the previous study, a suitable pitch difference between the bolt-nut was obtained as α = 33 μm for M16 JIS bolt- nut through loosening experiment and FEM simulation for tightening process. However, other bolt-nut diameters have not been considered yet. In this paper, therefore, suitable pitch difference is considered for various diameters to realize anti-loosening performance. Since bolt-nut thread geometries are different depending on the diameter, they are expressed as approximate formula. Then, loosening force and anti-loosening force are considered by varying the diameter. Finally, suitable pitch difference {α }minsuit< α < {α }maxsuit was determined from mechanical condition.

A dynamic viticultural zoning to explore the resilience of terroir concept under climate change.

PubMed

Bonfante, A; Monaco, E; Langella, G; Mercogliano, P; Bucchignani, E; Manna, P; Terribile, F

2018-05-15

Climate change (CC) directly influences agricultural sectors, presenting the need to identify both adaptation and mitigation actions that can make local farming communities and crop production more resilient. In this context, the viticultural sector is one of those most challenged by CC due to the need to combine grape quality, grapevine cultivar adaptation and therefore farmers' future incomes. Thus, understanding how suitability for viticulture is changing under CC is of primary interest in the development of adaptation strategies in traditional wine-growing regions. Considering that climate is an essential part of the terroir system, the expected variability in climate change could have a marked influence on terroir resilience with important effects on local farming communities in viticultural regions. From this perspective, the aim of this paper is to define a new dynamic viticultural zoning procedure that is able to integrate the effects of CC on grape quality responses and evaluate terroir resilience, providing a support tool for stakeholders involved in viticultural planning (winegrowers, winegrower consortiums, policy makers etc.). To achieve these aims, a Hybrid Land Evaluation System, combining qualitative (standard Land Evaluation) and quantitative (simulation model) approaches, was applied within a traditional region devoted to high quality wine production in Southern Italy (Valle Telesina, BN), for a specific grapevine cultivar (Aglianico). The work employed high resolution climate projections that were derived under two different IPCC scenarios, namely RCP 4.5 and RCP 8.5. The results obtained indicate that: (i) only 2% of the suitable area of Valle Telesina expresses the concept of terroir resilience orientated towards Aglianico ultra quality grape production; (ii) within 2010-2040, it is expected that 41% of the area suitable for Aglianico cultivation will need irrigation to achieve quality grape production; (iii) by 2100, climate change benefits for the cultivation of Aglianico will decrease, as well as the suitable areas. Copyright © 2017 Elsevier B.V. All rights reserved.

Electrodynamic properties of a hypercrystal with ferrite and semiconductor layers in an external magnetic field

NASA Astrophysics Data System (ADS)

Fedorin, Illia V.

2018-01-01

Electrodynamic properties of a photonic hypercrystal formed by periodically alternating two types of anisotropic metamaterials are studied. The first metamaterial consists of ferrite and dielectric layers, while the second metamaterial consists of semiconductor and dielectric layers. The system is assumed to be placed in an external magnetic field, which applied parallel to the boundaries of the layers. An effective medium theory which is suitable for calculation of properties of long-wavelength electromagnetic modes is applied in order to derive averaged expressions for effective constitutive parameters. It has been shown that providing a conscious choice of the constitutive parameters and material fractions of magnetic, semiconductor, and dielectric layers, the system under study shows hypercrystal properties for both TE and TM waves in the different frequency ranges.

A study of the nucleus-nucleus total reaction cross section of stable systems at intermediate energies: An application to 12C

NASA Astrophysics Data System (ADS)

Hu, Liyuan; Song, Yushou; Hou, Yingwei; Liu, Huilan; Li, Hui

2018-07-01

A semi-microscopic analytical expression of the nucleus-nucleus total reaction cross section (σR) was proposed based on the strong absorption model. It is suitable for stable nuclei at intermediate energies. The matter density distributions of nuclei and the nucleon-nucleon total cross section were both considered. Particularly, the Fermi motion effect of the nucleons in a nucleus was also taken into account. The parametrization of σR was applied to the colliding systems including 12C. The experimental data at energies from 30 to 1000 MeV/nucleon were well reproduced, according to which an approach of deriving σR without adjustable parameters was developed. The necessity of considering the Fermi motion effect in the parametrization was discussed.

Thermographic Sensing For On-Line Industrial Control

NASA Astrophysics Data System (ADS)

Holmsten, Dag

1986-10-01

It is today's emergence of thermoelectrically cooled, highly accurate infrared linescanners and imaging systems that has definitely made on-line Infraread Thermography (IRT) possible. Specifically designed for continuous use, these scanners are equipped with dedicated software capable of monitoring and controlling highly complex thermodynamic situations. This paper will outline some possible implications of using IRT on-line by describing some uses of this technology in the steel-making (hot rolling) and automotive industries (machine-vision). A warning is also expressed that IRT technology not originally designed for automated applications e.g. high resolution, imaging systems, should not be directly applied to an on-line measurement situation without having its measurement resolution, accuracy and especially its repeatability, reliably proven. Some suitable testing procedures are briefly outlined at the end of the paper.

N-Glycosylation of an IgG antibody secreted by Nicotiana tabacum BY-2 cells can be modulated through co-expression of human β-1,4-galactosyltransferase.

PubMed

Navarre, Catherine; Smargiasso, Nicolas; Duvivier, Laurent; Nader, Joseph; Far, Johann; De Pauw, Edwin; Boutry, Marc

2017-06-01

Nicotiana tabacum BY-2 suspension cells have several advantages that make them suitable for the production of full-size monoclonal antibodies which can be purified directly from the culture medium. Carbohydrate characterization of an antibody (Lo-BM2) expressed in N. tabacum BY-2 cells showed that the purified Lo-BM2 displays N-glycan homogeneity with a high proportion (>70%) of the complex GnGnXF glycoform. The stable co-expression of a human β-1,4-galactosyltransferase targeted to different Golgi sub-compartments altered Lo-BM2N-glycosylation and resulted in the production of an antibody that exhibited either hybrid structures containing a low abundance of the plant epitopes (α-1,3-fucose and β-1,2-xylose), or a large amount of galactose-extended N-glycan structures. These results demonstrate the suitability of stable N-glycoengineered N. tabacum BY-2 cell lines for the production of human-like antibodies.

A rule-based approach to model checking of UML state machines

NASA Astrophysics Data System (ADS)

Grobelna, Iwona; Grobelny, Michał; Stefanowicz, Łukasz

2016-12-01

In the paper a new approach to formal verification of control process specification expressed by means of UML state machines in version 2.x is proposed. In contrast to other approaches from the literature, we use the abstract and universal rule-based logical model suitable both for model checking (using the nuXmv model checker), but also for logical synthesis in form of rapid prototyping. Hence, a prototype implementation in hardware description language VHDL can be obtained that fully reflects the primary, already formally verified specification in form of UML state machines. Presented approach allows to increase the assurance that implemented system meets the user-defined requirements.

Selection of reference genes for gene expression studies in heart failure for left and right ventricles.

PubMed

Li, Mengmeng; Rao, Man; Chen, Kai; Zhou, Jianye; Song, Jiangping

2017-07-15

Real-time quantitative reverse transcriptase-PCR (qRT-PCR) is a feasible tool for determining gene expression profiles, but the accuracy and reliability of the results depends on the stable expression of selected housekeeping genes in different samples. By far, researches on stable housekeeping genes in human heart failure samples are rare. Moreover the effect of heart failure on the expression of housekeeping genes in right and left ventricles is yet to be studied. Therefore we aim to provide stable housekeeping genes for both ventricles in heart failure and normal heart samples. In this study, we selected seven commonly used housekeeping genes as candidates. By using the qRT-PCR, the expression levels of ACTB, RAB7A, GAPDH, REEP5, RPL5, PSMB4 and VCP in eight heart failure and four normal heart samples were assessed. The stability of candidate housekeeping genes was evaluated by geNorm and Normfinder softwares. GAPDH showed the least variation in all heart samples. Results also indicated the difference of gene expression existed in heart failure left and right ventricles. GAPDH had the highest expression stability in both heart failure and normal heart samples. We also propose using different sets of housekeeping genes for left and right ventricles respectively. The combination of RPL5, GAPDH and PSMB4 is suitable for the right ventricle and the combination of GAPDH, REEP5 and RAB7A is suitable for the left ventricle. Copyright © 2017 Elsevier B.V. All rights reserved.

Isolation and selection of suitable reference genes for real-time PCR analyses in the skeletal muscle of the fine flounder in response to nutritional status: assessment and normalization of gene expression of growth-related genes.

PubMed

Fuentes, Eduardo N; Safian, Diego; Valdés, Juan Antonio; Molina, Alfredo

2013-08-01

In the present study, different reference genes were isolated, and their stability in the skeletal muscle of fine flounder subjected to different nutritional states was assessed using geNorm and NormFinder. The combinations between 18S and ActB; Fau and 18S; and Fau and Tubb were chosen as the most stable gene combinations in feeding, long-term fasting and refeeding, and short-term refeeding conditions, respectively. In all periods, ActB was identified as the single least stable gene. Subsequently, the expression of the myosin heavy chain (MYH) and the insulin-like growth factor-I receptor (IGF-IR) was assessed. A large variation in MYH and IGF-IR expression was found depending on the reference gene that was chosen for normalizing the expression of both genes. Using the most stable reference genes, mRNA levels of MYH decreased and IGF-IR increased during fasting, with both returning to basal levels during refeeding. However, the drop in mRNA levels for IGF-IR occurred during short-term refeeding, in contrast with the observed events in the expression of MYH, which occurred during long-term refeeding. The present study highlights the vast differences incurred when using unsuitable versus suitable reference genes for normalizing gene expression, pointing out that normalization without proper validation could result in a bias of gene expression.

10 CFR 963.16 - Postclosure suitability evaluation method.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 963.16 Energy DEPARTMENT OF ENERGY YUCCA MOUNTAIN SITE SUITABILITY GUIDELINES Site Suitability... assessment to evaluate the ability of the Yucca Mountain disposal system to limit radiological doses and... the performance of the Yucca Mountain disposal system using the method described in paragraph (b) of...

Research on Fault Characteristics and Line Protections Within a Large-scale Photovoltaic Power Plant

NASA Astrophysics Data System (ADS)

Zhang, Chi; Zeng, Jie; Zhao, Wei; Zhong, Guobin; Xu, Qi; Luo, Pandian; Gu, Chenjie; Liu, Bohan

2017-05-01

Centralized photovoltaic (PV) systems have different fault characteristics from distributed PV systems due to the different system structures and controls. This makes the fault analysis and protection methods used in distribution networks with distributed PV not suitable for a centralized PV power plant. Therefore, a consolidated expression for the fault current within a PV power plant under different controls was calculated considering the fault response of the PV array. Then, supported by the fault current analysis and the on-site testing data, the overcurrent relay (OCR) performance was evaluated in the collection system of an 850 MW PV power plant. It reveals that the OCRs at downstream side on overhead lines may malfunction. In this case, a new relay scheme was proposed using directional distance elements. In the PSCAD/EMTDC, a detailed PV system model was built and verified using the on-site testing data. Simulation results indicate that the proposed relay scheme could effectively solve the problems under variant fault scenarios and PV plant output levels.

Deriving the exact nonadiabatic quantum propagator in the mapping variable representation.

PubMed

Hele, Timothy J H; Ananth, Nandini

2016-12-22

We derive an exact quantum propagator for nonadiabatic dynamics in multi-state systems using the mapping variable representation, where classical-like Cartesian variables are used to represent both continuous nuclear degrees of freedom and discrete electronic states. The resulting Liouvillian is a Moyal series that, when suitably approximated, can allow for the use of classical dynamics to efficiently model large systems. We demonstrate that different truncations of the exact Liouvillian lead to existing approximate semiclassical and mixed quantum-classical methods and we derive an associated error term for each method. Furthermore, by combining the imaginary-time path-integral representation of the Boltzmann operator with the exact Liouvillian, we obtain an analytic expression for thermal quantum real-time correlation functions. These results provide a rigorous theoretical foundation for the development of accurate and efficient classical-like dynamics to compute observables such as electron transfer reaction rates in complex quantized systems.

Research on the Orientation and Application of Distributed Energy Storage in Energy Internet

NASA Astrophysics Data System (ADS)

Zeng, Ming; Zhou, Pengcheng; Li, Ran; Zhou, Jingjing; Chen, Tao; Li, Zhe

2018-01-01

Energy storage is indispensable resources to achieve a high proportion of new energy power consumption in electric power system. As an important support to energy Internet, energy storage system can achieve a variety of energy integration operation to ensure maximum energy efficiency. In this paper, firstly, the SWOT analysis method is used to express the internal and external advantages and disadvantages of distributed energy storage participating in the energy Internet. Secondly, the function orientation of distributed energy storage in energy Internet is studied, based on which the application modes of distributed energy storage in virtual power plant, community energy storage and auxiliary services are deeply studied. Finally, this paper puts forward the development strategy of distributed energy storage which is suitable for the development of China’s energy Internet, and summarizes and prospects the application of distributed energy storage system.

Model and Comparative Study for Flow of Viscoelastic Nanofluids with Cattaneo-Christov Double Diffusion

PubMed Central

Hayat, Tasawar; Aziz, Arsalan; Muhammad, Taseer; Alsaedi, Ahmed

2017-01-01

Here two classes of viscoelastic fluids have been analyzed in the presence of Cattaneo-Christov double diffusion expressions of heat and mass transfer. A linearly stretched sheet has been used to create the flow. Thermal and concentration diffusions are characterized firstly by introducing Cattaneo-Christov fluxes. Novel features regarding Brownian motion and thermophoresis are retained. The conversion of nonlinear partial differential system to nonlinear ordinary differential system has been taken into place by using suitable transformations. The resulting nonlinear systems have been solved via convergent approach. Graphs have been sketched in order to investigate how the velocity, temperature and concentration profiles are affected by distinct physical flow parameters. Numerical values of skin friction coefficient and heat and mass transfer rates at the wall are also computed and discussed. Our observations demonstrate that the temperature and concentration fields are decreasing functions of thermal and concentration relaxation parameters. PMID:28046011

Development and evaluation of habitat suitability criteria for use in the instream flow incremental methodology

USGS Publications Warehouse

Bovee, Ken D.

1986-01-01

The Instream Flow Incremental Methodology (IFIM) is a habitat-based tool used to evaluate the environmental consequences of various water and land use practices. As such, knowledge about the conditions that provide favorable habitat for a species, and those that do not, is necessary for successful implementation of the methodology. In the context of IFIM, this knowledge is defined as habitat suitability criteria: characteristic behavioral traits of a species that are established as standards for comparison in the decision-making process. Habitat suitability criteria may be expressed in a variety of types and formats. The type, or category, refers to the procedure used to develop the criteria. Category I criteria are based on professional judgment, with little or no empirical data. Category II criteria have as their source, microhabitat data collected at locations where target organisms are observed or collected. These are called “utilization” functions because they are based on observed locations that were used by the target organism. These functions tend to be biased by the environmental conditions that were available to the fish or invertebrates at the time they were observed. Correction of the utilization function for environmental availability creates category III, or “preference” criteria, which tend to be much less site specific than category II criteria. There are also several ways to express habitat suitability in graphical form. The binary format establishes a suitable range for each variable as it pertains to a life stage of interest, and is presented graphically as a step function. The quality rating for a variable is 1.0 if it falls within the range of the criteria, and 0.0 if it falls outside the range. The univariate curve format established both the usable range and the optimum range for each variable, with conditions of intermediate usability expressed along the portion between the tails and the peak of the curve. Multivariate probability density functions, which can be used to compute suitability for several variables simultaneously, are conveyed as three dimensional figures with suitability on the z-axis, and two independent variables on the x-y plane. These functions are useful for incorporating interactive terms between two or more variable. Such interactions can also be demonstrated using conditional criteria, which are stratified by cover type or substrate size. Conditional criteria may be of any category or format, but are distinguishable by two or more sets of functional relationships for each life stage.

Identification of appropriate reference genes for normalizing transcript expression by quantitative real-time PCR in Litsea cubeba.

PubMed

Lin, Liyuan; Han, Xiaojiao; Chen, Yicun; Wu, Qingke; Wang, Yangdong

2013-12-01

Quantitative real-time PCR has emerged as a highly sensitive and widely used method for detection of gene expression profiles, via which accurate detection depends on reliable normalization. Since no single control is appropriate for all experimental treatments, it is generally advocated to select suitable internal controls prior to use for normalization. This study reported the evaluation of the expression stability of twelve potential reference genes in different tissue/organs and six fruit developmental stages of Litsea cubeba in order to screen the superior internal reference genes for data normalization. Two softwares-geNorm, and NormFinder-were used to identify stability of these candidate genes. The cycle threshold difference and coefficient of variance were also calculated to evaluate the expression stability of candidate genes. F-BOX, EF1α, UBC, and TUA were selected as the most stable reference genes across 11 sample pools. F-BOX, EF1α, and EIF4α exhibited the highest expression stability in different tissue/organs and different fruit developmental stages. Besides, a combination of two stable reference genes would be sufficient for gene expression normalization in different fruit developmental stages. In addition, the relative expression profiles of DXS and DXR were evaluated by EF1α, UBC, and SAMDC. The results further validated the reliability of stable reference genes and also highlighted the importance of selecting suitable internal controls for L. cubeba. These reference genes will be of great importance for transcript normalization in future gene expression studies on L. cubeba.

Using RNA-seq data to select reference genes for normalizing gene expression in apple roots.

PubMed

Zhou, Zhe; Cong, Peihua; Tian, Yi; Zhu, Yanmin

2017-01-01

Gene expression in apple roots in response to various stress conditions is a less-explored research subject. Reliable reference genes for normalizing quantitative gene expression data have not been carefully investigated. In this study, the suitability of a set of 15 apple genes were evaluated for their potential use as reliable reference genes. These genes were selected based on their low variance of gene expression in apple root tissues from a recent RNA-seq data set, and a few previously reported apple reference genes for other tissue types. Four methods, Delta Ct, geNorm, NormFinder and BestKeeper, were used to evaluate their stability in apple root tissues of various genotypes and under different experimental conditions. A small panel of stably expressed genes, MDP0000095375, MDP0000147424, MDP0000233640, MDP0000326399 and MDP0000173025 were recommended for normalizing quantitative gene expression data in apple roots under various abiotic or biotic stresses. When the most stable and least stable reference genes were used for data normalization, significant differences were observed on the expression patterns of two target genes, MdLecRLK5 (MDP0000228426, a gene encoding a lectin receptor like kinase) and MdMAPK3 (MDP0000187103, a gene encoding a mitogen-activated protein kinase). Our data also indicated that for those carefully validated reference genes, a single reference gene is sufficient for reliable normalization of the quantitative gene expression. Depending on the experimental conditions, the most suitable reference genes can be specific to the sample of interest for more reliable RT-qPCR data normalization.

Using RNA-seq data to select reference genes for normalizing gene expression in apple roots

PubMed Central

Zhou, Zhe; Cong, Peihua; Tian, Yi

2017-01-01

Gene expression in apple roots in response to various stress conditions is a less-explored research subject. Reliable reference genes for normalizing quantitative gene expression data have not been carefully investigated. In this study, the suitability of a set of 15 apple genes were evaluated for their potential use as reliable reference genes. These genes were selected based on their low variance of gene expression in apple root tissues from a recent RNA-seq data set, and a few previously reported apple reference genes for other tissue types. Four methods, Delta Ct, geNorm, NormFinder and BestKeeper, were used to evaluate their stability in apple root tissues of various genotypes and under different experimental conditions. A small panel of stably expressed genes, MDP0000095375, MDP0000147424, MDP0000233640, MDP0000326399 and MDP0000173025 were recommended for normalizing quantitative gene expression data in apple roots under various abiotic or biotic stresses. When the most stable and least stable reference genes were used for data normalization, significant differences were observed on the expression patterns of two target genes, MdLecRLK5 (MDP0000228426, a gene encoding a lectin receptor like kinase) and MdMAPK3 (MDP0000187103, a gene encoding a mitogen-activated protein kinase). Our data also indicated that for those carefully validated reference genes, a single reference gene is sufficient for reliable normalization of the quantitative gene expression. Depending on the experimental conditions, the most suitable reference genes can be specific to the sample of interest for more reliable RT-qPCR data normalization. PMID:28934340

Siting a municipal solid waste disposal facility, part II: the effects of external criteria on the final decision.

PubMed

Korucu, M Kemal; Karademir, Aykan

2014-02-01

The procedure of a multi-criteria decision analysis supported by the geographic information systems was applied to the site selection process of a planning municipal solid waste management practice based on twelve different scenarios. The scenarios included two different decision tree modes and two different weighting models for three different area requirements. The suitability rankings of the suitable sites obtained from the application of the decision procedure for the scenarios were assessed by a factorial experimental design concerning the effect of some external criteria on the final decision of the site selection process. The external criteria used in the factorial experimental design were defined as "Risk perception and approval of stakeholders" and "Visibility". The effects of the presence of these criteria in the decision trees were evaluated in detail. For a quantitative expression of the differentiations observed in the suitability rankings, the ranking data were subjected to ANOVA test after a normalization process. Then the results of these tests were evaluated by Tukey test to measure the effects of external criteria on the final decision. The results of Tukey tests indicated that the involvement of the external criteria into the decision trees produced statistically meaningful differentiations in the suitability rankings. Since the external criteria could cause considerable external costs during the operation of the disposal facilities, the presence of these criteria in the decision tree in addition to the other criteria related to environmental and legislative requisites could prevent subsequent external costs in the first place.

Reliable reference genes for normalization of gene expression data in tea plants (Camellia sinensis) exposed to metal stresses.

PubMed

Wang, Ming-Le; Li, Qing-Hui; Xin, Hua-Hong; Chen, Xuan; Zhu, Xu-Jun; Li, Xing-Hui

2017-01-01

Tea plants [Camellia sinensis (L.) O. Kuntze] are an important leaf-type crop that are widely used for the production of non-alcoholic beverages in the world. Exposure to excessive amounts of heavy metals adversely affects the quality and yield of tea leaves. To analyze the molecular responses of tea plants to heavy metals, a reliable quantification of gene expression is important and of major importance herein is the normalization of the measured expression levels for the target genes. Ideally, stably expressed reference genes should be evaluated in all experimental systems. In this study, 12 candidate reference genes (i.e., 18S rRNA, Actin, CYP, EF-1α, eIF-4α, GAPDH, MON1, PP2AA3, TBP, TIP41, TUA, and UBC) were cloned from tea plants, and the stability of their expression was examined systematically in 60 samples exposed to diverse heavy metals (i.e., manganese, aluminum, copper, iron, and zinc). Three Excel-based algorithms (geNorm, NormFinder, and BestKeeper) were used to evaluate the expression stability of these genes. PP2AA3 and 18S rRNA were the most stably expressed genes, even though their expression profiles exhibited some variability. Moreover, commonly used reference genes (i.e., GAPDH and TBP) were the least appropriate reference genes for most samples. To further validate the suitability of the analyzed reference genes, the expression level of a phytochelatin synthase gene (i.e., CsPCS1) was determined using the putative reference genes for data normalizations. Our results may be beneficial for future studies involving the quantification of relative gene expression levels in tea plants.

Development of a baculovirus vector carrying a small hairpin RNA for suppression of sf-caspase-1 expression and improvement of recombinant protein production.

PubMed

Zhang, Xiaoyue; Xu, Keyan; Ou, Yanmei; Xu, Xiaodong; Chen, Hongying

2018-05-02

The Baculovirus expression vector system (BEVS) is a transient expression platform for recombinant protein production in insect cells. Baculovirus infection of insect cells will shutoff host translation and induce apoptosis and lead to the termination of protein expression. Previous reports have demonstrated the enhancement of protein yield in BEVS using stable insect cell lines expressing interference RNA to suppress the expression of caspase-1. In this study, short-hairpin RNA (shRNA) expression cassettes targeting Spodoptera frugiperda caspase-1 (Sf-caspase-1) were constructed and inserted into an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) vector. Using the recombinant baculovirus vectors, we detected the suppression of Sf-caspase-1 expression and cell apoptosis. Green fluorescent protein (GFP), Discosoma sp. Red (DsRed) and firefly luciferase were then expressed as reporter proteins. The results showed that suppression of apoptosis enhanced the accumulation of exogenous proteins at 2 and 3 days post infection. After 4 days post infection, the activity of the reporter proteins remained higher in BEVS using the baculovirus carrying shRNA in comparison with the control without shRNA, but the accumulated protein levels showed no obvious difference between them, suggesting that apoptosis suppression resulted in improved protein folding rather than translation efficiency at the very late stage of baculovirus infection. The baculovirus vector developed in this study would be a useful tool for the production of active proteins suitable for structural and functional studies or pharmaceutical applications in Sf9 cells, and it also has the potential to be adapted for the improvement of protein expression in different insect cell lines that can be infected by AcMNPV.

Reliable reference genes for normalization of gene expression data in tea plants (Camellia sinensis) exposed to metal stresses

PubMed Central

Wang, Ming-Le; Li, Qing-Hui; Xin, Hua-Hong; Chen, Xuan; Zhu, Xu-Jun

2017-01-01

Tea plants [Camellia sinensis (L.) O. Kuntze] are an important leaf-type crop that are widely used for the production of non-alcoholic beverages in the world. Exposure to excessive amounts of heavy metals adversely affects the quality and yield of tea leaves. To analyze the molecular responses of tea plants to heavy metals, a reliable quantification of gene expression is important and of major importance herein is the normalization of the measured expression levels for the target genes. Ideally, stably expressed reference genes should be evaluated in all experimental systems. In this study, 12 candidate reference genes (i.e., 18S rRNA, Actin, CYP, EF-1α, eIF-4α, GAPDH, MON1, PP2AA3, TBP, TIP41, TUA, and UBC) were cloned from tea plants, and the stability of their expression was examined systematically in 60 samples exposed to diverse heavy metals (i.e., manganese, aluminum, copper, iron, and zinc). Three Excel-based algorithms (geNorm, NormFinder, and BestKeeper) were used to evaluate the expression stability of these genes. PP2AA3 and 18S rRNA were the most stably expressed genes, even though their expression profiles exhibited some variability. Moreover, commonly used reference genes (i.e., GAPDH and TBP) were the least appropriate reference genes for most samples. To further validate the suitability of the analyzed reference genes, the expression level of a phytochelatin synthase gene (i.e., CsPCS1) was determined using the putative reference genes for data normalizations. Our results may be beneficial for future studies involving the quantification of relative gene expression levels in tea plants. PMID:28453515

The Human EST Ontology Explorer: a tissue-oriented visualization system for ontologies distribution in human EST collections.

PubMed

Merelli, Ivan; Caprera, Andrea; Stella, Alessandra; Del Corvo, Marcello; Milanesi, Luciano; Lazzari, Barbara

2009-10-15

The NCBI dbEST currently contains more than eight million human Expressed Sequenced Tags (ESTs). This wide collection represents an important source of information for gene expression studies, provided it can be inspected according to biologically relevant criteria. EST data can be browsed using different dedicated web resources, which allow to investigate library specific gene expression levels and to make comparisons among libraries, highlighting significant differences in gene expression. Nonetheless, no tool is available to examine distributions of quantitative EST collections in Gene Ontology (GO) categories, nor to retrieve information concerning library-dependent EST involvement in metabolic pathways. In this work we present the Human EST Ontology Explorer (HEOE) http://www.itb.cnr.it/ptp/human_est_explorer, a web facility for comparison of expression levels among libraries from several healthy and diseased tissues. The HEOE provides library-dependent statistics on the distribution of sequences in the GO Direct Acyclic Graph (DAG) that can be browsed at each GO hierarchical level. The tool is based on large-scale BLAST annotation of EST sequences. Due to the huge number of input sequences, this BLAST analysis was performed with the aid of grid computing technology, which is particularly suitable to address data parallel task. Relying on the achieved annotation, library-specific distributions of ESTs in the GO Graph were inferred. A pathway-based search interface was also implemented, for a quick evaluation of the representation of libraries in metabolic pathways. EST processing steps were integrated in a semi-automatic procedure that relies on Perl scripts and stores results in a MySQL database. A PHP-based web interface offers the possibility to simultaneously visualize, retrieve and compare data from the different libraries. Statistically significant differences in GO categories among user selected libraries can also be computed. The HEOE provides an alternative and complementary way to inspect EST expression levels with respect to approaches currently offered by other resources. Furthermore, BLAST computation on the whole human EST dataset was a suitable test of grid scalability in the context of large-scale bioinformatics analysis. The HEOE currently comprises sequence analysis from 70 non-normalized libraries, representing a comprehensive overview on healthy and unhealthy tissues. As the analysis procedure can be easily applied to other libraries, the number of represented tissues is intended to increase.

Improved quantitative and qualitative production of single-domain intrabodies mediated by the co-expression of Erv1p sulfhydryl oxidase.

PubMed

Veggiani, Gianluca; de Marco, Ario

2011-09-01

Camelidae single domain antibodies (VHHs) have structural and binding features that render them suitable alternatives to conventional IgG antibodies. VHHs are usually easier to produce as recombinant proteins than other antibody fragments. However, for some of the biotechnological applications for which they have been proposed, such as immunochromatography and assisted-crystallography, large amounts of purified antibodies are necessary, whereas some VHH-fusions with common tags such as GFP and SNAP are poorly expressed in the bacterial periplasm. Here we have shown that the co-expression of Erv1p sulfhydryl oxidase resulted in an astonishing yield increase of VHH-SNAP constructs expressed in the bacterial cytoplasm. The resulting recombinant antibodies were also more stable than the antibodies produced using the same plasmid, but in wild-type bacteria. Using this approach, it was possible to obtain tens of milligram of purified fusion antibodies using a basic flask fermentation protocol. Therefore, the described method represents a valid solution to produce inexpensively large amounts of single domain antibodies for in vitro applications and we expect it will be suitable for the production of other antibody fragments. Copyright © 2011 Elsevier Inc. All rights reserved.

Identification of suitable qPCR reference genes in leaves of Brassica oleracea under abiotic stresses.

PubMed

Brulle, Franck; Bernard, Fabien; Vandenbulcke, Franck; Cuny, Damien; Dumez, Sylvain

2014-04-01

Real-time quantitative PCR is nowadays a standard method to study gene expression variations in various samples and experimental conditions. However, to interpret results accurately, data normalization with appropriate reference genes appears to be crucial. The present study describes the identification and the validation of suitable reference genes in Brassica oleracea leaves. Expression stability of eight candidates was tested following drought and cold abiotic stresses by using three different softwares (BestKeeper, NormFinder and geNorm). Four genes (BolC.TUB6, BolC.SAND1, BolC.UBQ2 and BolC.TBP1) emerged as the most stable across the tested conditions. Further gene expression analysis of a drought- and a cold-responsive gene (BolC.DREB2A and BolC.ELIP, respectively), confirmed the stability and the reliability of the identified reference genes when used for normalization in the leaves of B. oleracea. These four genes were finally tested upon a benzene exposure and all appeared to be useful reference genes along this toxicological condition. These results provide a good starting point for future studies involving gene expression measurement on leaves of B. oleracea exposed to environmental modifications.

Validation of endogenous internal real-time PCR controls in renal tissues.

PubMed

Cui, Xiangqin; Zhou, Juling; Qiu, Jing; Johnson, Martin R; Mrug, Michal

2009-01-01

Endogenous internal controls ('reference' or 'housekeeping' genes) are widely used in real-time PCR (RT-PCR) analyses. Their use relies on the premise of consistently stable expression across studied experimental conditions. Unfortunately, none of these controls fulfills this premise across a wide range of experimental conditions; consequently, none of them can be recommended for universal use. To determine which endogenous RT-PCR controls are suitable for analyses of renal tissues altered by kidney disease, we studied the expression of 16 commonly used 'reference genes' in 7 mildly and 7 severely affected whole kidney tissues from a well-characterized cystic kidney disease model. Expression levels of these 16 genes, determined by TaqMan RT-PCR analyses and Affymetrix GeneChip arrays, were normalized and tested for overall variance and equivalence of the means. Both statistical approaches and both TaqMan- and GeneChip-based methods converged on 3 out of the 4 top-ranked genes (Ppia, Gapdh and Pgk1) that had the most constant expression levels across the studied phenotypes. A combination of the top-ranked genes will provide a suitable endogenous internal control for similar studies of kidney tissues across a wide range of disease severity. Copyright 2009 S. Karger AG, Basel.

Unstable Expression of Commonly Used Reference Genes in Rat Pancreatic Islets Early after Isolation Affects Results of Gene Expression Studies.

PubMed

Kosinová, Lucie; Cahová, Monika; Fábryová, Eva; Týcová, Irena; Koblas, Tomáš; Leontovyč, Ivan; Saudek, František; Kříž, Jan

2016-01-01

The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3) in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0-120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48-120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information from 48 hrs onwards.

Characterization of mercury bioremediation by transgenic bacteria expressing metallothionein and polyphosphate kinase.

PubMed

Ruiz, Oscar N; Alvarez, Derry; Gonzalez-Ruiz, Gloriene; Torres, Cesar

2011-08-12

The use of transgenic bacteria has been proposed as a suitable alternative for mercury remediation. Ideally, mercury would be sequestered by metal-scavenging agents inside transgenic bacteria for subsequent retrieval. So far, this approach has produced limited protection and accumulation. We report here the development of a transgenic system that effectively expresses metallothionein (mt-1) and polyphosphate kinase (ppk) genes in bacteria in order to provide high mercury resistance and accumulation. In this study, bacterial transformation with transcriptional and translational enhanced vectors designed for the expression of metallothionein and polyphosphate kinase provided high transgene transcript levels independent of the gene being expressed. Expression of polyphosphate kinase and metallothionein in transgenic bacteria provided high resistance to mercury, up to 80 μM and 120 μM, respectively. Here we show for the first time that metallothionein can be efficiently expressed in bacteria without being fused to a carrier protein to enhance mercury bioremediation. Cold vapor atomic absorption spectrometry analyzes revealed that the mt-1 transgenic bacteria accumulated up to 100.2 ± 17.6 μM of mercury from media containing 120 μM Hg. The extent of mercury remediation was such that the contaminated media remediated by the mt-1 transgenic bacteria supported the growth of untransformed bacteria. Cell aggregation, precipitation and color changes were visually observed in mt-1 and ppk transgenic bacteria when these cells were grown in high mercury concentrations. The transgenic bacterial system described in this study presents a viable technology for mercury bioremediation from liquid matrices because it provides high mercury resistance and accumulation while inhibiting elemental mercury volatilization. This is the first report that shows that metallothionein expression provides mercury resistance and accumulation in recombinant bacteria. The high accumulation of mercury in the transgenic cells could present the possibility of retrieving the accumulated mercury for further industrial applications.

Characterization of mercury bioremediation by transgenic bacteria expressing metallothionein and polyphosphate kinase

PubMed Central

2011-01-01

Background The use of transgenic bacteria has been proposed as a suitable alternative for mercury remediation. Ideally, mercury would be sequestered by metal-scavenging agents inside transgenic bacteria for subsequent retrieval. So far, this approach has produced limited protection and accumulation. We report here the development of a transgenic system that effectively expresses metallothionein (mt-1) and polyphosphate kinase (ppk) genes in bacteria in order to provide high mercury resistance and accumulation. Results In this study, bacterial transformation with transcriptional and translational enhanced vectors designed for the expression of metallothionein and polyphosphate kinase provided high transgene transcript levels independent of the gene being expressed. Expression of polyphosphate kinase and metallothionein in transgenic bacteria provided high resistance to mercury, up to 80 μM and 120 μM, respectively. Here we show for the first time that metallothionein can be efficiently expressed in bacteria without being fused to a carrier protein to enhance mercury bioremediation. Cold vapor atomic absorption spectrometry analyzes revealed that the mt-1 transgenic bacteria accumulated up to 100.2 ± 17.6 μM of mercury from media containing 120 μM Hg. The extent of mercury remediation was such that the contaminated media remediated by the mt-1 transgenic bacteria supported the growth of untransformed bacteria. Cell aggregation, precipitation and color changes were visually observed in mt-1 and ppk transgenic bacteria when these cells were grown in high mercury concentrations. Conclusion The transgenic bacterial system described in this study presents a viable technology for mercury bioremediation from liquid matrices because it provides high mercury resistance and accumulation while inhibiting elemental mercury volatilization. This is the first report that shows that metallothionein expression provides mercury resistance and accumulation in recombinant bacteria. The high accumulation of mercury in the transgenic cells could present the possibility of retrieving the accumulated mercury for further industrial applications. PMID:21838857

A probe-based qRT-PCR method to profile immunological gene expression in blood of captive beluga whales (Delphinapterus leucas)

PubMed Central

Wang, Jiann-Hsiung; Chou, Shih-Jen; Li, Tsung-Hsien; Leu, Ming-Yih; Ho, Hsiao-Kuan

2017-01-01

Cytokines are fundamental for a functioning immune system, and thus potentially serve as important indicators of animal health. Quantitation of mRNA using quantitative reverse transcription polymerase chain reaction (qRT-PCR) is an established immunological technique. It is particularly suitable for detecting the expression of proteins against which monoclonal antibodies are not available. In this study, we developed a probe-based quantitative gene expression assay for immunological assessment of captive beluga whales (Delphinapterus leucas) that is one of the most common cetacean species on display in aquariums worldwide. Six immunologically relevant genes (IL-2Rα, -4, -10, -12, TNFα, and IFNγ) were selected for analysis, and two validated housekeeping genes (PGK1 and RPL4) with stable expression were used as reference genes. Sixteen blood samples were obtained from four animals with different health conditions and stored in RNAlater™ solution. These samples were used for RNA extraction followed by qRT-PCR analysis. Analysis of gene transcripts was performed by relative quantitation using the comparative Cq method with the integration of amplification efficiency and two reference genes. The expression levels of each gene in the samples from clinically healthy animals were normally distributed. Transcript outliers for IL-2Rα, IL-4, IL-12, TNFα, and IFNγ were noticed in four samples collected from two clinically unhealthy animals. This assay has the potential to identify immune system deviation from normal state, which is caused by health problems. Furthermore, knowing the immune status of captive cetaceans could help both trainers and veterinarians in implementing preventive approaches prior to disease onset. PMID:28970970

A probe-based qRT-PCR method to profile immunological gene expression in blood of captive beluga whales (Delphinapterus leucas).

PubMed

Tsai, Ming-An; Chen, I-Hua; Wang, Jiann-Hsiung; Chou, Shih-Jen; Li, Tsung-Hsien; Leu, Ming-Yih; Ho, Hsiao-Kuan; Yang, Wei Cheng

2017-01-01

Cytokines are fundamental for a functioning immune system, and thus potentially serve as important indicators of animal health. Quantitation of mRNA using quantitative reverse transcription polymerase chain reaction (qRT-PCR) is an established immunological technique. It is particularly suitable for detecting the expression of proteins against which monoclonal antibodies are not available. In this study, we developed a probe-based quantitative gene expression assay for immunological assessment of captive beluga whales ( Delphinapterus leucas ) that is one of the most common cetacean species on display in aquariums worldwide. Six immunologically relevant genes (IL-2Rα, -4, -10, -12, TNFα, and IFNγ) were selected for analysis, and two validated housekeeping genes (PGK1 and RPL4) with stable expression were used as reference genes. Sixteen blood samples were obtained from four animals with different health conditions and stored in RNA later ™ solution. These samples were used for RNA extraction followed by qRT-PCR analysis. Analysis of gene transcripts was performed by relative quantitation using the comparative Cq method with the integration of amplification efficiency and two reference genes. The expression levels of each gene in the samples from clinically healthy animals were normally distributed. Transcript outliers for IL-2Rα, IL-4, IL-12, TNFα, and IFNγ were noticed in four samples collected from two clinically unhealthy animals. This assay has the potential to identify immune system deviation from normal state, which is caused by health problems. Furthermore, knowing the immune status of captive cetaceans could help both trainers and veterinarians in implementing preventive approaches prior to disease onset.

Animal component-free Agrobacterium tumefaciens cultivation media for better GMP-compliance increases biomass yield and pharmaceutical protein expression in Nicotiana benthamiana.

PubMed

Houdelet, Marcel; Galinski, Anna; Holland, Tanja; Wenzel, Kathrin; Schillberg, Stefan; Buyel, Johannes Felix

2017-04-01

Transient expression systems allow the rapid production of recombinant proteins in plants. Such systems can be scaled up to several hundred kilograms of biomass, making them suitable for the production of pharmaceutical proteins required at short notice, such as emergency vaccines. However, large-scale transient expression requires the production of recombinant Agrobacterium tumefaciens strains with the capacity for efficient gene transfer to plant cells. The complex media often used for the cultivation of this species typically include animal-derived ingredients that can contain human pathogens, thus conflicting with the requirements of good manufacturing practice (GMP). We replaced all the animal-derived components in yeast extract broth (YEB) cultivation medium with soybean peptone, and then used a design-of-experiments approach to optimize the medium composition, increasing the biomass yield while maintaining high levels of transient expression in subsequent infiltration experiments. The resulting plant peptone Agrobacterium medium (PAM) achieved a two-fold increase in OD 600 compared to YEB medium during a 4-L batch fermentation lasting 18 h. Furthermore, the yields of the monoclonal antibody 2G12 and the fluorescent protein DsRed were maintained when the cells were cultivated in PAM rather than YEB. We have thus demonstrated a simple, efficient and scalable method for medium optimization that reduces process time and costs. The final optimized medium for the cultivation of A. tumefaciens completely lacks animal-derived components, thus facilitating the GMP-compliant large-scale transient expression of recombinant proteins in plants. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Automated, Miniaturized Instrument for Measuring Gene Expression in Space

NASA Technical Reports Server (NTRS)

Pohorille, A.; Peyvan, K.; Danley, D.; Ricco, A. J.

2010-01-01

To facilitate astrobiological studies on the survival and adaptation of microorganisms and mixed microbial cultures to space environment, we have been developing a fully automated, miniaturized system for measuring their gene expression on small spacecraft. This low-cost, multi-purpose instrument represents a major scientific and technological advancement in our ability to study the impact of the space environment on biological systems by providing data on cellular metabolism and regulation orders of magnitude richer than what is currently available. The system supports growth of the organism, lyse it to release the expressed RNA, label the RNA, read the expression levels of a large number of genes by microarray analysis of labeled RNA and transmit the measurements to Earth. To measure gene expression we use microarray technology developed by CombiMatrix, which is based on electrochemical reactions on arrays of electrodes on a semiconductor substrate. Since the electrical integrity of the microarray remains intact after probe synthesis, the circuitry can be employed to sense nucleic acid binding at each electrode. CombiMatrix arrays can be sectored to allow multiple samples per chip. In addition, a single array can be used for several assays. The array has been integrated into an automated microfluidic cartridge that uses flexible reagent blisters and pinch pumping to move liquid reagents between chambers. The proposed instrument will help to understand adaptation of terrestrial life to conditions beyond the planet of origin, identify deleterious effects of the space environment, develop effective countermeasures against these effects, and test our ability to sustain and grow in space organisms that can be used for life support and in situ resource utilization during long-duration space exploration. The instrument is suitable for small satellite platforms, which provide frequent, low cost access to space. It can be also used on any other platform in space, including the ISS. It can be replicated and used with only small modifications in multiple biological experiments with a broad range of goals in mind.

Automated, Miniaturized Instrument for Measuring Gene Expression in Space

NASA Astrophysics Data System (ADS)

Pohorille, Andrew; Danley, David; Payvan, Kia; Ricco, Antonio

To facilitate astrobiological studies on the survival and adaptation of microorganisms and mixed microbial cultures to space environment, we have been developing a fully automated, minia-turized system for measuring their gene expression on small spacecraft. This low-cost, multi-purpose instrument represents a major scientific and technological advancement in our ability to study the impact of the space environment on biological systems by providing data on cel-lular metabolism and regulation orders of magnitude richer than what is currently available. The system supports growth of the organism, lyse it to release the expressed RNA, label the RNA, read the expression levels of a large number of genes by microarray analysis of labeled RNA and transmit the measurements to Earth. To measure gene expression we use microarray technology developed by CombiMatrix, which is based on electrochemical reactions on arrays of electrodes on a semiconductor substrate. Since the electrical integrity of the microarray re-mains intact after probe synthesis, the circuitry can be employed to sense nucleic acid binding at each electrode. CombiMatrix arrays can be sectored to allow multiple samples per chip. In addition, a single array can be used for several assays. The array has been integrated into an automated microfluidic cartridge that uses flexible reagent blisters and pinch pumping to move liquid reagents between chambers. The proposed instrument will help to understand adaptation of terrestrial life to conditions be-yond the planet of origin, identify deleterious effects of the space environment, develop effective countermeasures against these effects, and test our ability to sustain and grow in space organ-isms that can be used for life support and in situ resource utilization during long-duration space exploration. The instrument is suitable for small satellite platforms, which provide frequent, low cost access to space. It can be also used on any other platform in space, including the ISS. It can be replicated and used with only small modifications in multiple biological experiments with a broad range of goals in mind.

Expression of genes of the cardiac and renal renin-angiotensin systems in preterm piglets: is this system a suitable target for therapeutic intervention?

PubMed

Kim, Eleanor; Eiby, Yvonne; Lumbers, Eugenie; Boyce, Amanda; Gibson, Karen; Lingwood, Barbara

2015-10-01

The newborn circulating, cardiac and renal renin-angiotensin systems (RASs) are essential for blood pressure control, and for cardiac and renal development. If cardiac and renal RASs are immature this may contribute to cardiovascular compromise in preterm infants. This study measured mRNA expression of cardiac and renal RAS components in preterm, glucocorticoid (GC) exposed preterm, and term piglets. Renal and cardiac RAS mRNA levels were measured using real-time polymerase chain reaction (PCR). Genes studied were: (pro)renin receptor, renin, angiotensinogen, angiotensin converting enzyme (ACE), ACE2, angiotensin type 1 receptor (AT1R) and angiotensin type 2 receptor (AT2R). All the genes studied were expressed in the kidney; neither renin nor AT2R mRNA were detected in the heart. There were no gestational changes in (pro)renin receptor, renin, ACE or AT1R mRNA levels. Right ventricular angiotensinogen mRNA levels in females were lower in preterm animals than at term, and GC exposure increased levels in male piglets. Renal angiotensinogen mRNA levels in female term piglets were lower than females from both preterm groups, and lower than male term piglets. Left ventricular ACE2 mRNA expression was lower in GC treated preterm piglets. Renal AT2R mRNA abundance was highest in GC treated preterm piglets, and the AT1R/AT2R ratio was increased at term. Preterm cardiac and renal RAS mRNA levels were similar to term piglets, suggesting that immaturity of these RASs does not contribute to preterm cardiovascular compromise. Since preterm expression of both renal and cardiac angiotensin II-AT1R is similar to term animals, cardiovascular dysfunction in the sick preterm human neonate might be effectively treated by agents acting on their RASs. © The Author(s), 2015.

Effect of hypergravity on expression of the immediate early gene, c-fos, in central nervous system of medaka (Oryzias latipes)

NASA Astrophysics Data System (ADS)

Sayaka, Shimomura-Umemura; Ijiri, Kenichi

2006-01-01

Immediate-early genes serve as useful neurobiological tools for mapping brain activity induced by a sensory stimulation. In this study, we have examined brain activity related to gravity perception of medaka (Oryzias latipes) by use of c-fos. The gene, which is homologous to the c-fos genes of other vertebrates, was identified in medaka. Functionally important domains are highly conserved among all the vertebrate species analyzed. Intraperitoneal administration of kainic acid transiently induced the c-fos mRNAs in medaka brains. The results indicate that the expression of c-fos can be utilized as a suitable anatomical marker for the increased neural activities in the central nervous system of medaka. Fish were continuously exposed to 3 g hypergravity by centrifugation. Investigation of c-fos mRNA expression indicated that c-fos mRNA significantly increased 30 min after a start of 3 g exposure. The distribution of its transcripts within the brains was analyzed by an in situ hybridization method. The 3-g treated medakas displayed c-fos positive cells in their brainstem regions, which are related to vestibular function, such as torus semicircularis, nucleus tangentialis, posterior octavu nucleus, and inferior olive. Our results established a method to follow the effect of gravity stimulation, which can be used to investigate gravity perception.

Expression of c-fos gene in central nervous system of adult medaka (Oryzias latipes) after hypergravity stimulation

NASA Astrophysics Data System (ADS)

Shimomura, S.; Ijiri, K.

The immediate-early genes serve as useful neurobiological tools for mapping brain activity induced by a sensory stimulation. In this study, we have examined brain activity related to gravity perception of medaka (Oryzias latipes) by use of c-fos. The gene, which is homologous to the c-fos genes of other vertebrates, was identified in medaka. Functionally important domains are highly conserved among all the vertebrate species analyzed. Intraperitoneal administration of kainic acid transiently induced the c-fos mRNAs in medaka brain. The results indicate that the expression of c-fos can be utilized as a suitable anatomical marker for the increased neural activities in the central nervous system of medaka. Fish were continuously exposed to 3G hypergravity by centrifugation. Investigation of c-fos mRNA expression showed that c-fos mRNA significantly increased 30 minutes after a start of 3G exposure. The distribution of its transcripts within brains was analyzed by an in situ hybridization method. The 3G-treated medakas displayed c-fos positive cells in their brainstem regions, which are related to vestibular function, such as torus semicircularis, posterior octavu nucleus, nucleus tangentialis and inferior olive. Our results established the method to trace the activated area in the fish brain following gravity stimulation. The method will be a useful tool for understanding gravity perception in the brain.

FluBlok, a next generation influenza vaccine manufactured in insect cells.

PubMed

Cox, Manon M J; Hollister, Jason R

2009-06-01

FluBlok, a recombinant trivalent hemagglutinin (rHA) vaccine produced in insect cell culture using the baculovirus expression system, provides an attractive alternative to the current egg-based trivalent inactivated influenza vaccine (TIV). Its manufacturing process presents the possibility for safe and expeditious vaccine production. FluBlok contains three times more HA than TIV and does not contain egg-protein or preservatives. The high purity of the antigen enables administration at higher doses without a significant increase in side-effects in human subjects. The insect cell-baculovirus production technology is particularly suitable for influenza where annual adjustment of the vaccine is required. The baculovirus-insect expression system is generally considered a safe production system, with limited growth potential for adventitious agents. Still regulators question and challenge the safety of this novel cell substrate as FluBlok continues to advance toward product approval. This review provides an overview of cell substrate characterization for expresSF cell line used for the manufacturing of FluBlok. In addition, this review includes an update on the clinical development of FluBlok. The highly purified protein vaccine, administered at three times higher antigen content than TIV, is well tolerated and results in stronger immunogenicity, a long lasting immune response and provides cross-protection against drift influenza viruses.

Identification of suitable genes contributes to lung adenocarcinoma clustering by multiple meta-analysis methods.

PubMed

Yang, Ze-Hui; Zheng, Rui; Gao, Yuan; Zhang, Qiang

2016-09-01

With the widespread application of high-throughput technology, numerous meta-analysis methods have been proposed for differential expression profiling across multiple studies. We identified the suitable differentially expressed (DE) genes that contributed to lung adenocarcinoma (ADC) clustering based on seven popular multiple meta-analysis methods. Seven microarray expression profiles of ADC and normal controls were extracted from the ArrayExpress database. The Bioconductor was used to perform the data preliminary preprocessing. Then, DE genes across multiple studies were identified. Hierarchical clustering was applied to compare the classification performance for microarray data samples. The classification efficiency was compared based on accuracy, sensitivity and specificity. Across seven datasets, 573 ADC cases and 222 normal controls were collected. After filtering out unexpressed and noninformative genes, 3688 genes were remained for further analysis. The classification efficiency analysis showed that DE genes identified by sum of ranks method separated ADC from normal controls with the best accuracy, sensitivity and specificity of 0.953, 0.969 and 0.932, respectively. The gene set with the highest classification accuracy mainly participated in the regulation of response to external stimulus (P = 7.97E-04), cyclic nucleotide-mediated signaling (P = 0.01), regulation of cell morphogenesis (P = 0.01) and regulation of cell proliferation (P = 0.01). Evaluation of DE genes identified by different meta-analysis methods in classification efficiency provided a new perspective to the choice of the suitable method in a given application. Varying meta-analysis methods always present varying abilities, so synthetic consideration should be taken when providing meta-analysis methods for particular research. © 2015 John Wiley & Sons Ltd.

Porphyria: A Suitable Case for Teaching.

ERIC Educational Resources Information Center

Hawkey, Roy

1990-01-01

The porphyrias are a family of genetic disorders whose genetics and biochemistry are largely identified. Background information on these diseases are discussed including porphyrins, gene expression, population genetics, and historical significance. (CW)

Construction of an Expression System for Bioactive IL-18 and Generation of Recombinant Canine Distemper Virus Expressing IL-18

PubMed Central

LIU, Yuxiu; SATO, Hiroki; HAMANA, Masahiro; MOONAN, Navita Anisia; YONEDA, Misako; XIA, Xianzhu; KAI, Chieko

2014-01-01

ABSTRACT Interleukin 18 (IL-18) plays an important role in the T-helper-cell type 1 immune response against intracellular parasites, bacteria and viral infections. It has been widely used as an adjuvant for vaccines and as an anticancer agent. However, IL-18 protein lacks a typical signal sequence and requires cleavage into its mature active form by caspase 1. In this study, we constructed mammalian expression vectors carrying cDNA encoding mature canine IL-18 (cIL-18) or mouse IL-18 (mIL-18) fused to the human IL-2 (hIL-2) signal sequence. The expressed proIL-18 proteins were processed to their mature forms in the cells. The supernatants of cells transfected with these plasmids induced high interferon-γ production in canine peripheral blood mononuclear cells or mouse splenocytes, respectively, indicating the secretion of bioactive IL-18. Using reverse genetics, we also generated a recombinant canine distemper virus that expresses cIL-18 or mIL-18 fused to the hIL-2 signal sequence. As expected, both recombinant viruses produced mature IL-18 in the infected cells, which secreted bioactive IL-18. These results indicate that the signal sequence from hIL-2 is suitable for the secretion of mature IL-18. These recombinant viruses can also potentially be used as immunoadjuvants and agents for anticancer therapies in vivo. PMID:24898077

The amyR-deletion strain of Aspergillus niger CICC2462 is a suitable host strain to express secreted protein with a low background.

PubMed

Zhang, Hui; Wang, Shuang; Zhang, Xiang Xiang; Ji, Wei; Song, Fuping; Zhao, Yue; Li, Jie

2016-04-28

The filamentous fungus Aspergillus niger is widely exploited as an important expression host for industrial production. The glucoamylase high-producing strain A. niger CICC2462 has been used as a host strain for the establishment of a secretion expression system. It expresses recombinant xylanase, mannase and asparaginase at a high level, but some high secretory background proteins in these recombinant strains still remain, such as alpha-amylase and alpha-glucosidase; lead to a low-purity of fermentation products. The aim was to construct an A. niger host strain with a low background of protein secretion. The transcription factor amyR was deleted in A. niger CICC2462, and the results from enzyme activity assays and SDS-PAGE analysis showed that the glucoamylase and amylase activities of the ∆amyR strains were significantly lower than those of the wild-type strain. High-throughput RNA-sequencing and shotgun LC-MS/MS proteomic technology analysis demonstrated that the expression of amylolytic enzymes was decreased at both the transcriptional and translational levels in the ∆amyR strain. Interestingly, the ∆amyR strain growth rate better than the wild-type strain. Our findings clearly indicated that the ∆amyR strain of A. niger CICC2462 can be used as a host strain with a low background of protein secretion.

Construction of an expression system for bioactive IL-18 and generation of recombinant canine distemper virus expressing IL-18.

PubMed

Liu, Yuxiu; Sato, Hiroki; Hamana, Masahiro; Moonan, Navita Anisia; Yoneda, Misako; Xia, Xianzhu; Kai, Chieko

2014-09-01

Interleukin 18 (IL-18) plays an important role in the T-helper-cell type 1 immune response against intracellular parasites, bacteria and viral infections. It has been widely used as an adjuvant for vaccines and as an anticancer agent. However, IL-18 protein lacks a typical signal sequence and requires cleavage into its mature active form by caspase 1. In this study, we constructed mammalian expression vectors carrying cDNA encoding mature canine IL-18 (cIL-18) or mouse IL-18 (mIL-18) fused to the human IL-2 (hIL-2) signal sequence. The expressed proIL-18 proteins were processed to their mature forms in the cells. The supernatants of cells transfected with these plasmids induced high interferon-γ production in canine peripheral blood mononuclear cells or mouse splenocytes, respectively, indicating the secretion of bioactive IL-18. Using reverse genetics, we also generated a recombinant canine distemper virus that expresses cIL-18 or mIL-18 fused to the hIL-2 signal sequence. As expected, both recombinant viruses produced mature IL-18 in the infected cells, which secreted bioactive IL-18. These results indicate that the signal sequence from hIL-2 is suitable for the secretion of mature IL-18. These recombinant viruses can also potentially be used as immunoadjuvants and agents for anticancer therapies in vivo.

Microarray-based comparison of three amplification methods for nanogram amounts of total RNA

PubMed Central

Singh, Ruchira; Maganti, Rajanikanth J.; Jabba, Sairam V.; Wang, Martin; Deng, Glenn; Heath, Joe Don; Kurn, Nurith; Wangemann, Philine

2007-01-01

Gene expression profiling using microarrays requires microgram amounts of RNA, which limits its direct application for the study of nanogram RNA samples obtained using microdissection, laser capture microscopy, or needle biopsy. A novel system based on Ribo-SPIA technology (RS, Ovation-Biotin amplification and labeling system) was recently introduced. The utility of the RS system, an optimized prototype system for picogram RNA samples (pRS), and two T7-based systems involving one or two rounds of amplification (OneRA, Standard Protocol, or TwoRA, Small Sample Prototcol, version II) were evaluated in the present study. Mouse kidney (MK) and mouse universal reference (MUR) RNA samples, 0.3 ng to 10 μg, were analyzed using high-density Affymetrix Mouse Genome 430 2.0 GeneChip arrays. Call concordance between replicates, correlations of signal intensity, signal intensity ratios, and minimal fold increase necessary for significance were determined. All systems amplified partially overlapping sets of genes with similar signal intensity correlations. pRS amplified the highest number of genes from 10-ng RNA samples. We detected 24 of 26 genes verified by RT-PCR in samples prepared using pRS. TwoRA yielded somewhat higher call concordances than did RS and pRS (91.8% vs. 89.3% and 88.1%, respectively). Although all target preparation methods were suitable, pRS amplified the highest number of targets and was found to be suitable for amplification of as little as 0.3 ng of total RNA. In addition, RS and pRS were faster and simpler to use than the T7-based methods and resulted in the generation of cDNA, which is more stable than cRNA. PMID:15613496

Using fluorometry and ion-sensitive microelectrodes to study the functional expression of heterologously-expressed ion channels and transporters in Xenopus oocytes

PubMed Central

Musa-Aziz, Raif; Boron, Walter F.; Parker, Mark D.

2010-01-01

The Xenopus laevis oocyte is a model system for the electrophysiological study of exogenous ion transporters. Three main reasons make the oocyte suitable for this purpose: (a) it has a large cell size (~1 mm diameter), (b) it has an established capacity to produce—from microinjected mRNAs or cRNAs—exogenous ion transporters with close-to-physiological post-translational modifications and actions, and (c) its membranes contain endogenous ion-transport activities which are usually smaller in magnitude than the activities of exogenously-expressed ion transporters. The expression of ion-transporters as green-fluorescent-protein fusions allows the fluorometric assay of transporter yield in living oocytes. Monitoring of transporter-mediated movement of ions such as Cl−, H+ (and hence base equivalents like OH−1 and HCO3−), K+, and Na+ is achieved by positioning the tips of ion-sensitive microelectrodes inside the oocyte and/or at the surface of the oocyte plasma membrane. The use of ion-sensitive electrodes is critical for studying net ion-movements mediated by electroneutral transporters. The combined use of fluorometry and electrophysiology expedites transporter study by allowing measurement of transporter yield prior to electrophysiological study and correlation of relative transporter yield with transport rates. PMID:20051266

Cloning, expression and characterisation of P450-Hal1 (CYP116B62) from Halomonas sp. NCIMB 172: A self-sufficient P450 with high expression and diverse substrate scope.

PubMed

Porter, Joanne L; Sabatini, Selina; Manning, Jack; Tavanti, Michele; Galman, James L; Turner, Nicholas J; Flitsch, Sabine L

2018-06-01

Cytochrome P450 monooxygenases are able to catalyse a range of synthetically challenging reactions ranging from hydroxylation and demethylation to sulfoxidation and epoxidation. As such they have great potential for biocatalytic applications but are underutilised due to often-poor expression, stability and solubility in recombinant bacterial hosts. The use of self-sufficient P450 s with fused haem and reductase domains has already contributed heavily to improving catalytic efficiency and simplifying an otherwise more complex multi-component system of P450 and redox partners. Herein, we present a new addition to the class VII family with the cloning, sequencing and characterisation of the self-sufficient CYP116B62 Hal1 from Halomonas sp. NCIMB 172, the genome of which has not yet been sequenced. Hal1 exhibits high levels of expression in a recombinant E. coli host and can be utilised from cell lysate or used in purified form. Hal1 favours NADPH as electron donor and displays a diverse range of activities including hydroxylation, demethylation and sulfoxidation. These properties make Hal1 suitable for future biocatalytic applications or as a template for optimisation through engineering. Copyright © 2018 Elsevier Inc. All rights reserved.

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

PubMed

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

2015-12-01

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

Proteomic and cellular localisation studies suggest non-tight junction cytoplasmic and nuclear roles for occludin in astrocytes.

PubMed

Morgan, Sarah V; Garwood, Claire J; Jennings, Luke; Simpson, Julie E; Castelli, Lydia M; Heath, Paul R; Mihaylov, Simeon R; Vaquéz-Villaseñor, Irina; Minshull, Thomas C; Ince, Paul G; Dickman, Mark J; Hautbergue, Guillaume M; Wharton, Stephen B

2018-05-08

Occludin is a component of tight junctions, which are essential structural components of the blood-brain barrier. However, occludin is expressed in cells without tight junctions, implying additional functions. We determined the expression and localisation of occludin in astrocytes in cell culture and in human brain tissue, and sought novel binding partners using a proteomic approach. Expression was investigated by immunocytochemistry and immunoblotting in the 1321N1 astrocytoma cell line and ScienCell human primary astrocytes, and by immunohistochemistry in human autopsy brain tissue. Recombinant N- and C-terminal occludin was used to pull-down proteins from 1321N1 cell lysates and protein-binding partners identified by mass spectrometry analysis. Occludin was expressed in both the cytoplasm and nucleus of astrocytes in vitro and in vivo. Mass spectrometry identified binding to nuclear and cytoplasmic proteins, particularly those related to RNA metabolism and nuclear function. Occludin is expressed in several subcellular compartments of brain cell-types that do not form tight junctions and the expression patterns in cell culture reflect those in human brain tissue, indicating they are suitable model systems. Proteomic analysis suggests that occludin has novel functions in neuroepithelial cells that are unrelated to tight junction formation. Further research will establish the roles of these functions in both cellular physiology and in disease states. © 2018 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Three-Dimensional Imaging of the Intracellular Fate of Plasmid DNA and Transgene Expression: ZsGreen1 and Tissue Clearing Method CUBIC Are an Optimal Combination for Multicolor Deep Imaging in Murine Tissues.

PubMed

Fumoto, Shintaro; Nishimura, Koyo; Nishida, Koyo; Kawakami, Shigeru

2016-01-01

Evaluation methods for determining the distribution of transgene expression in the body and the in vivo fate of viral and non-viral vectors are necessary for successful development of in vivo gene delivery systems. Here, we evaluated the spatial distribution of transgene expression using tissue clearing methods. After hydrodynamic injection of plasmid DNA into mice, whole tissues were subjected to tissue clearing. Tissue clearing followed by confocal laser scanning microscopy enabled evaluation of the three-dimensional distribution of transgene expression without preparation of tissue sections. Among the tested clearing methods (ClearT2, SeeDB, and CUBIC), CUBIC was the most suitable method for determining the spatial distribution of transgene expression in not only the liver but also other tissues such as the kidney and lung. In terms of the type of fluorescent protein, the observable depth for green fluorescent protein ZsGreen1 was slightly greater than that for red fluorescent protein tdTomato. We observed a depth of ~1.5 mm for the liver and 500 μm for other tissues without preparation of tissue sections. Furthermore, we succeeded in multicolor deep imaging of the intracellular fate of plasmid DNA in the murine liver. Thus, tissue clearing would be a powerful approach for determining the spatial distribution of plasmid DNA and transgene expression in various murine tissues.

Subsurface storage of freshwater in south Florida; a prospectus

USGS Publications Warehouse

Merritt, M.L.; Meyer, F.W.; Sonntag, W.H.; Fitzpatrick, D.J.

1983-01-01

A method of increasing storage capacity for freshwater in south Florida is to use brackish artesian aquifers as reservoirs. In this way, water deficiencies occurring during the annual dry season can be offset by surplus water obtained during the wet season and injected underground. Most of south Florida is underlain by several deep, confined, carbonate waterbearing zones which might be suitable for freshwater storage. These zones are in the Avon Park, Ocala, Suwannee, Tampa, and Hawthorn Formations. Experimental freshwater injection systems have been operated at five locations with promising, but not fully definitive, results. A determination of the feasibility of freshwater injection at a selected site begins with an assessment of the local geologic suitability. Verification of feasibility, however, requires injection and recovery tests to be performed at the site. Recovery efficiency, a measure of the success of the operation, is the amount of potable water, expressed as a percentage of the volume injected, which can be recovered before its salinity, or the concentration of other chemical constituents present in the native aquifer water, increases to the point that the recovered water is no longer useable. (USGS)

An advanced real-time digital signal processing system for linear systems emulation, with special emphasis on network and acoustic response characterization

NASA Astrophysics Data System (ADS)

Gaydecki, Patrick; Fernandes, Bosco

2003-11-01

A fast digital signal processing (DSP) system is described that can perform real-time emulation of a wide variety of linear audio-bandwidth systems and networks, such as reverberant spaces, musical instrument bodies and very high order filter networks. The hardware design is based upon a Motorola DSP56309 operating at 110 million multiplication-accumulations per second and a dual-channel 24 bit codec with a maximum sampling frequency of 192 kHz. High level software has been developed to express complex vector frequency responses as both infinite impulse response (IIR) and finite impulse response (FIR) coefficients, in a form suitable for real-time convolution by the firmware installed in the DSP system memory. An algorithm has also been devised to express IIR filters as equivalent FIR structures, thereby obviating the potential instabilities associated with recursive equations and negating the traditional deficiencies of FIR filters respecting equivalent analogue designs. The speed and dynamic range of the system is such that, when sampling at 48 kHz, the frequency response can be specified to a spectral precision of 22 Hz when sampling at 10 kHz, this resolution increases to 0.9 Hz. Moreover, it is also possible to control the phase of any frequency band with a theoretical precision of 10-5 degrees in all cases. The system has been applied in the study of analogue filter networks, real-time Hilbert transformation, phase-shift systems and musical instrument body emulation, where it is providing valuable new insights into the understanding of psychoacoustic mechanisms.

Properties of Dental Pulp-derived Mesenchymal Stem Cells and the Effects of Culture Conditions.

PubMed

Kawashima, Nobuyuki; Noda, Sonoko; Yamamoto, Mioko; Okiji, Takashi

2017-09-01

Dental pulp mesenchymal stem cells (DPMSCs) highly express mesenchymal stem cell markers and possess the potential to differentiate into neural cells, osteoblasts, adipocytes, and chondrocytes. Thus, DPMSCs are considered suitable for tissue regeneration. The colony isolation method has commonly been used to collect relatively large amounts of heterogeneous DPMSCs. Homogenous DPMSCs can be isolated by fluorescence-activated cell sorting using antibodies against mesenchymal stem cell markers, although this method yields a limited number of cells. Both quality and quantity of DPMSCs are critical to regenerative therapy, and cell culture methods need to be improved. We thus investigated the properties of DPMSCs cultured with different methods. DPMSCs in a three-dimensional spheroid culture system, which is similar to the hanging drop culture for differentiation of embryonic stem cells, showed upregulation of odonto-/osteoblastic markers and mineralized nodule formation. This suggests that this three-dimensional spheroid culturing system for DPMSCs may be suitable for inducing hard tissues. We further examined the effect of cell culture density on the properties of DPMSCs because the properties of stem cells can be altered depending on the cell density. DPMSCs cultured under the confluent cell density condition showed slight downregulation of some mesenchymal stem cell markers compared with those under the sparse condition. The ability of DPMSCs to differentiate into hard tissue-forming cells was found to be enhanced in the confluent condition, suggesting that the confluent culture condition may not be suitable for maintaining the stemness of DPMSCs. When DPMSCs are to be used for hard tissue regeneration, dense followed by sparse cell culture conditions may be a better alternative strategy. Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Normalizing gene expression by quantitative PCR during somatic embryogenesis in two representative conifer species: Pinus pinaster and Picea abies.

PubMed

de Vega-Bartol, José J; Santos, Raquen Raissa; Simões, Marta; Miguel, Célia M

2013-05-01

Suitable internal control genes to normalize qPCR data from different stages of embryo development and germination were identified in two representative conifer species. Clonal propagation by somatic embryogenesis has a great application potentiality in conifers. Quantitative PCR (qPCR) is widely used for gene expression analysis during somatic embryogenesis and embryo germination. No single reference gene is universal, so a systematic characterization of endogenous genes for concrete conditions is fundamental for accuracy. We identified suitable internal control genes to normalize qPCR data obtained at different steps of somatic embryogenesis (embryonal mass proliferation, embryo maturation and germination) in two representative conifer species, Pinus pinaster and Picea abies. Candidate genes included endogenous genes commonly used in conifers, genes previously tested in model plants, and genes with a lower variation of the expression along embryo development according to genome-wide transcript profiling studies. Three different algorithms were used to evaluate expression stability. The geometric average of the expression values of elongation factor-1α, α-tubulin and histone 3 in P. pinaster, and elongation factor-1α, α-tubulin, adenosine kinase and CAC in P. abies were adequate for expression studies throughout somatic embryogenesis. However, improved accuracy was achieved when using other gene combinations in experiments with samples at a single developmental stage. The importance of studies selecting reference genes to use in different tissues or developmental stages within one or close species, and the instability of commonly used reference genes, is highlighted.

Isoflurane is a suitable alternative to ether for anesthetizing rats prior to euthanasia for gene expression analysis.

PubMed

Nakatsu, Noriyuki; Igarashi, Yoshinobu; Aoshi, Taiki; Hamaguchi, Isao; Saito, Masumichi; Mizukami, Takuo; Momose, Haruka; Ishii, Ken J; Yamada, Hiroshi

2017-01-01

Diethyl ether (ether) had been widely used in Japan for anesthesia, despite its explosive properties and toxicity to both humans and animals. We also had used ether as an anesthetic for euthanizing rats for research in the Toxicogenomics Project (TGP). Because the use of ether for these purposes will likely cease, it is required to select an alternative anesthetic which is validated for consistency with existing TGP data acquired under ether anesthesia. We therefore compared two alternative anesthetic candidates, isoflurane and pentobarbital, with ether in terms of hematological findings, serum biochemical parameters, and gene expressions. As a result, few differences among the three agents were observed. In hematological and serum biochemistry analysis, no significant changes were found. In gene expression analysis, four known genes were extracted as differentially expressed genes in the liver of rats anesthetized with ether, isoflurane, or pentobarbital. However, no significant relationships were detected using gene ontology, pathway, or gene enrichment analyses by DAVID and TargetMine. Surprisingly, although it was expected that the lung would be affected by administration via inhalation, only one differentially expressed gene was extracted in the lung. Taken together, our data indicate that there are no significant differences among ether, isoflurane, and pentobarbital with respect to effects on hematological parameters, serum biochemistry parameters, and gene expression. Based on its smallest affect to existing data and its safety profile for humans and animals, we suggest isoflurane as a suitable alternative anesthetic for use in rat euthanasia in toxicogenomics analysis.

An event-version-based spatio-temporal modeling approach and its application in the cadastral management

NASA Astrophysics Data System (ADS)

Li, Yangdong; Han, Zhen; Liao, Zhongping

2009-10-01

Spatiality, temporality, legality, accuracy and continuality are characteristic of cadastral information, and the cadastral management demands that the cadastral data should be accurate, integrated and updated timely. It's a good idea to build an effective GIS management system to manage the cadastral data which are characterized by spatiality and temporality. Because no sound spatio-temporal data models have been adopted, however, the spatio-temporal characteristics of cadastral data are not well expressed in the existing cadastral management systems. An event-version-based spatio-temporal modeling approach is first proposed from the angle of event and version. Then with the help of it, an event-version-based spatio-temporal cadastral data model is built to represent spatio-temporal cadastral data. At last, the previous model is used in the design and implementation of a spatio-temporal cadastral management system. The result of the application of the system shows that the event-version-based spatio-temporal data model is very suitable for the representation and organization of cadastral data.

Progress in Development of the ITER Plasma Control System Simulation Platform

NASA Astrophysics Data System (ADS)

Walker, Michael; Humphreys, David; Sammuli, Brian; Ambrosino, Giuseppe; de Tommasi, Gianmaria; Mattei, Massimiliano; Raupp, Gerhard; Treutterer, Wolfgang; Winter, Axel

2017-10-01

We report on progress made and expected uses of the Plasma Control System Simulation Platform (PCSSP), the primary test environment for development of the ITER Plasma Control System (PCS). PCSSP will be used for verification and validation of the ITER PCS Final Design for First Plasma, to be completed in 2020. We discuss the objectives of PCSSP, its overall structure, selected features, application to existing devices, and expected evolution over the lifetime of the ITER PCS. We describe an archiving solution for simulation results, methods for incorporating physics models of the plasma and physical plant (tokamak, actuator, and diagnostic systems) into PCSSP, and defining characteristics of models suitable for a plasma control development environment such as PCSSP. Applications of PCSSP simulation models including resistive plasma equilibrium evolution are demonstrated. PCSSP development supported by ITER Organization under ITER/CTS/6000000037. Resistive evolution code developed under General Atomics' Internal funding. The views and opinions expressed herein do not necessarily reflect those of the ITER Organization.

[A new method for the classification of neonates based on maturity and somatic development].

PubMed

Berkö, P

1992-03-01

The author points out the sad fact that the methods of estimating classifying, comparative examining of the maturity, somatic development of newborns and the methods of marking off the retarded newborns used up to now are essentially centering round the birth-weight. He exposes the errors, deficiencies of these methods and confronts them with the possibilities of the NDN-system (newborn's somatic development and nutritional state) worked out earlier by him. He thinks the NDF-system to be able to express simultaneously and exactly the gestational age, weight- and length-development, state of being fed of the newborns and the relation to the populational average, the fact and type of retardation, as well. The informational means of NDF-system in NDF-index. The NDF-system makes it possible to break down the birth-weight centric view as it offers a more suitable and qualified method than used before to describe the maturity and somatic development and the classifying of the newborns on the basis of these.

Identification of suitable reference genes for hepatic microRNA quantitation.

PubMed

Lamba, Vishal; Ghodke-Puranik, Yogita; Guan, Weihua; Lamba, Jatinder K

2014-03-07

MicroRNAs (miRNAs) are short (~22 nt) endogenous RNAs that play important roles in regulating expression of a wide variety of genes involved in different cellular processes. Alterations in microRNA expression patterns have been associated with a number of human diseases. Accurate quantitation of microRNA levels is important for their use as biomarkers and in determining their functions. Real time PCR is the gold standard and the most frequently used technique for miRNA quantitation. Real time PCR data analysis includes normalizing the amplification data to suitable endogenous control/s to ensure that microRNA quantitation is not affected by the variability that is potentially introduced at different experimental steps. U6 (RNU6A) and RNU6B are two commonly used endogenous controls in microRNA quantitation. The present study was designed to investigate inter-individual variability and gender differences in hepatic microRNA expression as well as to identify the best endogenous control/s that could be used for normalization of real-time expression data in liver samples. We used Taqman based real time PCR to quantitate hepatic expression levels of 22 microRNAs along with U6 and RNU6B in 50 human livers samples (25 M, 25 F). To identify the best endogenous controls for use in data analysis, we evaluated the amplified candidates for their stability (least variability) in expression using two commonly used software programs: Normfinder and GeNormplus, Both Normfinder and GeNormplus identified U6 to be among the least stable of all the candidates analyzed, and RNU6B was also not among the top genes in stability. mir-152 and mir-23b were identified to be the two most stable candidates by both Normfinder and GeNormplus in our analysis, and were used as endogenous controls for normalization of hepatic miRNA levels. Measurements of microRNA stability indicate that U6 and RNU6B are not suitable for use as endogenous controls for normalizing microRNA relative quantitation data in hepatic tissue, and their use can led to possibly erroneous conclusions.

Motion and Emotional Behavior Design for Pet Robot Dog

NASA Astrophysics Data System (ADS)

Cheng, Chi-Tai; Yang, Yu-Ting; Miao, Shih-Heng; Wong, Ching-Chang

A pet robot dog with two ears, one mouth, one facial expression plane, and one vision system is designed and implemented so that it can do some emotional behaviors. Three processors (Inter® Pentium® M 1.0 GHz, an 8-bit processer 8051, and embedded soft-core processer NIOS) are used to control the robot. One camera, one power detector, four touch sensors, and one temperature detector are used to obtain the information of the environment. The designed robot with 20 DOF (degrees of freedom) is able to accomplish the walking motion. A behavior system is built on the implemented pet robot so that it is able to choose a suitable behavior for different environmental situation. From the practical test, we can see that the implemented pet robot dog can do some emotional interaction with the human.

Review of deployment technology for tethered satellite systems

NASA Astrophysics Data System (ADS)

Yu, B. S.; Wen, H.; Jin, D. P.

2018-03-01

Tethered satellite systems (TSSs) have attracted significant attention due to their potential and valuable applications for scientific research. With the development of various launched on-orbit missions, the deployment of tethers is considered a crucial technology for operation of a TSS. Both past orbiting experiments and numerical results have shown that oscillations of the deployed tether due to the Coriolis force and environmental perturbations are inevitable and that the impact between the space tether and end-body at the end of the deployment process leads to complicated nonlinear phenomena. Hence, a set of suitable control methods plays a fundamental role in tether deployment. This review article summarizes previous work on aspects of the dynamics, control, and ground-based experiments of tether deployment. The relevant basic principles, analytical expressions, simulation cases, and experimental results are presented as well.

Rapid generation of recombinant Pseudomonas putida secondary metabolite producers using yTREX.

PubMed

Domröse, Andreas; Weihmann, Robin; Thies, Stephan; Jaeger, Karl-Erich; Drepper, Thomas; Loeschcke, Anita

2017-12-01

Microbial secondary metabolites represent a rich source of valuable compounds with a variety of applications in medicine or agriculture. Effective exploitation of this wealth of chemicals requires the functional expression of the respective biosynthetic genes in amenable heterologous hosts. We have previously established the TREX system which facilitates the transfer, integration and expression of biosynthetic gene clusters in various bacterial hosts. Here, we describe the yTREX system, a new tool adapted for one-step yeast recombinational cloning of gene clusters. We show that with yTREX, Pseudomonas putida secondary metabolite production strains can rapidly be constructed by random targeting of chromosomal promoters by Tn5 transposition. Feasibility of this approach was corroborated by prodigiosin production after yTREX cloning, transfer and expression of the respective biosynthesis genes from Serratia marcescens . Furthermore, the applicability of the system for effective pathway rerouting by gene cluster adaptation was demonstrated using the violacein biosynthesis gene cluster from Chromobacterium violaceum , producing pathway metabolites violacein, deoxyviolacein, prodeoxyviolacein, and deoxychromoviridans. Clones producing both prodigiosin and violaceins could be readily identified among clones obtained after random chromosomal integration by their strong color-phenotype. Finally, the addition of a promoter-less reporter gene enabled facile detection also of phenazine-producing clones after transfer of the respective phenazine-1-carboxylic acid biosynthesis genes from Pseudomonas aeruginosa . All compounds accumulated to substantial titers in the mg range. We thus corroborate here the suitability of P. putida for the biosynthesis of diverse natural products, and demonstrate that the yTREX system effectively enables the rapid generation of secondary metabolite producing bacteria by activation of heterologous gene clusters, applicable for natural compound discovery and combinatorial biosynthesis.

Genetics of solvent-producing clostridia. Final technical report

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

NONE

Specific Aims 1 and 2 of the original project proposal were specifically addressed during this project period. This involved the development of the pCAK1 phagemid delivery vector, refinement of the C. acetobutylicum electroporation protocol, selection and characterization of the engB cellulase gene from C. cellulovorans and the introduction and successful expression of this heterologous engB gene from C. cellulovorans in C. acetobutylicum. The successful expression of a heterologous engB gene from C. cellulovorans in C. acetobutylicum ATCC 824 has important industrial significance for the utilization of cellulose by this ABE fermentation microorganism. Conversion efficiency testing of the developed recombinant strainsmore » in batch and continuous culture (Specific Aim 3) will be carried out once suitable strains have been developed which can utilize cellulose as sole carbon source. The functionality of pCAK1 in the E. coli host system, especially in generating ssDNA, in the absence of impairing E. coli cell viability, together with successful introduction of pCAK1 into C. acetobutylicum and C. perfringens is the basis for the construction of a M13-like genetic system for the genus Clostridium and is expected to allow for more sophisticated molecular genetic analysis of this genus.« less

Light-regulated promoters for tunable, temporal, and affordable control of fungal gene expression.

PubMed

Fuller, Kevin K; Dunlap, Jay C; Loros, Jennifer J

2018-05-01

Regulatable promoters are important genetic tools, particularly for assigning function to essential and redundant genes. They can also be used to control the expression of enzymes that influence metabolic flux or protein secretion, thereby optimizing product yield in bioindustry. This review will focus on regulatable systems for use in filamentous fungi, an important group of organisms whose members include key research models, devastating pathogens of plants and animals, and exploitable cell factories. Though we will begin by cataloging those promoters that are controlled by nutritional or chemical means, our primary focus will rest on those who can be controlled by a literal flip-of-the-switch: promoters of light-regulated genes. The vvd promoter of Neurospora will first serve as a paradigm for how light-driven systems can provide tight, robust, tunable, and temporal control of either autologous or heterologous fungal proteins. We will then discuss a theoretical approach to, and practical considerations for, the development of such promoters in other species. To this end, we have compiled genes from six previously published light-regulated transcriptomic studies to guide the search for suitable photoregulatable promoters in your fungus of interest.

Validation of housekeeping genes as internal controls for studying biomarkers of endocrine-disrupting chemicals in disk abalone by real-time PCR.

PubMed

Wan, Qiang; Whang, Ilson; Choi, Cheol Young; Lee, Jae-Seong; Lee, Jehee

2011-04-01

Our experiments were designed to identify suitable housekeeping genes (HKGs) in disk abalone as internal controls to quantify biomarker expression following endocrine disrupting chemicals (EDCs). Relative expression levels of twelve candidate HKGs were examined by real-time reverse transcription PCR (qRT-PCR) in gill and hepatopancreas of abalone following a 7-day challenge with either tributyltin chloride (TBT) or 17β-estradiol (E2). The expression levels of several conventional HKGs, such as 18s rRNA, glyceraldehyde-3-phosphate dehydrogenase and β-actin, were significantly altered by the challenges, indicating that they might not be suitable internal controls. Instead, the geNorm analysis pinpointed ribosomal protein L-5/ elongation factor 1 and ribosomal protein L-5/ succinate dehydrogenase as the most stable HKGs under TBT and E2 challenges, respectively. Moreover, these three HKGs also showed the highest stabilities overall amongst different tissues, genders and EDC challenges. The expression of a biomarker gene, cytochrome P450 4B (CYP4), was also investigated and exhibited a significant increase after the challenges. Importantly, when unsuitable HKGs were used for normalization, the influence of two EDCs on CYP4 expression was imprecisely overestimated or underestimated, which strongly emphasized the importance of selecting appropriately validated HKGs as internal controls in biomarker studies. Copyright © 2010 Elsevier Inc. All rights reserved.

Validation of reference genes aiming accurate normalization of qRT-PCR data in Dendrocalamus latiflorus Munro.

PubMed

Liu, Mingying; Jiang, Jing; Han, Xiaojiao; Qiao, Guirong; Zhuo, Renying

2014-01-01

Dendrocalamus latiflorus Munro distributes widely in subtropical areas and plays vital roles as valuable natural resources. The transcriptome sequencing for D. latiflorus Munro has been performed and numerous genes especially those predicted to be unique to D. latiflorus Munro were revealed. qRT-PCR has become a feasible approach to uncover gene expression profiling, and the accuracy and reliability of the results obtained depends upon the proper selection of stable reference genes for accurate normalization. Therefore, a set of suitable internal controls should be validated for D. latiflorus Munro. In this report, twelve candidate reference genes were selected and the assessment of gene expression stability was performed in ten tissue samples and four leaf samples from seedlings and anther-regenerated plants of different ploidy. The PCR amplification efficiency was estimated, and the candidate genes were ranked according to their expression stability using three software packages: geNorm, NormFinder and Bestkeeper. GAPDH and EF1α were characterized to be the most stable genes among different tissues or in all the sample pools, while CYP showed low expression stability. RPL3 had the optimal performance among four leaf samples. The application of verified reference genes was illustrated by analyzing ferritin and laccase expression profiles among different experimental sets. The analysis revealed the biological variation in ferritin and laccase transcript expression among the tissues studied and the individual plants. geNorm, NormFinder, and BestKeeper analyses recommended different suitable reference gene(s) for normalization according to the experimental sets. GAPDH and EF1α had the highest expression stability across different tissues and RPL3 for the other sample set. This study emphasizes the importance of validating superior reference genes for qRT-PCR analysis to accurately normalize gene expression of D. latiflorus Munro.

Dermal-epidermal membrane systems by using human keratinocytes and mesenchymal stem cells isolated from dermis.

PubMed

Salerno, Simona; Messina, Antonietta; Giordano, Francesca; Bader, Augustinus; Drioli, Enrico; De Bartolo, Loredana

2017-02-01

Dermal-epidermal membrane systems were developed by co-culturing human keratinocytes with Skin derived Stem Cells (SSCs), which are Mesenchymal Stem Cells (MSCs) isolated from dermis, on biodegradable membranes of chitosan (CHT), polycaprolactone (PCL) and a polymeric blend of CHT and PCL. The membranes display physico-chemical, morphological, mechanical and biodegradation properties that could satisfy and fulfil specific requirements in skin tissue engineering. CHT membrane exhibits an optimal biodegradation rate for acute wounds; CHT-PCL for the chronic ones. On the other hand, PCL membrane in spite of its very slow biodegradation rate exhibits mechanical properties similar to in vivo dermis, a lower hydrophilic character, and a surface roughness, all properties that make it able to sustain cell adhesion and proliferation for in vitro skin models. Both CHT-PCL and PCL membranes guided epidermal and dermal differentiation of SSCs as pointed out by the expression of cytokeratins and the deposition of the ECM protein fibronectin, respectively. In the dermal-epidermal membrane systems, a more suitable microenvironment for the SSCs differentiation was promoted by the interactions and the mutual interplay with keratinocytes. Being skin tissue-biased stem cells committed to their specific final dermal and/or epidermal cell differentiation, SSCs are more suitable for skin tissue engineering than other adult MSCs with different origin. For this reason, they represent a useful autologous cell source for engineering skin substitutes for both in vivo and in vitro applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Production of human interferon alfa 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression.

PubMed

Sindarovska, Y R; Gerasymenko, I M; Sheludko, Y V; Olevinskaya, Z M; Spivak, N Y; Kuchuk, N V

2010-01-01

Human interferon alpha2b gene was transiently expressed in Nicotiana excelsior plants. Fusion with N. plumbaginifolia calreticulin signal peptide for improved apoplast targeting and carrying out the expression under optimized conditions resulted in maximal interferon activity of 3.2 x 10(3) IU/g fresh weight (FW) with an average of 2.1 +/- 0.8 x 10(3) IU/g FW. It proves that N. excelsior is a suitable host for Agrobacterium-mediated transient expression of genes encoding physiologically active human proteins. The transient expression conditions optimized for GFP marker protein were confirmed to be preferable for hIFN alpha2b.

An innovative three-dimensional gelatin foam culture system for improved study of glioblastoma stem cell behavior.

PubMed

Yang, Meng-Yin; Chiao, Ming-Tsang; Lee, Hsu-Tung; Chen, Chien-Min; Yang, Yi-Chin; Shen, Chiung-Chyi; Ma, Hsin-I

2015-04-01

Three-dimensional (3-D) tissue engineered constructs provide a platform for examining how the local extracellular matrix contributes to the malignancy of various cancers, including human glioblastoma multiforme. Here, we describe a simple and innovative 3-D culture environment and assess its potential for use with glioblastoma stem cells (GSCs) to examine the diversification inside the cell mass in the 3-D culture system. The dissociated human GSCs were cultured using gelatin foam. These cells were subsequently identified by immunohistochemical staining, reverse transcriptase-polymerase chain reaction, and Western blot assay. We demonstrate that the gelatin foam provides a suitable microenvironment, as a 3-D culture system, for GSCs to maintain their stemness. The gelatin foam culture system contributes a simplified assessment of cell blocks for immunohistochemistry assay. We show that the significant transcription activity of hypoxia and the protein expression of inflammatory responses are detected at the inside of the cell mass in vitro, while robust expression of PROM1/CD133 and hypoxia-induced factor-1 alpha are detected at the xenografted tumor in vivo. We also examine the common clinical trials under this culture platform and characterized a significant difference of drug resistance. The 3-D gelatin foam culture system can provide a more realistic microenvironment through which to study the in vivo behavior of GSCs to evaluate the role that biophysical factors play in the hypoxia, inflammatory responses and subsequent drug resistance. © 2014 Wiley Periodicals, Inc.

Regulation of host-pathogen interactions via the post-transcriptional Csr/Rsm system.

PubMed

Kusmierek, Maria; Dersch, Petra

2018-02-01

A successful colonization of specific hosts requires a rapid and efficient adaptation of the virulence-relevant gene expression program by bacterial pathogens. An important element in this endeavor is the Csr/Rsm system. This multi-component, post-transcriptional control system forms a central hub within complex regulatory networks and coordinately adjusts virulence properties with metabolic and physiological attributes of the pathogen. A key function is elicited by the RNA-binding protein CsrA/RsmA. CsrA/RsmA interacts with numerous target mRNAs, many of which encode crucial virulence factors, and alters their translation, stability or elongation of transcription. Recent studies highlighted that important colonization factors, toxins, and bacterial secretion systems are under CsrA/RsmA control. CsrA/RsmA deficiency impairs host colonization and attenuates virulence, making this post-transcriptional regulator a suitable drug target. The CsrA/RsmA protein can be inactivated through sequestration by non-coding RNAs, or via binding to specific highly abundant mRNAs and interacting proteins. The wide range of interaction partners and RNA targets, as well as the overarching, interlinked genetic control circuits illustrate the complexity of this regulatory system in the different pathogens. Future work addressing spatio-temporal changes of Csr/Rsm-mediated control during the course of an infection will help us to understand how bacteria reprogram their expression profile to cope with continuous changes experienced in colonized niches. Copyright © 2017 Elsevier Ltd. All rights reserved.

MicroRNAs are suitable for assessment as biomarkers from formalin-fixed paraffin-embedded tissue, and miR-24 represents an appropriate reference microRNA for diffuse large B-cell lymphoma studies.

PubMed

Culpin, Rachel Emily; Sieniawski, Michal; Proctor, Stephen John; Menon, Geetha; Mainou-Fowler, Tryfonia

2013-03-01

Tissue biopsy specimens in the form of formalin-fixed paraffin-embedded tissue (FFPET) represent a valuable resource for biomarker identification and validation. However, to date, they remain an underused asset due to uncertainty regarding RNA extraction and the reliability of downstream techniques, including quantitative RT-PCR. Recently, much interest has emerged in the study of microRNAs; small single-stranded RNAs with a role in transcriptional regulation, that are thought to be well preserved in FFPET. In this study, we show that microRNA expression is comparable between FFPET and matched fresh-frozen samples (miR-17-5p: p=0.01, miR-92: p=0.003), and demonstrate that no significant deterioration in expression occurs over prolonged FFPET storage (p=0.06). Furthermore, microRNA expression is equivalent dependant on RNA extraction method (p<0.001) or DNAse treatment of total RNA (p<0.001). Finally, we validate miR-24 as a suitable reference microRNA for diffuse large B-cell lymphoma (DLBCL) FFPET studies.

A discrete-time adaptive control scheme for robot manipulators

NASA Technical Reports Server (NTRS)

Tarokh, M.

1990-01-01

A discrete-time model reference adaptive control scheme is developed for trajectory tracking of robot manipulators. The scheme utilizes feedback, feedforward, and auxiliary signals, obtained from joint angle measurement through simple expressions. Hyperstability theory is utilized to derive the adaptation laws for the controller gain matrices. It is shown that trajectory tracking is achieved despite gross robot parameter variation and uncertainties. The method offers considerable design flexibility and enables the designer to improve the performance of the control system by adjusting free design parameters. The discrete-time adaptation algorithm is extremely simple and is therefore suitable for real-time implementation. Simulations and experimental results are given to demonstrate the performance of the scheme.

Comment on "Defocusing complex short-pulse equation and its multi-dark-soliton solution"

NASA Astrophysics Data System (ADS)

Youssoufa, Saliou; Kuetche, Victor K.; Kofane, Timoleon C.

2017-08-01

In their recent paper, Feng et al. [Phys. Rev. E 93, 052227 (2016), 10.1103/PhysRevE.93.052227] proposed a complex short-pulse equation of both focusing and defocusing types. They studied in detail the defocusing case and derived its multi-dark-soliton solutions. Nonetheless, from a physical viewpoint in order to better and deeply understand their genuine implications, we find it useful to provide a real and proper background for the derivation of the previous evolution system while showing that the expression of the nonlinear electric polarization the above authors used in their scheme is not suitable for getting the defocusing complex short-pulse equation.

Comment on "Defocusing complex short-pulse equation and its multi-dark-soliton solution".

PubMed

Youssoufa, Saliou; Kuetche, Victor K; Kofane, Timoleon C

2017-08-01

In their recent paper, Feng et al. [Phys. Rev. E 93, 052227 (2016)PREHBM2470-004510.1103/PhysRevE.93.052227] proposed a complex short-pulse equation of both focusing and defocusing types. They studied in detail the defocusing case and derived its multi-dark-soliton solutions. Nonetheless, from a physical viewpoint in order to better and deeply understand their genuine implications, we find it useful to provide a real and proper background for the derivation of the previous evolution system while showing that the expression of the nonlinear electric polarization the above authors used in their scheme is not suitable for getting the defocusing complex short-pulse equation.

Visualizing and quantifying the in vivo structure and dynamics of the Arabidopsis cortical cytoskeleton using CLSM and VAEM.

PubMed

Rosero, Amparo; Zárský, Viktor; Cvrčková, Fatima

2014-01-01

The cortical microtubules, and to some extent also the actin meshwork, play a central role in the shaping of plant cells. Transgenic plants expressing fluorescent protein markers specifically tagging the two main cytoskeletal systems are available, allowing noninvasive in vivo studies. Advanced microscopy techniques, in particular confocal laser scanning microscopy (CLSM) and variable angle epifluorescence microscopy (VAEM), can be nowadays used for imaging the cortical cytoskeleton of living cells with unprecedented spatial and temporal resolution. With the aid of suitable computing techniques, quantitative information can be extracted from microscopic images and video sequences, providing insight into both architecture and dynamics of the cortical cytoskeleton.

From microscopic taxation and redistribution models to macroscopic income distributions

NASA Astrophysics Data System (ADS)

Bertotti, Maria Letizia; Modanese, Giovanni

2011-10-01

We present here a general framework, expressed by a system of nonlinear differential equations, suitable for the modeling of taxation and redistribution in a closed society. This framework allows one to describe the evolution of income distribution over the population and to explain the emergence of collective features based on knowledge of the individual interactions. By making different choices of the framework parameters, we construct different models, whose long-time behavior is then investigated. Asymptotic stationary distributions are found, which enjoy similar properties as those observed in empirical distributions. In particular, they exhibit power law tails of Pareto type and their Lorenz curves and Gini indices are consistent with some real world ones.

Sensor selection cost optimisation for tracking structurally cyclic systems: a P-order solution

NASA Astrophysics Data System (ADS)

Doostmohammadian, M.; Zarrabi, H.; Rabiee, H. R.

2017-08-01

Measurements and sensing implementations impose certain cost in sensor networks. The sensor selection cost optimisation is the problem of minimising the sensing cost of monitoring a physical (or cyber-physical) system. Consider a given set of sensors tracking states of a dynamical system for estimation purposes. For each sensor assume different costs to measure different (realisable) states. The idea is to assign sensors to measure states such that the global cost is minimised. The number and selection of sensor measurements need to ensure the observability to track the dynamic state of the system with bounded estimation error. The main question we address is how to select the state measurements to minimise the cost while satisfying the observability conditions. Relaxing the observability condition for structurally cyclic systems, the main contribution is to propose a graph theoretic approach to solve the problem in polynomial time. Note that polynomial time algorithms are suitable for large-scale systems as their running time is upper-bounded by a polynomial expression in the size of input for the algorithm. We frame the problem as a linear sum assignment with solution complexity of ?.

46 CFR 95.05-1 - Fire detecting, manual alarm, and supervised patrol systems.

Code of Federal Regulations, 2014 CFR

2014-10-01

... provided a smoke detecting or other suitable type fire detecting system. (c) Enclosed spaces which are “specially suitable for vehicles” shall be fitted with an approved fire or smoke detecting system. [CGFR 66...

46 CFR 95.05-1 - Fire detecting, manual alarm, and supervised patrol systems.

Code of Federal Regulations, 2013 CFR

2013-10-01

... provided a smoke detecting or other suitable type fire detecting system. (c) Enclosed spaces which are “specially suitable for vehicles” shall be fitted with an approved fire or smoke detecting system. [CGFR 66...

Small, synthetic, GC-rich mRNA stem-loop modules 5' proximal to the AUG start-codon predictably tune gene expression in yeast.

PubMed

Lamping, Erwin; Niimi, Masakazu; Cannon, Richard D

2013-07-29

A large range of genetic tools has been developed for the optimal design and regulation of complex metabolic pathways in bacteria. However, fewer tools exist in yeast that can precisely tune the expression of individual enzymes in novel metabolic pathways suitable for industrial-scale production of non-natural compounds. Tuning expression levels is critical for reducing the metabolic burden of over-expressed proteins, the accumulation of toxic intermediates, and for redirecting metabolic flux from native pathways involving essential enzymes without negatively affecting the viability of the host. We have developed a yeast membrane protein hyper-expression system with critical advantages over conventional, plasmid-based, expression systems. However, expression levels are sometimes so high that they adversely affect protein targeting/folding or the growth and/or phenotype of the host. Here we describe the use of small synthetic mRNA control modules that allowed us to predictably tune protein expression levels to any desired level. Down-regulation of expression was achieved by engineering small GC-rich mRNA stem-loops into the 5' UTR that inhibited translation initiation of the yeast ribosomal 43S preinitiation complex (PIC). Exploiting the fact that the yeast 43S PIC has great difficulty scanning through GC-rich mRNA stem-loops, we created yeast strains containing 17 different RNA stem-loop modules in the 5' UTR that expressed varying amounts of the fungal multidrug efflux pump reporter Cdr1p from Candida albicans. Increasing the length of mRNA stem-loops (that contained only GC-pairs) near the AUG start-codon led to a surprisingly large decrease in Cdr1p expression; ~2.7-fold for every additional GC-pair added to the stem, while the mRNA levels remained largely unaffected. An mRNA stem-loop of seven GC-pairs (∆G = -15.8 kcal/mol) reduced Cdr1p expression levels by >99%, and even the smallest possible stem-loop of only three GC-pairs (∆G = -4.4 kcal/mol) inhibited Cdr1p expression by ~50%. We have developed a simple cloning strategy to fine-tune protein expression levels in yeast that has many potential applications in metabolic engineering and the optimization of protein expression in yeast. This study also highlights the importance of considering the use of multiple cloning-sites carefully to preclude unwanted effects on gene expression.

Small, synthetic, GC-rich mRNA stem-loop modules 5′ proximal to the AUG start-codon predictably tune gene expression in yeast

PubMed Central

2013-01-01

Background A large range of genetic tools has been developed for the optimal design and regulation of complex metabolic pathways in bacteria. However, fewer tools exist in yeast that can precisely tune the expression of individual enzymes in novel metabolic pathways suitable for industrial-scale production of non-natural compounds. Tuning expression levels is critical for reducing the metabolic burden of over-expressed proteins, the accumulation of toxic intermediates, and for redirecting metabolic flux from native pathways involving essential enzymes without negatively affecting the viability of the host. We have developed a yeast membrane protein hyper-expression system with critical advantages over conventional, plasmid-based, expression systems. However, expression levels are sometimes so high that they adversely affect protein targeting/folding or the growth and/or phenotype of the host. Here we describe the use of small synthetic mRNA control modules that allowed us to predictably tune protein expression levels to any desired level. Down-regulation of expression was achieved by engineering small GC-rich mRNA stem-loops into the 5′ UTR that inhibited translation initiation of the yeast ribosomal 43S preinitiation complex (PIC). Results Exploiting the fact that the yeast 43S PIC has great difficulty scanning through GC-rich mRNA stem-loops, we created yeast strains containing 17 different RNA stem-loop modules in the 5′ UTR that expressed varying amounts of the fungal multidrug efflux pump reporter Cdr1p from Candida albicans. Increasing the length of mRNA stem-loops (that contained only GC-pairs) near the AUG start-codon led to a surprisingly large decrease in Cdr1p expression; ~2.7-fold for every additional GC-pair added to the stem, while the mRNA levels remained largely unaffected. An mRNA stem-loop of seven GC-pairs (∆G = −15.8 kcal/mol) reduced Cdr1p expression levels by >99%, and even the smallest possible stem-loop of only three GC-pairs (∆G = −4.4 kcal/mol) inhibited Cdr1p expression by ~50%. Conclusion We have developed a simple cloning strategy to fine-tune protein expression levels in yeast that has many potential applications in metabolic engineering and the optimization of protein expression in yeast. This study also highlights the importance of considering the use of multiple cloning-sites carefully to preclude unwanted effects on gene expression. PMID:23895661

Characterization and assembly of a GFP-tagged cylindriform silk into hexameric complexes.

PubMed

Öster, Carl; Svensson Bonde, Johan; Bülow, Leif; Dicko, Cedric

2014-04-01

Spider silk has been studied extensively for its attractive mechanical properties and potential applications in medicine and industry. The production of spider silk, however, has been lagging behind for lack of suitable systems. Our approach focuses on solving the production of spider silk by designing, expressing, purifying and characterizing the silk from cylindriform glands. We show that the cylindriform silk protein, in contrast to the commonly used dragline silk protein, is fully folded and stable in solution. With the help of GFP as a fusion tag we enhanced the expression of the silk protein in Escherichia coli and could optimize the downstream processing. Secondary structures analysis by circular dichroism and FTIR shows that the GFP-silk fusion protein is predominantly α-helical, and that pH can trigger a α- to β-transition resulting in aggregation. Structural analysis by small angle X-ray scattering suggests that the GFP-Silk exists in the form of a hexamer in solution. Copyright © 2013 Wiley Periodicals, Inc.

High-level expression of Camelid nanobodies in Nicotiana benthamiana.

PubMed

Teh, Yi-Hui Audrey; Kavanagh, Tony A

2010-08-01

Nanobodies (or VHHs) are single-domain antigen-binding fragments derived from Camelid heavy chain-only antibodies. Their small size, monomeric behaviour, high stability and solubility, and ability to bind epitopes not accessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. Here we describe high-level expression, in Nicotiana benthamiana, of three versions of an anti-hen egg white lysozyme (HEWL) nanobody which include the original VHH from an immunized library (cAbLys3), a codon-optimized derivative, and a codon-optimized hybrid nanobody comprising the CDRs of cAbLys3 grafted onto an alternative 'universal' nanobody framework. His6- and StrepII-tagged derivatives of each nanobody were targeted for accumulation in the cytoplasm, chloroplast and apoplast using different pre-sequences. When targeted to the apoplast, intact functional nanobodies accumulated at an exceptionally high level (up to 30% total leaf protein), demonstrating the great potential of plants as a nanobody production system.

Primer sets for cloning the human repertoire of T cell Receptor Variable regions.

PubMed

Boria, Ilenia; Cotella, Diego; Dianzani, Irma; Santoro, Claudio; Sblattero, Daniele

2008-08-29

Amplification and cloning of naïve T cell Receptor (TR) repertoires or antigen-specific TR is crucial to shape immune response and to develop immuno-based therapies. TR variable (V) regions are encoded by several genes that recombine during T cell development. The cloning of expressed genes as large diverse libraries from natural sources relies upon the availability of primers able to amplify as many V genes as possible. Here, we present a list of primers computationally designed on all functional TR V and J genes listed in the IMGT, the ImMunoGeneTics information system. The list consists of unambiguous or degenerate primers suitable to theoretically amplify and clone the entire TR repertoire. We show that it is possible to selectively amplify and clone expressed TR V genes in one single RT-PCR step and from as little as 1000 cells. This new primer set will facilitate the creation of more diverse TR libraries than has been possible using currently available primer sets.

Ebola virus encodes a miR-155 analog to regulate importin-α5 expression.

PubMed

Liu, Yuanwu; Sun, Jing; Zhang, Hongwen; Wang, Mingming; Gao, George Fu; Li, Xiangdong

2016-10-01

The 2014 outbreak of Ebola virus caused more than 10,000 human deaths. Current knowledge of suitable drugs, clinical diagnostic biomarkers and molecular mechanisms of Ebola virus infection is either absent or insufficient. By screening stem-loop structures from the viral genomes of four virulence species, we identified a novel, putative viral microRNA precursor that is specifically expressed by the Ebola virus. The sequence of the microRNA precursor was further confirmed by mining the existing RNA-Seq database. Two putative mature microRNAs were predicted and subsequently validated in human cell lines. Combined with this prediction of the microRNA target, we identified importin-α5, which is a key regulator of interferon signaling following Ebola virus infection, as one putative target. We speculate that this microRNA could facilitate the evasion of the host immune system by the virus. Moreover, this microRNA might be a potential clinical therapeutic target or a diagnostic biomarker for Ebola virus.

Membrane contactors in the beverage industry for controlling the water gas composition.

PubMed

Criscuoli, Alessandra; Drioli, Enrico; Moretti, Ugo

2003-03-01

In the work described here, membrane contactors are used for coupling the removal of species (oxygen and hydrogen sulfide) present in the water with the water carbonation process. We include both experiments and a theoretical study devoted to the analysis of the transport phenomena that occur in the membrane contactor. The main resistance to mass transport was located at the liquid side. Correlations between Sherwood and Reynolds numbers on the shell side that are suitable for the membrane contactor used to carry out our experiments have been determined. In particular, for Re > 1.6, the expression proposed by Yang and Cussler in 1986: Sh = 0.90 Re(0.40) Sc(0.33) describes the behavior of the system; whereas, for Re between 0.03 and 0.3, a new expression is proposed: Sh = 0.435 Re(1.2)Sc(0.33). A comparison with traditional equipment is also furnished. Membrane contactors offer reduced size, CO(2) consumption, and capital costs.

An analytical method for assessing the spatial and temporal variation of juvenile Atlantic salmon habitat in an upland Scottish river.

NASA Astrophysics Data System (ADS)

Buddendorf, B.; Fabris, L.; Malcolm, I.; Lazzaro, G.; Tetzlaff, D.; Botter, G.; Soulsby, C.

2016-12-01

Wild Atlantic salmon populations in Scottish rivers constitute an important economic and recreational resource, as well as being a key component of biodiversity. Salmon have specific habitat requirements at different life stages and their distribution is therefore strongly influenced by a complex suite of biological and physical controls. Stream hydrodynamics have a strong influence on habitat quality and affect the distribution and density of juvenile salmon. As stream hydrodynamics directly relate to stream flow variability and channel morphology, the effects of hydroclimatic drivers on the spatial and temporal variability of habitat suitability can be assessed. Critical Displacement Velocity (CDV), which describes the velocity at which fish can no longer hold station, is one potential approach for characterising habitat suitability. CDV is obtained using an empirical formula that depends on fish size and stream temperature. By characterising the proportion of a reach below CDV it is possible to assess the suitable area. We demonstrate that a generic analytical approach based on field survey and hydraulic modelling can provide insights on the interactions between flow regime and average suitable area (SA) for juvenile salmon that could be extended to other aquatic species. Analytical functions are used to model the pdf of stream flow p(q) and the relationship between flow and suitable area SA(q). Theoretically these functions can assume any form. Here we used a gamma distribution to model p(q) and a gamma function to model SA(q). Integrating the product of these functions we obtain an analytical expression of SA. Since parameters of p(q) can be estimated from meteorological and flow measurements, they can be used directly to predict the effect of flow regime on SA. We show the utility of the approach with reference to 6 electrofishing sites in a single river system where long term (50 years) data on spatially distributed juvenile salmon densities are available.

Selection of Single-Chain Antibodies against the VP8* Subunit of Rotavirus VP4 Outer Capsid Protein and Their Expression in Lactobacillus casei

PubMed Central

Monedero, Vicente; Rodríguez-Díaz, Jesús; Viana, Rosa; Buesa, Javier; Pérez-Martínez, Gaspar

2004-01-01

Single-chain antibodies (scFv) recognizing the VP8* fraction of rotavirus outer capsid and blocking rotavirus infection in vitro were isolated by phage display. Vectors for the extracellular expression in Lactobacillus casei of one of the scFv were constructed. L. casei was able to secrete active scFv to the growth medium, showing the potential of probiotic bacteria to be engineered to express molecules suitable for in vivo antirotavirus therapies. PMID:15528568

An Efficient Visual Screen for CRISPR/Cas9 Activity in Arabidopsis thaliana.

PubMed

Hahn, Florian; Mantegazza, Otho; Greiner, André; Hegemann, Peter; Eisenhut, Marion; Weber, Andreas P M

2017-01-01

The CRISPR/Cas9 system enables precision editing of the genome of the model plant Arabidopsis thaliana and likely of any other organism. Tools and methods for further developing and optimizing this widespread and versatile system in Arabidopsis would hence be welcomed. Here, we designed a generic vector system that can be used to clone any sgRNA sequence in a plant T-DNA vector containing an ubiquitously expressed Cas9 gene. With this vector, we explored two alternative marker systems for tracking Cas9-mediated gene-editing in vivo : BIALAPHOS RESISTANCE ( BAR ) and GLABROUS1 ( GL1 ). BAR confers resistance to glufosinate and is widely used as a positive selection marker; GL1 is required for the formation of trichomes. Reversion of a frameshift null BAR allele to a functional one by Cas9-mediated gene editing yielded a higher than expected number of plants that are resistant to glufosinate. Surprisingly, many of those plants did not display reversion of the BAR gene through the germline. We hypothesize that few BAR revertant cells in a highly chimeric plant likely provide system-wide resistance to glufosinate and thus we suggest that BAR is not suitable as marker for tracking Cas9-mediated gene-editing. Targeting the GL1 gene for disruption with Cas9 provided clearly visible phenotypes of partially and completely glabrous plants. 50% of the analyzed T1 plants produced descendants with a chimeric phenotype and we could recover fully homozygous plants in the T3 generation with high efficiency. We propose that targeting of GL1 is suitable for assessing and optimizing Cas9-mediated gene-editing in Arabidopsis .

Live-cell imaging to compare the transfection and gene silencing efficiency of calcium phosphate nanoparticles and a liposomal transfection agent.

PubMed

Chernousova, S; Epple, M

2017-05-01

The processing of DNA (for transfection) and short interfering RNA (siRNA; for gene silencing), introduced into HeLa cells by triple-shell calcium phosphate nanoparticles, was followed by live-cell imaging. For comparison, the commercial liposomal transfection agent Lipofectamine was used. The cells were incubated with these delivery systems, carrying either enhanced green fluorescent protein (eGFP)-encoding DNA or siRNA against eGFP. In the latter case, HeLa cells that stably expressed eGFP were used. The expression of eGFP started after 5 h in the case of nanoparticles and after 4 h in the case of Lipofectamine. The corresponding times for gene silencing were 5 h (nanoparticles) and immediately after incubation (Lipofectamine). The expression of eGFP was notably enhanced 2-3 h after cell division (mitosis). In general, the transfection and gene silencing efficiencies of the nanoparticles were lower than those of Lipofectamime, even at a substantially higher dose (factor 20) of nucleic acids. However, the cytotoxicity of the nanoparticles was lower than that of Lipofectamine, making them suitable vectors for in vivo application.

Live-cell imaging to compare the transfection and gene silencing efficiency of calcium phosphate nanoparticles and a liposomal transfection agent

PubMed Central

Chernousova, S; Epple, M

2017-01-01

The processing of DNA (for transfection) and short interfering RNA (siRNA; for gene silencing), introduced into HeLa cells by triple-shell calcium phosphate nanoparticles, was followed by live-cell imaging. For comparison, the commercial liposomal transfection agent Lipofectamine was used. The cells were incubated with these delivery systems, carrying either enhanced green fluorescent protein (eGFP)-encoding DNA or siRNA against eGFP. In the latter case, HeLa cells that stably expressed eGFP were used. The expression of eGFP started after 5 h in the case of nanoparticles and after 4 h in the case of Lipofectamine. The corresponding times for gene silencing were 5 h (nanoparticles) and immediately after incubation (Lipofectamine). The expression of eGFP was notably enhanced 2–3 h after cell division (mitosis). In general, the transfection and gene silencing efficiencies of the nanoparticles were lower than those of Lipofectamime, even at a substantially higher dose (factor 20) of nucleic acids. However, the cytotoxicity of the nanoparticles was lower than that of Lipofectamine, making them suitable vectors for in vivo application. PMID:28218744

Isolation and partial characterization of a root-specific promoter for stacking multiple traits into cassava (Manihot esculenta CRANTZ).

PubMed

Gbadegesin, M A; Beeching, J R

2011-06-07

Cassava can be cultivated on impoverished soils with minimum inputs, and its storage roots are a staple food for millions in Africa. However, these roots are low in bioavailable nutrients and in protein content, contain cyanogenic glycosides, and suffer from a very short post-harvest shelf-life, and the plant is susceptible to viral and bacterial diseases prevalent in Africa. The demand for improvement of cassava with respect to these traits comes from both farmers and national agricultural institutions. Genetic improvement of cassava cultivars by molecular biology techniques requires the availability of appropriate genes, a system to introduce these genes into cassava, and the use of suitable gene promoters. Cassava root-specific promoter for auxin-repressed protein was isolated using the gene walking approach, starting with a cDNA sequence. In silico analysis of promoter sequences revealed putative cis-acting regulatory elements, including root-specific elements, which may be required for gene expression in vascular tissues. Research on the activities of this promoter is continuing, with the development of plant expression cassettes for transformation into major African elite lines and farmers' preferred cassava cultivars to enable testing of tissue-specific expression patterns in the field.

Synthesis and Characterization of Photoactivatable Doxycycline Analogues Bearing Two-Photon-Sensitive Photoremovable Groups Suitable for Light-Induced Gene Expression.

PubMed

Goegan, Bastien; Terzi, Firat; Bolze, Frédéric; Cambridge, Sidney; Specht, Alexandre

2018-06-18

We report the synthesis and photolytic properties of caged 9-aminodoxycycline derivatives modified with 2-{4'-bis-[2-(2methoxyethoxy)ethyl]-4-nitrobiphenyl-3-yl}prop-1-oxy (EANBP) and PEG7-ylated (7-diethylamino-2-oxo-2H-chromen-4-yl)methyl (PEG7-DEACM) groups. 9-Aminodoxycycline is a tetracycline analogue capable of activating transcription through the inducible TetOn transgene expression system and can be regioselectively coupled to two-photon-sensitive photo-removable protecting groups by carbamoylation. The EANBP-based caged 9-aminodoxycycline showed complex photochemical reactions but did release 10 % of 9-aminodoxycycline. However, 9-(PEG7-DEACMamino)doxycycline exhibited excellent photolysis efficiency at 405 nm with quantitative release of 9-aminodoxycycline and a 0.21 uncaging quantum yield. Thanks to the good two-photon sensitivity of the DEACM chromophore, 9-aminodoxycycline release by two-photon photolysis is possible, with calculated action cross-sections of up to 4.0 GM at 740 nm. Therefore, 9-(PEG7-DEACMamino)doxycycline represents a very attractive tool for the development of a light-induced gene expression method in living cells. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Suitability of virtual prototypes to support human factors/ergonomics evaluation during the design.

PubMed

Aromaa, Susanna; Väänänen, Kaisa

2016-09-01

In recent years, the use of virtual prototyping has increased in product development processes, especially in the assessment of complex systems targeted at end-users. The purpose of this study was to evaluate the suitability of virtual prototyping to support human factors/ergonomics evaluation (HFE) during the design phase. Two different virtual prototypes were used: augmented reality (AR) and virtual environment (VE) prototypes of a maintenance platform of a rock crushing machine. Nineteen designers and other stakeholders were asked to assess the suitability of the prototype for HFE evaluation. Results indicate that the system model characteristics and user interface affect the experienced suitability. The VE system was valued as being more suitable to support the assessment of visibility, reach, and the use of tools than the AR system. The findings of this study can be used as a guidance for the implementing virtual prototypes in the product development process. Copyright © 2016 Elsevier Ltd. All rights reserved.

Study of model systems to test the potential function of Artemia group 1 late embryogenesis abundant (LEA) proteins.

PubMed

Warner, Alden H; Guo, Zhi-hao; Moshi, Sandra; Hudson, John W; Kozarova, Anna

2016-01-01

Embryos of the brine shrimp, Artemia franciscana, are genetically programmed to develop either ovoviparously or oviparously depending on environmental conditions. Shortly upon their release from the female, oviparous embryos enter diapause during which time they undergo major metabolic rate depression while simultaneously synthesize proteins that permit them to tolerate a wide range of stressful environmental events including prolonged periods of desiccation, freezing, and anoxia. Among the known stress-related proteins that accumulate in embryos entering diapause are the late embryogenesis abundant (LEA) proteins. This large group of intrinsically disordered proteins has been proposed to act as molecular shields or chaperones of macromolecules which are otherwise intolerant to harsh conditions associated with diapause. In this research, we used two model systems to study the potential function of the group 1 LEA proteins from Artemia. Expression of the Artemia group 1 gene (AfrLEA-1) in Escherichia coli inhibited growth in proportion to the number of 20-mer amino acid motifs expressed. As well, clones of E. coli, transformed with the AfrLEA-1 gene, expressed multiple bands of LEA proteins, either intrinsically or upon induction with isopropyl-β-thiogalactoside (IPTG), in a vector-specific manner. Expression of AfrLEA-1 in E. coli did not overcome the inhibitory effects of high concentrations of NaCl and KCl but modulated growth inhibition resulting from high concentrations of sorbitol in the growth medium. In contrast, expression of the AfrLEA-1 gene in Saccharomyces cerevisiae did not alter the growth kinetics or permit yeast to tolerate high concentrations of NaCl, KCl, or sorbitol. However, expression of AfrLEA-1 in yeast improved its tolerance to drying (desiccation) and freezing. Under our experimental conditions, both E. coli and S. cerevisiae appear to be potentially suitable hosts to study the function of Artemia group 1 LEA proteins under environmentally stressful conditions.

PRT: A Suitable Transport System for Urban Areas in Sweden

DOT National Transportation Integrated Search

1998-03-18

This report summarizes a thematic research programme "Advanced transit systems" performed at Chalmers University ofTechnology during the years 1994-97. The programme aims to investigate if Personal R apid Transit (PRT) may be a suitable system for ur...

Expression, Purification, and Characterization of a Recombinant Flavin Reductase from the Luminescent Marine Bacterium "Photobacterium Leiognathi": A Set of Exercises for Students

ERIC Educational Resources Information Center

Crowley, Thomas E.

2010-01-01

In "Photobacterium," the flavin reductase encoded by "lux"G regenerates the reduced form of flavin mononucleotide (FMN). Reduced FMN is one of the substrates of the luciferase enzyme that catalyzes a light-emitting reaction. A set of experiments, that employs a "lux"G-expression plasmid construct (pGhis) and is suitable for an undergraduate…

Reference genes for normalization of gene expression studies in human osteoarthritic articular cartilage.

PubMed

Pombo-Suarez, Manuel; Calaza, Manuel; Gomez-Reino, Juan J; Gonzalez, Antonio

2008-01-29

Assessment of gene expression is an important component of osteoarthritis (OA) research, greatly improved by the development of quantitative real-time PCR (qPCR). This technique requires normalization for precise results, yet no suitable reference genes have been identified in human articular cartilage. We have examined ten well-known reference genes to determine the most adequate for this application. Analyses of expression stability in cartilage from 10 patients with hip OA, 8 patients with knee OA and 10 controls without OA were done with classical statistical tests and the software programs geNorm and NormFinder. Results from the three methods of analysis were broadly concordant. Some of the commonly used reference genes, GAPDH, ACTB and 18S RNA, performed poorly in our analysis. In contrast, the rarely used TBP, RPL13A and B2M genes were the best. It was necessary to use together several of these three genes to obtain the best results. The specific combination depended, to some extent, on the type of samples being compared. Our results provide a satisfactory set of previously unused reference genes for qPCR in hip and knee OA This confirms the need to evaluate the suitability of reference genes in every tissue and experimental situation before starting the quantitative assessment of gene expression by qPCR.

Validation of Endogenous Internal Real-Time PCR Controls in Renal Tissues

PubMed Central

Cui, Xiangqin; Zhou, Juling; Qiu, Jing; Johnson, Martin R.; Mrug, Michal

2009-01-01

Background Endogenous internal controls (‘reference’ or ‘housekeeping’ genes) are widely used in real-time PCR (RT-PCR) analyses. Their use relies on the premise of consistently stable expression across studied experimental conditions. Unfortunately, none of these controls fulfills this premise across a wide range of experimental conditions; consequently, none of them can be recommended for universal use. Methods To determine which endogenous RT-PCR controls are suitable for analyses of renal tissues altered by kidney disease, we studied the expression of 16 commonly used ‘reference genes’ in 7 mildly and 7 severely affected whole kidney tissues from a well-characterized cystic kidney disease model. Expression levels of these 16 genes, determined by TaqMan® RT-PCR analyses and Affymetrix GeneChip® arrays, were normalized and tested for overall variance and equivalence of the means. Results Both statistical approaches and both TaqMan- and GeneChip-based methods converged on 3 out of the 4 top-ranked genes (Ppia, Gapdh and Pgk1) that had the most constant expression levels across the studied phenotypes. Conclusion A combination of the top-ranked genes will provide a suitable endogenous internal control for similar studies of kidney tissues across a wide range of disease severity. PMID:19729889

In vitro culture of embryonic mouse intestinal epithelium: cell differentiation and introduction of reporter genes.

PubMed

Quinlan, Jonathan M; Yu, Wei-Yuan; Hornsey, Mark A; Tosh, David; Slack, Jonathan M W

2006-05-25

Study of the normal development of the intestinal epithelium has been hampered by a lack of suitable model systems, in particular ones that enable the introduction of exogenous genes. Production of such a system would advance our understanding of normal epithelial development and help to shed light on the pathogenesis of intestinal neoplasia. The criteria for a reliable culture system include the ability to perform real time observations and manipulations in vitro, the preparation of wholemounts for immunostaining and the potential for introducing genes. The new culture system involves growing mouse embryo intestinal explants on fibronectin-coated coverslips in basal Eagle's medium+20% fetal bovine serum. Initially the cultures maintain expression of the intestinal transcription factor Cdx2 together with columnar epithelial (cytokeratin 8) and mesenchymal (smooth muscle actin) markers. Over a few days of culture, differentiation markers appear characteristic of absorptive epithelium (sucrase-isomaltase), goblet cells (Periodic Acid Schiff positive), enteroendocrine cells (chromogranin A) and Paneth cells (lysozyme). Three different approaches were tested to express genes in the developing cultures: transfection, electroporation and adenoviral infection. All could introduce genes into the mesenchyme, but only to a small extent into the epithelium. However the efficiency of adenovirus infection can be greatly improved by a limited enzyme digestion, which makes accessible the lateral faces of cells bearing the Coxsackie and Adenovirus Receptor. This enables reliable delivery of genes into epithelial cells. We describe a new in vitro culture system for the small intestine of the mouse embryo that recapitulates its normal development. The system both provides a model for studying normal development of the intestinal epithelium and also allows for the manipulation of gene expression. The explants can be cultured for up to two weeks, they form the full repertoire of intestinal epithelial cell types (enterocytes, goblet cells, Paneth cells and enteroendocrine cells) and the method for gene introduction into the epithelium is efficient and reliable.

The Use of EST Expression Matrixes for the Quality Control of Gene Expression Data

PubMed Central

Milnthorpe, Andrew T.; Soloviev, Mikhail

2012-01-01

EST expression profiling provides an attractive tool for studying differential gene expression, but cDNA libraries' origins and EST data quality are not always known or reported. Libraries may originate from pooled or mixed tissues; EST clustering, EST counts, library annotations and analysis algorithms may contain errors. Traditional data analysis methods, including research into tissue-specific gene expression, assume EST counts to be correct and libraries to be correctly annotated, which is not always the case. Therefore, a method capable of assessing the quality of expression data based on that data alone would be invaluable for assessing the quality of EST data and determining their suitability for mRNA expression analysis. Here we report an approach to the selection of a small generic subset of 244 UniGene clusters suitable for identification of the tissue of origin for EST libraries and quality control of the expression data using EST expression information alone. We created a small expression matrix of UniGene IDs using two rounds of selection followed by two rounds of optimisation. Our selection procedures differ from traditional approaches to finding “tissue-specific” genes and our matrix yields consistency high positive correlation values for libraries with confirmed tissues of origin and can be applied for tissue typing and quality control of libraries as small as just a few hundred total ESTs. Furthermore, we can pick up tissue correlations between related tissues e.g. brain and peripheral nervous tissue, heart and muscle tissues and identify tissue origins for a few libraries of uncharacterised tissue identity. It was possible to confirm tissue identity for some libraries which have been derived from cancer tissues or have been normalised. Tissue matching is affected strongly by cancer progression or library normalisation and our approach may potentially be applied for elucidating the stage of normalisation in normalised libraries or for cancer staging. PMID:22412959

lpxC and yafS are the most suitable internal controls to normalize real time RT-qPCR expression in the phytopathogenic bacteria Dickeya dadantii.

PubMed

Hommais, Florence; Zghidi-Abouzid, Ouafa; Oger-Desfeux, Christine; Pineau-Chapelle, Emilie; Van Gijsegem, Frederique; Nasser, William; Reverchon, Sylvie

2011-01-01

Quantitative RT-PCR is the method of choice for studying, with both sensitivity and accuracy, the expression of genes. A reliable normalization of the data, using several reference genes, is critical for an accurate quantification of gene expression. Here, we propose a set of reference genes, of the phytopathogenic bacteria Dickeya dadantii and Pectobacterium atrosepticum, which are stable in a wide range of growth conditions. We extracted, from a D. dadantii micro-array transcript profile dataset comprising thirty-two different growth conditions, an initial set of 49 expressed genes with very low variation in gene expression. Out of these, we retained 10 genes representing different functional categories, different levels of expression (low, medium, and high) and with no systematic variation in expression correlating with growth conditions. We measured the expression of these reference gene candidates using quantitative RT-PCR in 50 different experimental conditions, mimicking the environment encountered by the bacteria in their host and directly during the infection process in planta. The two most stable genes (ABF-0017965 (lpxC) and ABF-0020529 (yafS) were successfully used for normalization of RT-qPCR data. Finally, we demonstrated that the ortholog of lpxC and yafS in Pectobacterium atrosepticum also showed stable expression in diverse growth conditions. We have identified at least two genes, lpxC (ABF-0017965) and yafS (ABF-0020509), whose expressions are stable in a wide range of growth conditions and during infection. Thus, these genes are considered suitable for use as reference genes for the normalization of real-time RT-qPCR data of the two main pectinolytic phytopathogenic bacteria D. dadantii and P. atrosepticum and, probably, of other Enterobacteriaceae. Moreover, we defined general criteria to select good reference genes in bacteria.

Gis-Based Crop Support System For Common Oatand Naked Oat in China

NASA Astrophysics Data System (ADS)

Wan, Fan; Wang, Zhen; Li, Fengmin; Cao, Huhua; Sun, Guojun

The identification of the suitable areas for common oat (Avena sativa L.) and naked oat (Avena nuda L.) in China using Multi-Criteria Evaluation (MCE) approach based on GIS is presented in the current article. Climate, topography, soil, land use and oat variety databases were created. Relevant criteria,suitability levels and their weights for each factor were defined. Then the criteria maps were obtained and turned into the MCE process, and suitability maps for common oat and naked oat were created. The land use and the suitability maps were crossed to identify the suitable areas for each crop. The results identified 397,720 km2 of suitable areas for common oats of forage purpose distributed in 744 counties in 17 provinces, and 556,232 km2 of suitable areas for naked oats of grain purpose distributed in 779 counties in 19 provinces. This result is in accordance with the distribution of farmingpastoral ecozones located in semi-arid regions of northern China. The mapped areas can help define the working limits and serve as indicative zones for oat in China. The created databases, mapped results, interface of expert system and relevant hardware facilities could construct a complete crop support system for oats.

Genetically Engineered Theranostic Mesenchymal Stem Cells for the Evaluation of the Anticancer Efficacy of Enzyme/Prodrug Systems

PubMed Central

Nouri, Faranak Salman; Wang, Xing; Hatefi, Arash

2015-01-01

Over the past decade, various enzyme/prodrug systems such as thymidine kinase/ganciclovir (TK/GCV), yeast cytosine deaminase/5-fluorocytosine (yCD/5-FC) and nitroreductase/CB1954 (NTR/CB1954) have been used for stem cell mediated suicide gene therapy of cancer. Yet, no study has been conducted to compare and demonstrate the advantages and disadvantages of using one system over another. Knowing that each enzyme/prodrug system has its own strengths and weaknesses, we utilized mesenchymal stem cells (MSCs) as a medium to perform for the first time a comparative study that illustrated the impact of subtle differences among these systems on the therapeutic outcome. For therapeutic purposes, we first genetically modified MSCs to stably express a panel of four suicide genes including TK (TK007 and TKSR39 mutants), yeast cytosine deaminase: uracil phosphoribosyltransferase (yCD:UPRT) and nitroreductase (NTR). Then, we evaluated the anticancer efficacies of the genetically engineered MSCs in vitro and in vivo by using SKOV3 cell line which is sensitive to all four enzyme/prodrug systems. In addition, all MSCs were engineered to stably express luciferase gene making them suitable for quantitative imaging and dose-response relationship studies in animals. Considering the limitations imposed by the prodrugs’ bystander effects, our findings show that yCD:UPRT/5-FC is the most effective enzyme/prodrug system among the ones tested. Our findings also demonstrate that theranostic MSCs are a reliable medium for the side-by-side evaluation and screening of the enzyme/prodrug systems at the preclinical level. The results of this study could help scientists who utilize cell-based, non-viral or viral vectors for suicide gene therapy of cancer make more informed decisions when choosing enzyme/prodrug systems. PMID:25575867

Corrosion evaluation of novel coatings for steel components of highway bridges.

DOT National Transportation Integrated Search

2015-03-01

The Florida Department of Transportation (FDOT) had expressed interest in gauging the available coating : technologies that may have suitable applications for steel components in highway bridges. The motivation was to : possibly identify coating syst...

Nonwoven-Based Gelatin/Polycaprolactone Membrane Proves Suitability in a Preclinical Assessment for Treatment of Soft Tissue Defects

PubMed Central

Schulz, Simon; Angarano, Marco; Fabritius, Martin; Mülhaupt, Rolf; Dard, Michel; Obrecht, Marcel; Tomakidi, Pascal

2014-01-01

Standard preclinical assessments in vitro often have limitations regarding their transferability to human beings, mainly evoked by their nonhuman and tissue-different/nontissue-specific source. Here, we aimed at employing tissue-authentic simple and complex interactive fibroblast-epithelial cell systems and their in vivo-relevant biomarkers for preclinical in vitro assessment of nonwoven-based gelatin/polycaprolactone membranes (NBMs) for treatment of soft tissue defects. NBMs were composed of electrospun gelatin and polycaprolactone nanofiber nonwovens. Scanning electron microscopy in conjunction with actin/focal contact integrin fluorescence revealed successful adhesion and proper morphogenesis of keratinocytes and fibroblasts, along with cells' derived extracellular matrix deposits. The “feel-good factor” of cells under study on the NBM was substantiated by forming a confluent connective tissue entity, which was concomitant with a stratified epithelial equivalent. Immunohistochemistry proved tissue authenticity over time by abundance of the biomarker vimentin in the connective tissue entity, and chronological increase of keratins KRT1/10 and involucrin expression in epithelial equivalents. Suitability of the novel NBM as wound dressing was evidenced by an almost completion of epithelial wound closure in a pilot mini-pig study, after a surgical intervention-caused gingival dehiscence. In summary, preclinical assessment by tissue-authentic cell systems and the animal pilot study revealed the NBM as an encouraging therapeutic medical device for prospective clinical applications. PMID:24494668

Application of the ideas and techniques of classical fluid mechanics to some problems in physical oceanography.

PubMed

Johnson, R S

2018-01-28

This review makes a case for describing many of the flows observed in our oceans, simply based on the Euler equation, with (piecewise) constant density and with suitable boundary conditions. The analyses start from the Euler and mass conservation equations, expressed in a rotating, spherical coordinate system (but the f -plane and β -plane approximations are also mentioned); five examples are discussed. For three of them, a suitable non-dimensionalization is introduced, and a single small parameter is identified in each case. These three examples lead straightforwardly and directly to new results for: waves on the Pacific Equatorial Undercurrent (EUC) with a thermocline (in the f -plane); a nonlinear, three-dimensional model for EUC-type flows (in the β -plane); and a detailed model for large gyres. The other two examples are exact solutions of the complete system: a flow which corresponds to the underlying structure of the Pacific EUC; and a flow based on the necessary requirement to use a non-conservative body force, which produces the type of flow observed in the Antarctic Circumpolar Current. (All these examples have been discussed in detail in the references cited.) This review concludes with a few comments on how these solutions can be extended and expanded.This article is part of the theme issue 'Nonlinear water waves'. © 2017 The Author(s).

A planning algorithm for quantifying decentralised water management opportunities in urban environments.

PubMed

Bach, Peter M; McCarthy, David T; Urich, Christian; Sitzenfrei, Robert; Kleidorfer, Manfred; Rauch, Wolfgang; Deletic, Ana

2013-01-01

With global change bringing about greater challenges for the resilient planning and management of urban water infrastructure, research has been invested in the development of a strategic planning tool, DAnCE4Water. The tool models how urban and societal changes impact the development of centralised and decentralised (distributed) water infrastructure. An algorithm for rigorous assessment of suitable decentralised stormwater management options in the model is presented and tested on a local Melbourne catchment. Following detailed spatial representation algorithms (defined by planning rules), the model assesses numerous stormwater options to meet water quality targets at a variety of spatial scales. A multi-criteria assessment algorithm is used to find top-ranking solutions (which meet a specific treatment performance for a user-defined percentage of catchment imperviousness). A toolbox of five stormwater technologies (infiltration systems, surface wetlands, bioretention systems, ponds and swales) is featured. Parameters that set the algorithm's flexibility to develop possible management options are assessed and evaluated. Results are expressed in terms of 'utilisation', which characterises the frequency of use of different technologies across the top-ranking options (bioretention being the most versatile). Initial results highlight the importance of selecting a suitable spatial resolution and providing the model with enough flexibility for coming up with different technology combinations. The generic nature of the model enables its application to other urban areas (e.g. different catchments, local municipal regions or entire cities).

Reference gene selection for quantitative reverse transcription-polymerase chain reaction normalization during in vitro adventitious rooting in Eucalyptus globulus Labill.

PubMed

de Almeida, Márcia R; Ruedell, Carolina M; Ricachenevsky, Felipe K; Sperotto, Raul A; Pasquali, Giancarlo; Fett-Neto, Arthur G

2010-09-20

Eucalyptus globulus and its hybrids are very important for the cellulose and paper industry mainly due to their low lignin content and frost resistance. However, rooting of cuttings of this species is recalcitrant and exogenous auxin application is often necessary for good root development. To date one of the most accurate methods available for gene expression analysis is quantitative reverse transcription-polymerase chain reaction (qPCR); however, reliable use of this technique requires reference genes for normalization. There is no single reference gene that can be regarded as universal for all experiments and biological materials. Thus, the identification of reliable reference genes must be done for every species and experimental approach. The present study aimed at identifying suitable control genes for normalization of gene expression associated with adventitious rooting in E. globulus microcuttings. By the use of two distinct algorithms, geNorm and NormFinder, we have assessed gene expression stability of eleven candidate reference genes in E. globulus: 18S, ACT2, EF2, EUC12, H2B, IDH, SAND, TIP41, TUA, UBI and 33380. The candidate reference genes were evaluated in microccuttings rooted in vitro, in presence or absence of auxin, along six time-points spanning the process of adventitious rooting. Overall, the stability profiles of these genes determined with each one of the algorithms were very similar. Slight differences were observed in the most stable pair of genes indicated by each program: IDH and SAND for geNorm, and H2B and TUA for NormFinder. Both programs identified UBI and 18S as the most variable genes. To validate these results and select the most suitable reference genes, the expression profile of the ARGONAUTE1 gene was evaluated in relation to the most stable candidate genes indicated by each algorithm. Our study showed that expression stability varied between putative reference genes tested in E. globulus. Based on the AGO1 relative expression profile obtained using the genes suggested by the algorithms, H2B and TUA were considered as the most suitable reference genes for expression studies in E. globulus adventitious rooting. UBI and 18S were unsuitable for use as controls in qPCR related to this process. These findings will enable more accurate and reliable normalization of qPCR results for gene expression studies in this economically important woody plant, particularly related to rooting and clonal propagation.

Reference gene selection for quantitative reverse transcription-polymerase chain reaction normalization during in vitro adventitious rooting in Eucalyptus globulus Labill

PubMed Central

2010-01-01

Background Eucalyptus globulus and its hybrids are very important for the cellulose and paper industry mainly due to their low lignin content and frost resistance. However, rooting of cuttings of this species is recalcitrant and exogenous auxin application is often necessary for good root development. To date one of the most accurate methods available for gene expression analysis is quantitative reverse transcription-polymerase chain reaction (qPCR); however, reliable use of this technique requires reference genes for normalization. There is no single reference gene that can be regarded as universal for all experiments and biological materials. Thus, the identification of reliable reference genes must be done for every species and experimental approach. The present study aimed at identifying suitable control genes for normalization of gene expression associated with adventitious rooting in E. globulus microcuttings. Results By the use of two distinct algorithms, geNorm and NormFinder, we have assessed gene expression stability of eleven candidate reference genes in E. globulus: 18S, ACT2, EF2, EUC12, H2B, IDH, SAND, TIP41, TUA, UBI and 33380. The candidate reference genes were evaluated in microccuttings rooted in vitro, in presence or absence of auxin, along six time-points spanning the process of adventitious rooting. Overall, the stability profiles of these genes determined with each one of the algorithms were very similar. Slight differences were observed in the most stable pair of genes indicated by each program: IDH and SAND for geNorm, and H2B and TUA for NormFinder. Both programs indentified UBI and 18S as the most variable genes. To validate these results and select the most suitable reference genes, the expression profile of the ARGONAUTE1 gene was evaluated in relation to the most stable candidate genes indicated by each algorithm. Conclusion Our study showed that expression stability varied between putative reference genes tested in E. globulus. Based on the AGO1 relative expression profile obtained using the genes suggested by the algorithms, H2B and TUA were considered as the most suitable reference genes for expression studies in E. globulus adventitious rooting. UBI and 18S were unsuitable for use as controls in qPCR related to this process. These findings will enable more accurate and reliable normalization of qPCR results for gene expression studies in this economically important woody plant, particularly related to rooting and clonal propagation. PMID:20854682

Quantification of heat shock protein mRNA expression in warm and cold anoxic turtles (Trachemys scripta) using an external RNA control for normalization.

PubMed

Stecyk, Jonathan A W; Couturier, Christine S; Fagernes, Cathrine E; Ellefsen, Stian; Nilsson, Göran E

2012-03-01

The mRNA expression of heat-shock protein 90 (HSP90) and heat-shock cognate 70 (HSC70) was examined in cardiac chambers and telencephalon of warm- (21°C) and cold-acclimated (5°C) turtles (Trachemys scripta) exposed to normoxia, prolonged anoxia or anoxia followed by reoxygenation. Additionally, the suitability of total RNA as well as mRNA from β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and cyclophilin A (PPIA) for normalizing gene expression data was assessed, as compared to the use of an external RNA control. Measurements of HSP90 and HSC70 mRNA expression revealed that anoxia and reoxygenation have tissue- and gene-specific effects. By and large, the alterations support previous investigations on HSP protein abundance in the anoxic turtle heart and brain, as well as the hypothesized roles of HSP90 and HSC70 during stress and non-stress conditions. However, more prominent was a substantially increased HSP90 and HSC70 mRNA expression in the cardiac chambers with cold acclimation. The finding provides support for the notion that cold temperature induces a number of adaptations in tissues of anoxia-tolerant vertebrates that precondition them for winter anoxia. β-actin, GAPDH and PPIA mRNA expression and total RNA also varied with oxygenation state and acclimation temperature in a tissue- and gene-specific manner, as well as among tissue types, thus disqualifying them as suitable for real-time RT-PCR normalization. Thus, the present data highlights the advantages of normalizing real-time RT-PCR data to an external RNA control, an approach that also allows inter-tissue and potentially inter-species comparisons of target gene expression. Copyright © 2011 Elsevier Inc. All rights reserved.

Caco-2 cells - expression, regulation and function of drug transporters compared with human jejunal tissue.

PubMed

Brück, S; Strohmeier, J; Busch, D; Drozdzik, M; Oswald, S

2017-03-01

Induction or inhibition of drug transporting proteins by concomitantly administered drugs can cause serious drug-drug interactions (DDIs). However, in vitro assays currently available are mostly for studying the inhibitory potential of drugs on intestinal transporter proteins, rather than induction. Therefore, this study investigated the suitability of the frequently used intestinal Caco-2 cell line to predict transporter-mediated DDIs as caused by induction via activation of nuclear receptors. TaqMan® low density arrays and LC-MS/MS based targeted proteomics were used to evaluate transporter expression in Caco-2 cells in comparison with jejunal tissue, in culture-time dependence studies and after incubation with different known inducers of drug metabolism and transport. Additionally, studies on ABCB1 function were performed using Transwell® assays with [ 3 H]-digoxin and [ 3 H]-talinolol as substrates after incubation with the prototypical inducers rifampicin, St John's wort, carbamazepine and efavirenz. The gene and protein expression pattern of drug transporters in Caco-2 cells and jejunal tissue differed considerably. For some transporters culture-time dependent differences in mRNA expression and/or protein abundance could be determined. Finally, none of the studied prototypical inducers showed an effect either on mRNA expression and protein abundance or on the function of ABCB1. Differences in transporter expression in Caco-2 cells compared with jejunal tissue, as well as expression dependence on culture time must be considered in in vitro studies to avoid under- or overestimation of certain transporters. The Caco-2 cell model is not suitable for the evaluation of DDIs caused by transporter induction. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Evaluation and Validation of Reference Genes for qRT-PCR Normalization in Frankliniella occidentalis (Thysanoptera:Thripidae)

PubMed Central

Zheng, Yu-Tao; Li, Hong-Bo; Lu, Ming-Xing; Du, Yu-Zhou

2014-01-01

Quantitative real time PCR (qRT-PCR) has emerged as a reliable and reproducible technique for studying gene expression analysis. For accurate results, the normalization of data with reference genes is particularly essential. Once the transcriptome sequencing of Frankliniella occidentalis was completed, numerous unigenes were identified and annotated. Unfortunately, there are no studies on the stability of reference genes used in F. occidentalis. In this work, seven candidate reference genes, including actin, 18S rRNA, H3, tubulin, GAPDH, EF-1 and RPL32, were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs BestKeeper, geNorm, Normfinder and the comparative ΔCt method. Because the rankings of the reference genes provided by each of the four programs were different, we chose a user-friendly web-based comprehensive tool RefFinder to get the final ranking. The result demonstrated that EF-1 and RPL32 displayed the most stable expression in different developmental stages; RPL32 and GAPDH showed the most stable expression at high temperatures, while 18S and EF-1 exhibited the most stable expression at low temperatures. In this study, we validated the suitable reference genes in F. occidentalis for gene expression profiling under different experimental conditions. The choice of internal standard is very important in the normalization of the target gene expression levels, thus validating and selecting the best genes will help improve the quality of gene expression data of F. occidentalis. What is more, these validated reference genes could serve as the basis for the selection of candidate reference genes in other insects. PMID:25356721

Evaluation and validation of reference genes for qRT-PCR normalization in Frankliniella occidentalis (Thysanoptera: Thripidae).

PubMed

Zheng, Yu-Tao; Li, Hong-Bo; Lu, Ming-Xing; Du, Yu-Zhou

2014-01-01

Quantitative real time PCR (qRT-PCR) has emerged as a reliable and reproducible technique for studying gene expression analysis. For accurate results, the normalization of data with reference genes is particularly essential. Once the transcriptome sequencing of Frankliniella occidentalis was completed, numerous unigenes were identified and annotated. Unfortunately, there are no studies on the stability of reference genes used in F. occidentalis. In this work, seven candidate reference genes, including actin, 18S rRNA, H3, tubulin, GAPDH, EF-1 and RPL32, were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs BestKeeper, geNorm, Normfinder and the comparative ΔCt method. Because the rankings of the reference genes provided by each of the four programs were different, we chose a user-friendly web-based comprehensive tool RefFinder to get the final ranking. The result demonstrated that EF-1 and RPL32 displayed the most stable expression in different developmental stages; RPL32 and GAPDH showed the most stable expression at high temperatures, while 18S and EF-1 exhibited the most stable expression at low temperatures. In this study, we validated the suitable reference genes in F. occidentalis for gene expression profiling under different experimental conditions. The choice of internal standard is very important in the normalization of the target gene expression levels, thus validating and selecting the best genes will help improve the quality of gene expression data of F. occidentalis. What is more, these validated reference genes could serve as the basis for the selection of candidate reference genes in other insects.

Intraoperative Sentinel Lymph Node Evaluation: Implications of Cytokeratin 19 Expression for the Adoption of OSNA in Oral Squamous Cell Carcinoma.

PubMed

Shaw, Richard; Christensen, Anders; Java, Kapil; Maddani, Rehab El; Liloglou, Triantafillos; Asterios, Triantafyllou; von Buchwald, Christian; Wessel, Irene; Kiss, Katalin; Kjaer, Andreas; Lelkaitis, Giedrius; Long, Anna; Risk, Janet; Robinson, Max

2016-11-01

Intraoperative analysis of sentinel lymph nodes would enhance the care of early-stage oral squamous cell carcinoma (OSCC). We determined the frequency and extent of cytokeratin 19 (CK19) expression in OSCC primary tumours and surrounding tissues to explore the feasibility of a "clinic-ready" intraoperative diagnostic test (one step nucleic acid amplification-OSNA, sysmex). Two cohorts were assembled: cohort 1, OSCC with stage and site that closely match cases suitable for sentinel lymph node biopsy (SLNB); cohort 2, HNSCC with sufficient fresh tumour tissue available for the OSNA assay (>50 mg). CK19 assays included qRT-PCR, RNA in situ hybridisation (ISH), and immunohistochemistry (IHC), as well as OSNA. CK19 mRNA expression was detected with variable sensitivity, depending on method, in 60-80% of primary OSCC tumours, while protein expression was observed in only 50% of tumours. Discordance between different techniques indicated that OSNA was more sensitive than qRT-PCR or RNA-ISH, which in turn were more sensitive than IHC. OSNA results showed CK19 expression in 80% of primary cases, so if used for diagnosis of lymph node metastasis would lead to a false-negative result in 20% of patients with cervical lymph node metastases. OSNA in its current form is not suitable for use in OSCC SLNB due to inadequate expression of the CK19 target in all case. However, the same assay technology would likely be very promising if applied using a more ubiquitous squamous epithelial target.

Information Management for Unmanned Systems: Combining DL-Reasoning with Publish/Subscribe

NASA Astrophysics Data System (ADS)

Moser, Herwig; Reichelt, Toni; Oswald, Norbert; Förster, Stefan

Sharing capabilities and information between collaborating entities by using modem information- and communication-technology is a core principle in complex distributed civil or military mission scenarios. Previous work proved the suitability of Service-oriented Architectures for modelling and sharing the participating entities' capabilities. Albeit providing a satisfactory model for capabilities sharing, pure service-orientation curtails expressiveness for information exchange as opposed to dedicated data-centric communication principles. In this paper we introduce an Information Management System which combines OWL-Ontologies and automated reasoning with Publish/Subscribe-Systems, providing for a shared but decoupled data model. While confirming existing related research results, we emphasise the novel application and lack of practical experience of using Semantic Web technologies in areas other than originally intended. That is, aiding decision support and software design in the context of a mission scenario for an unmanned system. Experiments within a complex simulation environment show the immediate benefits of a semantic information-management and -dissemination platform: Clear separation of concerns in code and data model, increased service re-usability and extensibility as well as regulation of data flow and respective system behaviour through declarative rules.

An In Vitro Expansion System for Generation of Human iPS Cell-Derived Hepatic Progenitor-Like Cells Exhibiting a Bipotent Differentiation Potential

PubMed Central

Yanagida, Ayaka; Ito, Keiichi; Chikada, Hiromi; Nakauchi, Hiromitsu; Kamiya, Akihide

2013-01-01

Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13highCD133+ cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development. PMID:23935837

An in vitro expansion system for generation of human iPS cell-derived hepatic progenitor-like cells exhibiting a bipotent differentiation potential.

PubMed

Yanagida, Ayaka; Ito, Keiichi; Chikada, Hiromi; Nakauchi, Hiromitsu; Kamiya, Akihide

2013-01-01

Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13(high)CD133(+) cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development.

Multi-Agent Architecture with Support to Quality of Service and Quality of Control

NASA Astrophysics Data System (ADS)

Poza-Luján, Jose-Luis; Posadas-Yagüe, Juan-Luis; Simó-Ten, Jose-Enrique

Multi Agent Systems (MAS) are one of the most suitable frameworks for the implementation of intelligent distributed control system. Agents provide suitable flexibility to give support to implied heterogeneity in cyber-physical systems. Quality of Service (QoS) and Quality of Control (QoC) parameters are commonly utilized to evaluate the efficiency of the communications and the control loop. Agents can use the quality measures to take a wide range of decisions, like suitable placement on the control node or to change the workload to save energy. This article describes the architecture of a multi agent system that provides support to QoS and QoC parameters to optimize de system. The architecture uses a Publish-Subscriber model, based on Data Distribution Service (DDS) to send the control messages. Due to the nature of the Publish-Subscribe model, the architecture is suitable to implement event-based control (EBC) systems. The architecture has been called FSACtrl.

Microencapsulated equine mesenchymal stromal cells promote cutaneous wound healing in vitro.

PubMed

Bussche, Leen; Harman, Rebecca M; Syracuse, Bethany A; Plante, Eric L; Lu, Yen-Chun; Curtis, Theresa M; Ma, Minglin; Van de Walle, Gerlinde R

2015-04-11

The prevalence of impaired cutaneous wound healing is high and treatment is difficult and often ineffective, leading to negative social and economic impacts for our society. Innovative treatments to improve cutaneous wound healing by promoting complete tissue regeneration are therefore urgently needed. Mesenchymal stromal cells (MSCs) have been reported to provide paracrine signals that promote wound healing, but (i) how they exert their effects on target cells is unclear and (ii) a suitable delivery system to supply these MSC-derived secreted factors in a controlled and safe way is unavailable. The present study was designed to provide answers to these questions by using the horse as a translational model. Specifically, we aimed to (i) evaluate the in vitro effects of equine MSC-derived conditioned medium (CM), containing all factors secreted by MSCs, on equine dermal fibroblasts, a cell type critical for successful wound healing, and (ii) explore the potential of microencapsulated equine MSCs to deliver CM to wounded cells in vitro. MSCs were isolated from the peripheral blood of healthy horses. Equine dermal fibroblasts from the NBL-6 (horse dermal fibroblast cell) line were wounded in vitro, and cell migration and expression levels of genes involved in wound healing were evaluated after treatment with MSC-CM or NBL-6-CM. These assays were repeated by using the CM collected from MSCs encapsulated in core-shell hydrogel microcapsules. Our salient findings were that equine MSC-derived CM stimulated the migration of equine dermal fibroblasts and increased their expression level of genes that positively contribute to wound healing. In addition, we found that equine MSCs packaged in core-shell hydrogel microcapsules had similar effects on equine dermal fibroblast migration and gene expression, indicating that microencapsulation of MSCs does not interfere with the release of bioactive factors. Our results demonstrate that the use of CM from MSCs might be a promising new therapy for impaired cutaneous wounds and that encapsulation may be a suitable way to effectively deliver CM to wounded cells in vivo.

The United States Revised Uniform Anatomical Gift Act (2006): new challenges to balancing patient rights and physician responsibilities.

PubMed

Verheijde, Joseph L; Rady, Mohamed Y; McGregor, Joan L

2007-09-12

Advance health care directives and informed consent remain the cornerstones of patients' right to self-determination regarding medical care and preferences at the end-of-life. However, the effectiveness and clinical applicability of advance health care directives to decision-making on the use of life support systems at the end-of-life is questionable. The Uniform Anatomical Gift Act (UAGA) has been revised in 2006 to permit the use of life support systems at or near death for the purpose of maximizing procurement opportunities of organs medically suitable for transplantation. Some states have enacted the Revised UAGA (2006) and a few of those have included amendments while attempting to preserve the uniformity of the revised Act. Other states have introduced the Revised UAGA (2006) for legislation and remaining states are likely to follow soon. The Revised UAGA (2006) poses challenges to the Patient Self Determination Act (PSDA) embodied in advance health care directives and individual expression about the use of life support systems at the end-of-life. The challenges are predicated on the UAGA revising the default choice to presumption of donation intent and the use of life support systems to ensure medical suitability of organs for transplantation. The default choice trumps the expressed intent in an individual's advance health care directive to withhold and/or withdraw life support systems at the end-of-life. The Revised UAGA (2006) overrides advance directives on utilitarian grounds, which is a serious ethical challenge to society. The subtle progression of the Revised UAGA (2006) towards the presumption about how to dispose of one's organs at death can pave the way for an affirmative "duty to donate". There are at least two steps required to resolve these challenges. First, physicians and hospitals must fulfill their responsibilities to educate patients on the new legislations and document their preferences about the use of life support systems for organ donation at the end-of-life. Second, a broad based societal discussion must be initiated to decide if the Revised UAGA (2006) infringes on the PSDA and the individual's right of autonomy. The discussion should also address other ethical concerns raised by the Revised UAGA (2006), including the moral stance on 1) the interpretation of the refusal of life support systems as not applicable to organ donation and 2) the disregarding of the diversity of cultural beliefs about end-of-life in a pluralistic society.

40 CFR 280.20 - Performance standards for new UST systems.

Code of Federal Regulations, 2010 CFR

2010-07-01

... protected in the following manner: (i) The tank is coated with a suitable dielectric material; (ii) Field... suitable dielectric material; (ii) Field-installed cathodic protection systems are designed by a corrosion...

40 CFR 280.20 - Performance standards for new UST systems.

Code of Federal Regulations, 2011 CFR

2011-07-01

... protected in the following manner: (i) The tank is coated with a suitable dielectric material; (ii) Field... suitable dielectric material; (ii) Field-installed cathodic protection systems are designed by a corrosion...

A lentiviral vector with a short troponin-I promoter for tracking cardiomyocyte differentiation of human embryonic stem cells.

PubMed

Gallo, P; Grimaldi, S; Latronico, M V G; Bonci, D; Pagliuca, A; Gallo, P; Ausoni, S; Peschle, C; Condorelli, G

2008-02-01

Human embryonic stem cells (hESCs) may become important for cardiac repair due to their potentially unlimited ability to generate cardiomyocytes (CMCs). Moreover, genetic manipulation of hESC-derived CMCs would be a very promising technique for curing myocardial disorders. At the present time, however, inducing the differentiation of hESCs into CMCs is extremely difficult and, therefore, an easy and standardizable technique is needed to evaluate differentiation strategies. Vectors driving cardiac-specific expression may represent an important tool not only for monitoring new cardiac-differentiation strategies, but also for the manipulation of cardiac differentiation of ESCs. To this aim, we generated cardiac-specific lentiviral vectors (LVVs) in which expression is driven by a short fragment of the cardiac troponin-I proximal promoter (TNNI3) with a human cardiac alpha-actin enhancer, and tested its suitability in inducing tissue-specific gene expression and ability to track the CMC lineage during differentiation of ESCs. We determined that (1) TNNI3-LVVs efficiently drive cardiac-specific gene expression and mark the cardiomyogenic lineage in human and mouse ESC differentiation systems (2) the cardiac alpha-actin enhancer confers a further increase in gene-expression specificity of TNNI3-LVVs in hESCs. Although this technique may not be useful in tracking small numbers of cells, data suggested that TNNI3-based LVVs are a powerful tool for manipulating human ESCs and modifying hESC-derived CMCs.

Plant-produced anti-dengue virus monoclonal antibodies exhibit reduced antibody-dependent enhancement of infection activity.

PubMed

Dent, Matthew; Hurtado, Jonathan; Paul, Amber M; Sun, Haiyan; Lai, Huafang; Yang, Ming; Esqueda, Adrian; Bai, Fengwei; Steinkellner, Herta; Chen, Qiang

2016-12-01

The mAb E60 has the potential to be a desirable therapeutic molecule since it efficiently neutralizes all four serotypes of dengue virus (DENV). However, mammalian-cell-produced E60 exhibits antibody-dependent enhancement of infection (ADE) activity, rendering it inefficacious in vivo, and treated animals more susceptible to developing more severe diseases during secondary infection. In this study, we evaluated a plant-based expression system for the production of therapeutically suitable E60. The mAb was transiently expressed in Nicotiana benthamianaWT and a ∆XFT line, a glycosylation mutant lacking plant-specific N-glycan residues. The mAb was efficiently expressed and assembled in leaves and exhibited highly homogenous N-glycosylation profiles, i.e. GnGnXF3 or GnGn structures, depending on the expression host. Both E60 glycovariants demonstrated equivalent antigen-binding specificity and in vitro neutralization potency against DENV serotypes 2 and 4 compared with their mammalian-cell-produced counterpart. By contrast, plant-produced E60 exhibited reduced ADE activity in Fc gamma receptor expressing human cells. Our results suggest the ability of plant-produced antibodies to minimize ADE, which may lead to the development of safe and highly efficacious antibody-based therapeutics against DENV and other ADE-prone viral diseases. Our study provides so far unknown insight into the relationship between mAb N-glycosylation and ADE, which contributes to our understanding of how sugar moieties of antibodies modulate Fc-mediated functions and viral pathogenesis.

Characterization of Human Aspartoacylase: the brain enzyme responsible for Canavan disease†

PubMed Central

Le Coq, Johanne; An, Hyun-Joo; Lebrilla, Carlito; Viola, Ronald E.

2008-01-01

Aspartoacylase catalyzes the deacetylation of N-acetylaspartic acid (NAA) to produce acetate and L-aspartate, and is the only brain enzyme that has been shown to effectively metabolize NAA. Although the exact role of this enzymatic reaction has not yet been completely elucidated, the metabolism of NAA appears to be necessary in the formation of myelin lipids and defects in this enzyme lead to Canavan disease, a fatal neurological disorder. The low catalytic activity and inherent instability observed with the Escherichia coli-expressed form of aspartoacylase suggested the need for a suitable eukaryotic expression system that would be capable of producing a fully functional, mature enzyme. Human aspartoacylase has now been successfully expressed in Pichia pastoris. While the expression yields are lower than in E. coli, the purified enzyme is significantly more stable. This enzyme form has the same substrate specificity, but is 150-fold more active than the E. coli-expressed enzyme. The molecular weight of the purified enzyme, measured by mass spectrometry, is higher than predicted, suggesting the presence of some posttranslational modifications. Deglycosylation of aspartoacylase or mutation at the glycosylation site causes decreased enzyme stability and diminished catalytic activity. A carbohydrate component has been removed and characterized by mass spectrometry. In addition to this carbohydrate moiety, the enzyme has also been shown to contain one zinc atom per subunit. Chelation studies to remove the zinc results in a reversible loss of catalytic activity, thus establishing aspartoacylase as a zinc metalloenzyme. PMID:16669630

Characterization of human aspartoacylase: the brain enzyme responsible for Canavan disease.

PubMed

Le Coq, Johanne; An, Hyun-Joo; Lebrilla, Carlito; Viola, Ronald E

2006-05-09

Aspartoacylase catalyzes the deacetylation of N-acetylaspartic acid (NAA) to produce acetate and L-aspartate and is the only brain enzyme that has been shown to effectively metabolize NAA. Although the exact role of this enzymatic reaction has not yet been completely elucidated, the metabolism of NAA appears to be necessary in the formation of myelin lipids, and defects in this enzyme lead to Canavan disease, a fatal neurological disorder. The low catalytic activity and inherent instability observed with the Escherichia coli-expressed form of aspartoacylase suggested the need for a suitable eukaryotic expression system that would be capable of producing a fully functional, mature enzyme. Human aspartoacylase has now been successfully expressed in Pichia pastoris. While the expression yields are lower than in E. coli, the purified enzyme is significantly more stable. This enzyme form has the same substrate specificity but is 150-fold more active than the E. coli-expressed enzyme. The molecular weight of the purified enzyme, measured by mass spectrometry, is higher than predicted, suggesting the presence of some post-translational modifications. Deglycosylation of aspartoacylase or mutation at the glycosylation site causes decreased enzyme stability and diminished catalytic activity. A carbohydrate component has been removed and characterized by mass spectrometry. In addition to this carbohydrate moiety, the enzyme has also been shown to contain one zinc atom per subunit. Chelation studies to remove the zinc result in a reversible loss of catalytic activity, thus establishing aspartoacylase as a zinc metalloenzyme.

Optical Silencing of C. elegans Cells with Arch Proton Pump

PubMed Central

Okazaki, Ayako; Sudo, Yuki; Takagi, Shin

2012-01-01

Background Optogenetic techniques using light-driven ion channels or ion pumps for controlling excitable cells have greatly facilitated the investigation of nervous systems in vivo. A model organism, C. elegans, with its small transparent body and well-characterized neural circuits, is especially suitable for optogenetic analyses. Methodology/Principal Findings We describe the application of archaerhodopsin-3 (Arch), a recently reported optical neuronal silencer, to C. elegans. Arch::GFP expressed either in all neurons or body wall muscles of the entire body by means of transgenes were localized, at least partially, to the cell membrane without adverse effects, and caused locomotory paralysis of worms when illuminated by green light (550 nm). Pan-neuronal expression of Arch endowed worms with quick and sustained responsiveness to such light. Worms reliably responded to repeated periods of illumination and non-illumination, and remained paralyzed under continuous illumination for 30 seconds. Worms expressing Arch in different subsets of motor neurons exhibited distinct defects in the locomotory behavior under green light: selective silencing of A-type motor neurons affected backward movement while silencing of B-type motor neurons affected forward movement more severely. Our experiments using a heat-shock-mediated induction system also indicate that Arch becomes fully functional only 12 hours after induction and remains functional for more than 24 hour. Conclusions/Sgnificance Arch can be used for silencing neurons and muscles, and may be a useful alternative to currently widely used halorhodopsin (NpHR) in optogenetic studies of C. elegans. PMID:22629299

Studying the effects of genistein on gene expression of fish embryos as an alternative testing approach for endocrine disruption.

PubMed

Schiller, Viktoria; Wichmann, Arne; Kriehuber, Ralf; Muth-Köhne, Elke; Giesy, John P; Hecker, Markus; Fenske, Martina

2013-01-01

Assessment of endocrine disruption currently relies on testing strategies involving adult vertebrates. In order to minimize the use of animal tests according to the 3Rs principle of replacement, reduction and refinement, we propose a transcriptomics and fish embryo based approach as an alternative to identify and analyze an estrogenic activity of environmental chemicals. For this purpose, the suitability of 48 h and 7 days post-fertilization zebrafish and medaka embryos to test for estrogenic disruption was evaluated. The embryos were exposed to the phytoestrogen genistein and subsequently analyzed by microarrays and quantitative real-time PCR. The functional analysis showed that the genes affected related to multiple metabolic and signaling pathways in the early fish embryo, which reflect the known components of genistein's mode of actions, like apoptosis, estrogenic response, hox gene expression and steroid hormone synthesis. Moreover, the transcriptomic data also suggested a thyroidal mode of action and disruption of the nervous system development. The parallel testing of two fish species provided complementary data on the effects of genistein at gene expression level and facilitated the separation of common from species-dependent effects. Overall, the study demonstrated that combining fish embryo testing with transcriptomics can deliver abundant information about the mechanistic effects of endocrine disrupting chemicals, rendering this strategy a promising alternative approach to test for endocrine disruption in a whole organism in-vitro scale system. Copyright © 2012 Elsevier Inc. All rights reserved.

Analytical results for the time-dependent current density distribution of expanding ultracold gases after a sudden change of the confining potential

NASA Astrophysics Data System (ADS)

Boumaza, R.; Bencheikh, K.

2017-12-01

Using the so-called operator product expansion to lowest order, we extend the work in Campbell et al (2015 Phys. Rev. Lett 114 125302) by deriving a simple analytical expression for the long-time asymptotic one-body reduced density matrix during free expansion for a one-dimensional system of bosons with large atom number interacting through a repulsive delta potential initially confined by a potential well. This density matrix allows direct access to the momentum distribution and also to the mass current density. For initially confining power-law potentials we give explicit expressions, in the limits of very weak and very strong interaction, for the current density distributions during the free expansion. In the second part of the work we consider the expansion of ultracold gas from a confining harmonic trap to another harmonic trap with a different frequency. For the case of a quantum impenetrable gas of bosons (a Tonks-Girardeau gas) with a given atom number, we present an exact analytical expression for the mass current distribution (mass transport) after release from one harmonic trap to another harmonic trap. It is shown that, for a harmonically quenched Tonks-Girardeau gas, the current distribution is a suitable collective observable and under the weak quench regime, it exhibits oscillations at the same frequencies as those recently predicted for the peak momentum distribution in the breathing mode. The analysis is extended to other possible quenched systems.

Role of indirect readout mechanism in TATA box binding protein-DNA interaction.

PubMed

Mondal, Manas; Choudhury, Devapriya; Chakrabarti, Jaydeb; Bhattacharyya, Dhananjay

2015-03-01

Gene expression generally initiates from recognition of TATA-box binding protein (TBP) to the minor groove of DNA of TATA box sequence where the DNA structure is significantly different from B-DNA. We have carried out molecular dynamics simulation studies of TBP-DNA system to understand how the DNA structure alters for efficient binding. We observed rigid nature of the protein while the DNA of TATA box sequence has an inherent flexibility in terms of bending and minor groove widening. The bending analysis of the free DNA and the TBP bound DNA systems indicate presence of some similar structures. Principal coordinate ordination analysis also indicates some structural features of the protein bound and free DNA are similar. Thus we suggest that the DNA of TATA box sequence regularly oscillates between several alternate structures and the one suitable for TBP binding is induced further by the protein for proper complex formation.

Dynamical phase transitions at finite temperature from fidelity and interferometric Loschmidt echo induced metrics

NASA Astrophysics Data System (ADS)

Mera, Bruno; Vlachou, Chrysoula; Paunković, Nikola; Vieira, Vítor R.; Viyuela, Oscar

2018-03-01

We study finite-temperature dynamical quantum phase transitions (DQPTs) by means of the fidelity and the interferometric Loschmidt echo (LE) induced metrics. We analyze the associated dynamical susceptibilities (Riemannian metrics), and derive analytic expressions for the case of two-band Hamiltonians. At zero temperature, the two quantities are identical, nevertheless, at finite temperatures they behave very differently. Using the fidelity LE, the zero-temperature DQPTs are gradually washed away with temperature, while the interferometric counterpart exhibits finite-temperature phase transitions. We analyze the physical differences between the two finite-temperature LE generalizations, and argue that, while the interferometric one is more sensitive and can therefore provide more information when applied to genuine quantum (microscopic) systems, when analyzing many-body macroscopic systems, the fidelity-based counterpart is a more suitable quantity to study. Finally, we apply the previous results to two representative models of topological insulators in one and two dimensions.

Research on channel characteristics of differential multi pulse position modulation without background noise

NASA Astrophysics Data System (ADS)

Gao, Zhuo; Zhan, Weida; Sun, Quan; Hao, Ziqiang

2018-04-01

Differential multi-pulse position modulation (DMPPM) is a new type of modulation technology. There is a fast transmission rate, high bandwidth utilization, high modulation rate characteristics. The study of DMPPM modulation has important scientific value and practical significance. Channel capacity is one of the important indexes to measure the communication capability of communication system, and studying the channel capacity of DMPPM without background noise is the key to analyze the characteristics of DMPPM. The DMPPM theoretical model is established. The symbol structure of DMPPM with guard time slot is analyzed, and the channel capacity expression of DMPPM is deduced. Simulation analysis by MATLAB. The curves of unit channel capacity and capacity efficiency at different pulse and photon counting rates are analyzed. The results show that DMPPM is more advantageous than multi-pulse position modulation (MPPM), and is more suitable for future wireless optical communication system.

Nest-Gallery Development and Caste Composition of Isolated Foraging Groups of the Drywood Termite, Incisitermes minor (Isoptera: Kalotermitidae)

PubMed Central

Himmi, S. Khoirul; Yoshimura, Tsuyoshi; Yanase, Yoshiyuki; Oya, Masao; Torigoe, Toshiyuki; Akada, Masanori; Imadzu, Setsuo

2016-01-01

An X-ray computed-tomographic examination of nest-gallery development from timbers naturally infested by foraging groups of Incisitermes minor colonies was conducted. This study documents the colonization process of I. minor to new timbers and how the isolated groups maintain their nest-gallery system. The results suggested that development of a nest-gallery within a suitable wood item is not random, but shows selection for softer substrate and other adaptations to the different timber environments. Stigmergic coordinations were expressed in dynamic changes of the nest-gallery system; indicated by fortification behavior in sealing and re-opening a tunnel approaching the outer edge of the timber, and accumulating fecal pellets in particular chambers located beneath the timber surface. The study also examines the caste composition of isolated groups to discover how I. minor sustains colonies with and without primary reproductives. PMID:27455332

Nonlinear stress waves in a perfectly flexible string. [for aerodynamic decelerating system

NASA Technical Reports Server (NTRS)

Fan, D.-N.; Mcgarvey, J. F.

1977-01-01

This paper discusses nonlinear stress-wave propagation in a perfectly flexible string obeying a quasilinear (rate-dependent) constitutive equation. Wave speeds and compatibility relations valid along various families of characteristics were determined. It was shown that the compatibility relations associated with the transverse as well as the longitudinal waves readily yield a physical interpretation when they are expressed in suitable variables and in vector form. Coding based on the present information was completed for the machine solution of a class of mixed initial- and boundary-value problems of practical interest. Computer simulation of the stress-wave interaction in the 40-foot lanyard in the Arcas 'Rocoz' system during deployment was carried out using a stress-strain relation for nylon at the strain rate of 30/second. A method for estimating the maximum tension and strain in a string during the initial loading phase is proposed.

Choice of fineness of pulverized coal

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

E.N. Tolchinskii; A.Yu. Lavrent'ev

2002-11-15

Various methods for choosing the fineness of power plant coal dust are reviewed and analytical expressions for determining the fineness are presented. It is shown that the use of the yield of combustibles as a parameter is not always suitable for evaluating the fineness of pulverized coal. The suggested expression for computing the fineness bears composite parameters that allow for the heat value of the volatiles and for the internal surface of the fuel particles.

Whole genome mRNA transcriptomics analysis reveals different modes of action of the diarrheic shellfish poisons okadaic acid and dinophysis toxin-1 versus azaspiracid-1 in Caco-2 cells.

PubMed

Bodero, Marcia; Hoogenboom, Ron L A P; Bovee, Toine F H; Portier, Liza; de Haan, Laura; Peijnenburg, Ad; Hendriksen, Peter J M

2018-02-01

A study with DNA microarrays was performed to investigate the effects of two diarrhetic and one azaspiracid shellfish poison, okadaic acid (OA), dinophysistoxin-1 (DTX-1) and azaspiracid-1 (AZA-1) respectively, on the whole-genome mRNA expression of undifferentiated intestinal Caco-2 cells. Previously, the most responding genes were used to develop a dedicated array tube test to screen shellfish samples on the presence of these toxins. In the present study the whole genome mRNA expression was analyzed in order to reveal modes of action and obtain hints on potential biomarkers suitable to be used in alternative bioassays. Effects on key genes in the most affected pathways and processes were confirmed by qPCR. OA and DTX-1 induced almost identical effects on mRNA expression, which strongly indicates that OA and DTX-1induce similar toxic effects. Biological interpretation of the microarray data indicates that both compounds induce hypoxia related pathways/processes, the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress. The gene expression profile of AZA-1 is different and shows increased mRNA expression of genes involved in cholesterol synthesis and glycolysis, suggesting a different mode of action for this toxin. Future studies should reveal whether identified pathways provide suitable biomarkers for rapid detection of DSPs in shellfish. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Transcriptome comparison of human neurons generated using induced pluripotent stem cells derived from dental pulp and skin fibroblasts.

PubMed

Chen, Jian; Lin, Mingyan; Foxe, John J; Pedrosa, Erika; Hrabovsky, Anastasia; Carroll, Reed; Zheng, Deyou; Lachman, Herbert M

2013-01-01

Induced pluripotent stem cell (iPSC) technology is providing an opportunity to study neuropsychiatric disorders through the capacity to grow patient-specific neurons in vitro. Skin fibroblasts obtained by biopsy have been the most reliable source of cells for reprogramming. However, using other somatic cells obtained by less invasive means would be ideal, especially in children with autism spectrum disorders (ASD) and other neurodevelopmental conditions. In addition to fibroblasts, iPSCs have been developed from cord blood, lymphocytes, hair keratinocytes, and dental pulp from deciduous teeth. Of these, dental pulp would be a good source for neurodevelopmental disorders in children because obtaining material is non-invasive. We investigated its suitability for disease modeling by carrying out gene expression profiling, using RNA-seq, on differentiated neurons derived from iPSCs made from dental pulp extracted from deciduous teeth (T-iPSCs) and fibroblasts (F-iPSCs). This is the first RNA-seq analysis comparing gene expression profiles in neurons derived from iPSCs made from different somatic cells. For the most part, gene expression profiles were quite similar with only 329 genes showing differential expression at a nominally significant p-value (p<0.05), of which 63 remained significant after correcting for genome-wide analysis (FDR <0.05). The most striking difference was the lower level of expression detected for numerous members of the all four HOX gene families in neurons derived from T-iPSCs. In addition, an increased level of expression was seen for several transcription factors expressed in the developing forebrain (FOXP2, OTX1, and LHX2, for example). Overall, pathway analysis revealed that differentially expressed genes that showed higher levels of expression in neurons derived from T-iPSCs were enriched for genes implicated in schizophrenia (SZ). The findings suggest that neurons derived from T-iPSCs are suitable for disease-modeling neuropsychiatric disorder and may have some advantages over those derived from F-iPSCs.

Three-dimensional culture of a mixed mullerian tumor of the ovary: expression of in vivo characteristics

NASA Technical Reports Server (NTRS)

Goodwin, T. J.; Prewett, T. L.; Spaulding, G. F.; Becker, J. L.

1997-01-01

The Rotating-Wall Vessel (RWV) is a novel in vitro cell culture system used to successfully culture a cell line derived from a heterologous mixed mullerian tumor cell of the ovary. Although the original tumor was comprised of both epithelial and mesodermal components, long-term culture in conventional flasks established a cell line from this tumor with homogeneous epitheliallike growth characteristics (1). Cells from Passage 36 were seeded into a Rotating-Wall Vessel containing Cytodex-3 microcarrier beads. Scanning electron micrographs of tumor cells cultured for 32 d in the RWV showed the presence of heterogeneous cell populations organized into three-dimensional tissuelike architecture. Immunocytochemical analysis confirmed the cellular heterogeneity, as demonstrated by expression of both epithelial and mesenchymal antigens. Reverse transcription polymerase chain reaction amplification demonstrated the presence of mRNA for cellular oncogenes HER-2/neu, H-ras, K-ras, and tumor suppressor p53. Thus, there are two advantages to propagation of tissue in the RWV culture system:(a) tissue diversification representing populations present in the original tumor, and (b) the three-dimensional freedom to organize tissues morphologically akin to those observed in vivo. These data indicate that the RWV culture system is suitable for generating large quantities of ovarian tumor cells in vitro that are amenable to immunocytochemical, oncogenic, morphologic characteristics demonstrated in vivo.

Design of a Type-1 Diabetes Vaccine Candidate Using Edible Plants Expressing a Major Autoantigen

PubMed Central

Bertini, Edoardo; Merlin, Matilde; Gecchele, Elisa; Puggia, Andrea; Brozzetti, Annalisa; Commisso, Mauro; Falorni, Alberto; Bini, Vittorio; Klymyuk, Victor; Pezzotti, Mario; Avesani, Linda

2018-01-01

Type-1 diabetes (T1D) is a metabolic disease involving the autoimmune destruction of insulin-producing pancreatic beta cells. It is often diagnosed by the detection of autoantibodies, typically those recognizing insulin itself or the 65-kDa isoform of glutamic acid decarboxylase (GAD65). Oral insulin can be used to induce systemic immunological tolerance and thus prevent or delay the onset of T1D, suggesting that combination treatments with other autoantigens such as GAD65 could be even more successful. GAD65 has induced oral tolerance and prevented T1D in preclinical studies but it is difficult to produce in sufficient quantities for clinical testing. Here we combined edible plant systems, namely spinach (Spinacia oleracea cv Industra) and red beet (Beta vulgaris cv Moulin Rouge), with the magnICON® expression system to develop a safe, cost-effective and environmentally sustainable platform for the large-scale production of GAD65. The superior red beet platform was extensively characterized in terms of recombinant protein yields and bioequivalence to wild-type plants, and the product was tested for its ability to resist simulated gastric digestion. Our results indicate that red beet plants are suitable for the production of a candidate oral vaccine based on GAD65 for the future preclinical and clinical testing of T1D immunotherapy approaches. PMID:29765386

Non-equilibrium thermodynamical description of rhythmic motion patterns of active systems: a canonical-dissipative approach.

PubMed

Dotov, D G; Kim, S; Frank, T D

2015-02-01

We derive explicit expressions for the non-equilibrium thermodynamical variables of a canonical-dissipative limit cycle oscillator describing rhythmic motion patterns of active systems. These variables are statistical entropy, non-equilibrium internal energy, and non-equilibrium free energy. In particular, the expression for the non-equilibrium free energy is derived as a function of a suitable control parameter. The control parameter determines the Hopf bifurcation point of the deterministic active system and describes the effective pumping of the oscillator. In analogy to the equilibrium free energy of the Landau theory, it is shown that the non-equilibrium free energy decays as a function of the control parameter. In doing so, a similarity between certain equilibrium and non-equilibrium phase transitions is pointed out. Data from an experiment on human rhythmic movements is presented. Estimates for pumping intensity as well as the thermodynamical variables are reported. It is shown that in the experiment the non-equilibrium free energy decayed when pumping intensity was increased, which is consistent with the theory. Moreover, pumping intensities close to zero could be observed at relatively slow intended rhythmic movements. In view of the Hopf bifurcation underlying the limit cycle oscillator model, this observation suggests that the intended limit cycle movements were actually more similar to trajectories of a randomly perturbed stable focus. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Strategies for engineering plant natural products: the iridoid-derived monoterpene indole alkaloids of Catharanthus roseus.

PubMed

O'Connor, Sarah E

2012-01-01

The manipulation of pathways to make unnatural variants of natural compounds, a process often termed combinatorial biosynthesis, has been robustly successful in prokaryotic systems. The development of approaches to generate new-to-nature compounds from plant-based pathways is, in comparison, much less advanced. Success will depend on the specific chemistry of the pathway, as well as on the suitability of the plant system for transformation and genetic manipulation. As plant pathways are elucidated, and can be heterologously expressed in hosts that are more amenable to genetic manipulation, biosynthetic production of new-to-nature compounds from plant pathways will become more widespread. In this chapter, some of the key strategies that have been developed for metabolic engineering of plant pathways, namely directed biosynthesis, mutasynthesis, and pathway incorporation of engineered enzymes are highlighted. The iridoid-derived monoterpene indole alkaloids from C. roseus, which are the focus of this chapter, provide an excellent system for developing these strategies. Copyright © 2012 Elsevier Inc. All rights reserved.

A mathematical representation of an advanced helicopter for piloted simulator investigations of control system and display variations

NASA Technical Reports Server (NTRS)

Aiken, E. W.

1980-01-01

A mathematical model of an advanced helicopter is described. The model is suitable for use in control/display research involving piloted simulation. The general design approach for the six degree of freedom equations of motion is to use the full set of nonlinear gravitational and inertial terms of the equations and to express the aerodynamic forces and moments as the reference values and first order terms of a Taylor series expansion about a reference trajectory defined as a function of longitudinal airspeed. Provisions for several different specific and generic flight control systems are included in the model. The logic required to drive various flight control and weapon delivery symbols on a pilot's electronic display is also provided. Finally, the model includes a simplified representation of low altitude wind and turbulence effects. This model was used in a piloted simulator investigation of the effects of control system and display variations for an attack helicopter mission.

Bioinspired sensory systems for local flow characterization

NASA Astrophysics Data System (ADS)

Colvert, Brendan; Chen, Kevin; Kanso, Eva

2016-11-01

Empirical evidence suggests that many aquatic organisms sense differential hydrodynamic signals.This sensory information is decoded to extract relevant flow properties. This task is challenging because it relies on local and partial measurements, whereas classical flow characterization methods depend on an external observer to reconstruct global flow fields. Here, we introduce a mathematical model in which a bioinspired sensory array measuring differences in local flow velocities characterizes the flow type and intensity. We linearize the flow field around the sensory array and express the velocity gradient tensor in terms of frame-independent parameters. We develop decoding algorithms that allow the sensory system to characterize the local flow and discuss the conditions under which this is possible. We apply this framework to the canonical problem of a circular cylinder in uniform flow, finding excellent agreement between sensed and actual properties. Our results imply that combining suitable velocity sensors with physics-based methods for decoding sensory measurements leads to a powerful approach for understanding and developing underwater sensory systems.

Porting the AVS/Express scientific visualization software to Cray XT4.

PubMed

Leaver, George W; Turner, Martin J; Perrin, James S; Mummery, Paul M; Withers, Philip J

2011-08-28

Remote scientific visualization, where rendering services are provided by larger scale systems than are available on the desktop, is becoming increasingly important as dataset sizes increase beyond the capabilities of desktop workstations. Uptake of such services relies on access to suitable visualization applications and the ability to view the resulting visualization in a convenient form. We consider five rules from the e-Science community to meet these goals with the porting of a commercial visualization package to a large-scale system. The application uses message-passing interface (MPI) to distribute data among data processing and rendering processes. The use of MPI in such an interactive application is not compatible with restrictions imposed by the Cray system being considered. We present details, and performance analysis, of a new MPI proxy method that allows the application to run within the Cray environment yet still support MPI communication required by the application. Example use cases from materials science are considered.

Mise en Scene: Conversion of Scenarios to CSP Traces for the Requirements-to-Design-to-Code Project

NASA Technical Reports Server (NTRS)

Carter. John D.; Gardner, William B.; Rash, James L.; Hinchey, Michael G.

2007-01-01

The "Requirements-to-Design-to-Code" (R2D2C) project at NASA's Goddard Space Flight Center is based on deriving a formal specification expressed in Communicating Sequential Processes (CSP) notation from system requirements supplied in the form of CSP traces. The traces, in turn, are to be extracted from scenarios, a user-friendly medium often used to describe the required behavior of computer systems under development. This work, called Mise en Scene, defines a new scenario medium (Scenario Notation Language, SNL) suitable for control-dominated systems, coupled with a two-stage process for automatic translation of scenarios to a new trace medium (Trace Notation Language, TNL) that encompasses CSP traces. Mise en Scene is offered as an initial solution to the problem of the scenarios-to-traces "D2" phase of R2D2C. A survey of the "scenario" concept and some case studies are also provided.

The effect of magnetic nanoparticles on neuronal differentiation of induced pluripotent stem cell-derived neural precursors

PubMed Central

Jiráková, Klára; Šeneklová, Monika; Jirák, Daniel; Turnovcová, Karolína; Vosmanská, Magda; Babič, Michal; Horák, Daniel; Veverka, Pavel; Jendelová, Pavla

2016-01-01

Introduction Magnetic resonance (MR) imaging is suitable for noninvasive long-term tracking. We labeled human induced pluripotent stem cell-derived neural precursors (iPSC-NPs) with two types of iron-based nanoparticles, silica-coated cobalt zinc ferrite nanoparticles (CZF) and poly-l-lysine-coated iron oxide superparamagnetic nanoparticles (PLL-coated γ-Fe2O3) and studied their effect on proliferation and neuronal differentiation. Materials and methods We investigated the effect of these two contrast agents on neural precursor cell proliferation and differentiation capability. We further defined the intracellular localization and labeling efficiency and analyzed labeled cells by MR. Results Cell proliferation was not affected by PLL-coated γ-Fe2O3 but was slowed down in cells labeled with CZF. Labeling efficiency, iron content and relaxation rates measured by MR were lower in cells labeled with CZF when compared to PLL-coated γ-Fe2O3. Cytoplasmic localization of both types of nanoparticles was confirmed by transmission electron microscopy. Flow cytometry and immunocytochemical analysis of specific markers expressed during neuronal differentiation did not show any significant differences between unlabeled cells or cells labeled with both magnetic nanoparticles. Conclusion Our results show that cells labeled with PLL-coated γ-Fe2O3 are suitable for MR detection, did not affect the differentiation potential of iPSC-NPs and are suitable for in vivo cell therapies in experimental models of central nervous system disorders. PMID:27920532

Degradable gene delivery systems based on Pluronics-modified low-molecular-weight polyethylenimine: preparation, characterization, intracellular trafficking, and cellular distribution

PubMed Central

Fan, Wei; Wu, Xin; Ding, Baoyue; Gao, Jing; Cai, Zhen; Zhang, Wei; Yin, Dongfeng; Wang, Xiang; Zhu, Quangang; Liu, Jiyong; Ding, Xueying; Gao, Shen

2012-01-01

Background Cationic copolymers consisting of polycations linked to nonionic amphiphilic block polymers have been evaluated as nonviral gene delivery systems, and a large number of different polymers and copolymers of linear, branched, and dendrimeric architectures have been tested in terms of their suitability and efficacy for in vitro and in vivo transfection. However, the discovery of new potent materials still largely relies on empiric approaches rather than a rational design. The authors investigated the relationship between the polymers’ structures and their biological performance, including DNA compaction, toxicity, transfection efficiency, and the effect of cellular uptake. Methods This article reports the synthesis and characterization of a series of cationic copolymers obtained by grafting polyethyleneimine with nonionic amphiphilic surfactant polyether-Pluronic® consisting of hydrophilic ethylene oxide and hydrophobic propylene oxide blocks. Transgene expression, cytotoxicity, localization of plasmids, and cellular uptake of these copolymers were evaluated following in vitro transfection of HeLa cell lines with various individual components of the copolymers. Results Pluronics can exhibit biological activity including effects on enhancing DNA cellular uptake, nuclear translocation, and gene expression. The Pluronics with a higher hydrophilic-lipophilic balance value lead to homogeneous distribution in the cytoplasm; those with a lower hydrophilic-lipophilic balance value prefer to localize in the nucleus. Conclusion This Pluronic-polyethyleneimine system may be worth exploring as components in the cationic copolymers as the DNA or small interfering RNA/microRNA delivery system in the near future. PMID:22403492

Suitability assessment of grey water quality treated with an upflow-downflow siliceous sand/marble waste filtration system for agricultural and industrial purposes.

PubMed

Chaabane, Safa; Riahi, Khalifa; Hamrouni, Hédi; Thayer, Béchir Ben

2017-04-01

The present study examines the suitability assessment of an upflow-downflow siliceous sand/marble waste filtration system for treatment and reuse of grey water collected from bathrooms of the student residential complex at the Higher Institute of Engineering Medjez El Bab (Tunisia). Once the optimization of grey water pre-treatment system has been determined, the filtration system was operated at different hydraulic loading rate and media filter proportions in order to assess the suitability of treated grey water for irrigational purpose according to salinity hazard, sodium hazard, magnesium hazard, permeability index, water infiltration rate, and widely used graphical methods. Suitability of the treated grey water for industrial purpose was evaluated in terms of foaming, corrosion, and scaling. Under optimal operational conditions, results reveals that treated grey water samples with an upflow-downflow siliceous sand/marble waste filtration system may be considered as a good and an excellent water quality suitable for irrigation purpose. However, treated grey water was found not appropriate for industrial purpose due to high concentrations of calcium and sodium that can generate foaming and scaling harm to boilers. These results suggest that treated grey water with an upflow-downflow siliceous sand/marble waste filtration system would support production when used as irrigation water.

Transcriptional signatures of parasitization and markers of colony decline in Varroa-infested honey bees (Apis mellifera).

PubMed

Zanni, Virginia; Galbraith, David A; Annoscia, Desiderato; Grozinger, Christina M; Nazzi, Francesco

2017-08-01

Extensive annual losses of honey bee colonies (Apis mellifera L.) reported in the northern hemisphere represent a global problem for agriculture and biodiversity. The parasitic mite Varroa destructor, in association with deformed wing virus (DWV), plays a key role in this phenomenon, but the underlying mechanisms are still unclear. To elucidate these mechanisms, we analyzed the gene expression profile of uninfested and mite infested bees, under laboratory and field conditions, highlighting the effects of parasitization on the bee's transcriptome under a variety of conditions and scenarios. Parasitization was significantly correlated with higher viral loads. Honey bees exposed to mite infestation exhibited an altered expression of genes related to stress response, immunity, nervous system function, metabolism and behavioural maturation. Additionally, mite infested young bees showed a gene expression profile resembling that of forager bees. To identify potential molecular markers of colony decline, the expression of genes that were commonly regulated across the experiments were subsequently assessed in colonies experiencing increasing mite infestation levels. These studies suggest that PGRP-2, hymenoptaecin, a glucan recognition protein, UNC93 and a p450 cytocrome maybe suitable general biomarkers of Varroa-induced colony decline. Furthermore, the reliability of vitellogenin, a yolk protein previously identified as a good marker of colony survival, was confirmed here. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tissue-specific expression of transgenic secreted ACE in vasculature can restore normal kidney functions, but not blood pressure, of Ace-/- mice.

PubMed

Chattopadhyay, Saurabh; Kessler, Sean P; Colucci, Juliana Almada; Yamashita, Michifumi; Senanayake, Preenie deS; Sen, Ganes C

2014-01-01

Angiotensin-converting enzyme (ACE) regulates normal blood pressure and fluid homeostasis through its action in the renin-angiotensin-system (RAS). Ace-/- mice are smaller in size, have low blood pressure and defective kidney structure and functions. All of these defects are cured by transgenic expression of somatic ACE (sACE) in vascular endothelial cells of Ace-/- mice. sACE is expressed on the surface of vascular endothelial cells and undergoes a natural cleavage secretion process to generate a soluble form in the body fluids. Both the tissue-bound and the soluble forms of ACE are enzymatically active, and generate the vasoactive octapeptide Angiotensin II (Ang II) with equal efficiency. To assess the relative physiological roles of the secreted and the cell-bound forms of ACE, we expressed, in the vascular endothelial cells of Ace-/- mice, the ectodomain of sACE, which corresponded to only the secreted form of ACE. Our results demonstrated that the secreted form of ACE could normalize kidney functions and RAS integrity, growth and development of Ace-/- mice, but not their blood pressure. This study clearly demonstrates that the secreted form of ACE cannot replace the tissue-bound ACE for maintaining normal blood pressure; a suitable balance between the tissue-bound and the soluble forms of ACE is essential for maintaining all physiological functions of ACE.

Transcriptional network inference from functional similarity and expression data: a global supervised approach.

PubMed

Ambroise, Jérôme; Robert, Annie; Macq, Benoit; Gala, Jean-Luc

2012-01-06

An important challenge in system biology is the inference of biological networks from postgenomic data. Among these biological networks, a gene transcriptional regulatory network focuses on interactions existing between transcription factors (TFs) and and their corresponding target genes. A large number of reverse engineering algorithms were proposed to infer such networks from gene expression profiles, but most current methods have relatively low predictive performances. In this paper, we introduce the novel TNIFSED method (Transcriptional Network Inference from Functional Similarity and Expression Data), that infers a transcriptional network from the integration of correlations and partial correlations of gene expression profiles and gene functional similarities through a supervised classifier. In the current work, TNIFSED was applied to predict the transcriptional network in Escherichia coli and in Saccharomyces cerevisiae, using datasets of 445 and 170 affymetrix arrays, respectively. Using the area under the curve of the receiver operating characteristics and the F-measure as indicators, we showed the predictive performance of TNIFSED to be better than unsupervised state-of-the-art methods. TNIFSED performed slightly worse than the supervised SIRENE algorithm for the target genes identification of the TF having a wide range of yet identified target genes but better for TF having only few identified target genes. Our results indicate that TNIFSED is complementary to the SIRENE algorithm, and particularly suitable to discover target genes of "orphan" TFs.

Tissue-Specific Expression of Transgenic Secreted ACE in Vasculature Can Restore Normal Kidney Functions, but Not Blood Pressure, of Ace-/- Mice

PubMed Central

Chattopadhyay, Saurabh; Kessler, Sean P.; Colucci, Juliana Almada; Yamashita, Michifumi; Senanayake, Preenie deS; Sen, Ganes C.

2014-01-01

Angiotensin-converting enzyme (ACE) regulates normal blood pressure and fluid homeostasis through its action in the renin-angiotensin-system (RAS). Ace-/- mice are smaller in size, have low blood pressure and defective kidney structure and functions. All of these defects are cured by transgenic expression of somatic ACE (sACE) in vascular endothelial cells of Ace-/- mice. sACE is expressed on the surface of vascular endothelial cells and undergoes a natural cleavage secretion process to generate a soluble form in the body fluids. Both the tissue-bound and the soluble forms of ACE are enzymatically active, and generate the vasoactive octapeptide Angiotensin II (Ang II) with equal efficiency. To assess the relative physiological roles of the secreted and the cell-bound forms of ACE, we expressed, in the vascular endothelial cells of Ace-/- mice, the ectodomain of sACE, which corresponded to only the secreted form of ACE. Our results demonstrated that the secreted form of ACE could normalize kidney functions and RAS integrity, growth and development of Ace-/- mice, but not their blood pressure. This study clearly demonstrates that the secreted form of ACE cannot replace the tissue-bound ACE for maintaining normal blood pressure; a suitable balance between the tissue-bound and the soluble forms of ACE is essential for maintaining all physiological functions of ACE. PMID:24475296

ASGR1 and ASGR2, the Genes that Encode the Asialoglycoprotein Receptor (Ashwell Receptor), Are Expressed in Peripheral Blood Monocytes and Show Interindividual Differences in Transcript Profile

PubMed Central

Harris, Rebecca Louise; van den Berg, Carmen Wilma; Bowen, Derrick John

2012-01-01

Background. The asialoglycoprotein receptor (ASGPR) is a hepatic receptor that mediates removal of potentially hazardous glycoconjugates from blood in health and disease. The receptor comprises two proteins, asialoglycoprotein receptor 1 and 2 (ASGR1 and ASGR2), encoded by the genes ASGR1 and ASGR2. Design and Methods. Using reverse transcription amplification (RT-PCR), expression of ASGR1 and ASGR2 was investigated in human peripheral blood monocytes. Results. Monocytes were found to express ASGR1 and ASGR2 transcripts. Correctly spliced transcript variants encoding different isoforms of ASGR1 and ASGR2 were present in monocytes. The profile of transcript variants from both ASGR1 and ASGR2 differed among individuals. Transcript expression levels were compared with the hepatocyte cell line HepG2 which produces high levels of ASGPR. Monocyte transcripts were 4 to 6 orders of magnitude less than in HepG2 but nonetheless readily detectable using standard RT-PCR. The monocyte cell line THP1 gave similar results to monocytes harvested from peripheral blood, indicating it may provide a suitable model system for studying ASGPR function in this cell type. Conclusions. Monocytes transcribe and correctly process transcripts encoding the constituent proteins of the ASGPR. Monocytes may therefore represent a mobile pool of the receptor, capable of reaching sites remote from the liver. PMID:22919488

DJ-1 Modulates Nuclear Erythroid 2-Related Factor-2-Mediated Protection in Human Primary Alveolar Type II Cells in Smokers.

PubMed

Bahmed, Karim; Messier, Elise M; Zhou, Wenbo; Tuder, Rubin M; Freed, Curt R; Chu, Hong Wei; Kelsen, Steven G; Bowler, Russell P; Mason, Robert J; Kosmider, Beata

2016-09-01

Cigarette smoke (CS) is a main source of oxidative stress and a key risk factor for emphysema, which consists of alveolar wall destruction. Alveolar type (AT) II cells are in the gas exchange regions of the lung. We isolated primary ATII cells from deidentified organ donors whose lungs were not suitable for transplantation. We analyzed the cell injury obtained from nonsmokers, moderate smokers, and heavy smokers. DJ-1 protects cells from oxidative stress and induces nuclear erythroid 2-related factor-2 (Nrf2) expression, which activates the antioxidant defense system. In ATII cells isolated from moderate smokers, we found DJ-1 expression by RT-PCR, and Nrf2 and heme oxygenase (HO)-1 translocation by Western blotting and immunocytofluorescence. In ATII cells isolated from heavy smokers, we detected Nrf2 and HO-1 cytoplasmic localization. Moreover, we found high oxidative stress, as detected by 4-hydroxynonenal (4-HNE) (immunoblotting), inflammation by IL-8 and IL-6 levels by ELISA, and apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in ATII cells obtained from heavy smokers. Furthermore, we detected early DJ-1 and late Nrf2 expression after ATII cell treatment with CS extract. We also overexpressed DJ-1 by adenovirus construct and found that this restored Nrf2 and HO-1 expression and induced nuclear translocation in heavy smokers. Moreover, DJ-1 overexpression also decreased ATII cell apoptosis caused by CS extract in vitro. Our results indicate that DJ-1 activates the Nrf2-mediated antioxidant defense system. Furthermore, DJ-1 overexpression can restore the impaired Nrf2 pathway, leading to ATII cell protection in heavy smokers. This suggests a potential therapeutic strategy for targeting DJ-1 in CS-related lung diseases.

DJ-1 Modulates Nuclear Erythroid 2–Related Factor-2–Mediated Protection in Human Primary Alveolar Type II Cells in Smokers

PubMed Central

Bahmed, Karim; Messier, Elise M.; Zhou, Wenbo; Tuder, Rubin M.; Freed, Curt R.; Chu, Hong Wei; Kelsen, Steven G.; Bowler, Russell P.; Mason, Robert J.

2016-01-01

Cigarette smoke (CS) is a main source of oxidative stress and a key risk factor for emphysema, which consists of alveolar wall destruction. Alveolar type (AT) II cells are in the gas exchange regions of the lung. We isolated primary ATII cells from deidentified organ donors whose lungs were not suitable for transplantation. We analyzed the cell injury obtained from nonsmokers, moderate smokers, and heavy smokers. DJ-1 protects cells from oxidative stress and induces nuclear erythroid 2–related factor-2 (Nrf2) expression, which activates the antioxidant defense system. In ATII cells isolated from moderate smokers, we found DJ-1 expression by RT-PCR, and Nrf2 and heme oxygenase (HO)-1 translocation by Western blotting and immunocytofluorescence. In ATII cells isolated from heavy smokers, we detected Nrf2 and HO-1 cytoplasmic localization. Moreover, we found high oxidative stress, as detected by 4-hydroxynonenal (4-HNE) (immunoblotting), inflammation by IL-8 and IL-6 levels by ELISA, and apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in ATII cells obtained from heavy smokers. Furthermore, we detected early DJ-1 and late Nrf2 expression after ATII cell treatment with CS extract. We also overexpressed DJ-1 by adenovirus construct and found that this restored Nrf2 and HO-1 expression and induced nuclear translocation in heavy smokers. Moreover, DJ-1 overexpression also decreased ATII cell apoptosis caused by CS extract in vitro. Our results indicate that DJ-1 activates the Nrf2-mediated antioxidant defense system. Furthermore, DJ-1 overexpression can restore the impaired Nrf2 pathway, leading to ATII cell protection in heavy smokers. This suggests a potential therapeutic strategy for targeting DJ-1 in CS-related lung diseases. PMID:27093578

Integrative functional analyses using rainbow trout selected for tolerance to plant diets reveal nutrigenomic signatures for soy utilization without the concurrence of enteritis.

PubMed

Abernathy, Jason; Brezas, Andreas; Snekvik, Kevin R; Hardy, Ronald W; Overturf, Ken

2017-01-01

Finding suitable alternative protein sources for diets of carnivorous fish species remains a major concern for sustainable aquaculture. Through genetic selection, we created a strain of rainbow trout that outperforms parental lines in utilizing an all-plant protein diet and does not develop enteritis in the distal intestine, as is typical with salmonids on long-term plant protein-based feeds. By incorporating this strain into functional analyses, we set out to determine which genes are critical to plant protein utilization in the absence of gut inflammation. After a 12-week feeding trial with our selected strain and a control trout strain fed either a fishmeal-based diet or an all-plant protein diet, high-throughput RNA sequencing was completed on both liver and muscle tissues. Differential gene expression analyses, weighted correlation network analyses and further functional characterization were performed. A strain-by-diet design revealed differential expression ranging from a few dozen to over one thousand genes among the various comparisons and tissues. Major gene ontology groups identified between comparisons included those encompassing central, intermediary and foreign molecule metabolism, associated biosynthetic pathways as well as immunity. A systems approach indicated that genes involved in purine metabolism were highly perturbed. Systems analysis among the tissues tested further suggests the interplay between selection for growth, dietary utilization and protein tolerance may also have implications for nonspecific immunity. By combining data from differential gene expression and co-expression networks using selected trout, along with ontology and pathway analyses, a set of 63 candidate genes for plant diet tolerance was found. Risk loci in human inflammatory bowel diseases were also found in our datasets, indicating rainbow trout selected for plant-diet tolerance may have added utility as a potential biomedical model.

Eucalyptus hairy roots, a fast, efficient and versatile tool to explore function and expression of genes involved in wood formation.

PubMed

Plasencia, Anna; Soler, Marçal; Dupas, Annabelle; Ladouce, Nathalie; Silva-Martins, Guilherme; Martinez, Yves; Lapierre, Catherine; Franche, Claudine; Truchet, Isabelle; Grima-Pettenati, Jacqueline

2016-06-01

Eucalyptus are of tremendous economic importance being the most planted hardwoods worldwide for pulp and paper, timber and bioenergy. The recent release of the Eucalyptus grandis genome sequence pointed out many new candidate genes potentially involved in secondary growth, wood formation or lineage-specific biosynthetic pathways. Their functional characterization is, however, hindered by the tedious, time-consuming and inefficient transformation systems available hitherto for eucalypts. To overcome this limitation, we developed a fast, reliable and efficient protocol to obtain and easily detect co-transformed E. grandis hairy roots using fluorescent markers, with an average efficiency of 62%. We set up conditions both to cultivate excised roots in vitro and to harden composite plants and verified that hairy root morphology and vascular system anatomy were similar to wild-type ones. We further demonstrated that co-transformed hairy roots are suitable for medium-throughput functional studies enabling, for instance, protein subcellular localization, gene expression patterns through RT-qPCR and promoter expression, as well as the modulation of endogenous gene expression. Down-regulation of the Eucalyptus cinnamoyl-CoA reductase1 (EgCCR1) gene, encoding a key enzyme in lignin biosynthesis, led to transgenic roots with reduced lignin levels and thinner cell walls. This gene was used as a proof of concept to demonstrate that the function of genes involved in secondary cell wall biosynthesis and wood formation can be elucidated in transgenic hairy roots using histochemical, transcriptomic and biochemical approaches. The method described here is timely because it will accelerate gene mining of the genome for both basic research and industry purposes. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system

PubMed Central

Szczesny, Roman J.; Kowalska, Katarzyna; Klosowska-Kosicka, Kamila; Chlebowski, Aleksander; Owczarek, Ewelina P.; Warkocki, Zbigniew; Kulinski, Tomasz M.; Adamska, Dorota; Affek, Kamila; Jedroszkowiak, Agata; Kotrys, Anna V.; Tomecki, Rafal; Krawczyk, Pawel S.; Borowski, Lukasz S.; Dziembowski, Andrzej

2018-01-01

Deciphering a function of a given protein requires investigating various biological aspects. Usually, the protein of interest is expressed with a fusion tag that aids or allows subsequent analyses. Additionally, downregulation or inactivation of the studied gene enables functional studies. Development of the CRISPR/Cas9 methodology opened many possibilities but in many cases it is restricted to non-essential genes. Recombinase-dependent gene integration methods, like the Flp-In system, are very good alternatives. The system is widely used in different research areas, which calls for the existence of compatible vectors and efficient protocols that ensure straightforward DNA cloning and generation of stable cell lines. We have created and validated a robust series of 52 vectors for streamlined generation of stable mammalian cell lines using the FLP recombinase-based methodology. Using the sequence-independent DNA cloning method all constructs for a given coding-sequence can be made with just three universal PCR primers. Our collection allows tetracycline-inducible expression of proteins with various tags suitable for protein localization, FRET, bimolecular fluorescence complementation (BiFC), protein dynamics studies (FRAP), co-immunoprecipitation, the RNA tethering assay and cell sorting. Some of the vectors contain a bidirectional promoter for concomitant expression of miRNA and mRNA, so that a gene can be silenced and its product replaced by a mutated miRNA-insensitive version. Our toolkit and protocols have allowed us to create more than 500 constructs with ease. We demonstrate the efficacy of our vectors by creating stable cell lines with various tagged proteins (numatrin, fibrillarin, coilin, centrin, THOC5, PCNA). We have analysed transgene expression over time to provide a guideline for future experiments and compared the effectiveness of commonly used inducers for tetracycline-responsive promoters. As proof of concept we examined the role of the exoribonuclease XRN2 in transcription termination by RNAseq. PMID:29590189

Generation of CMAHKO/GTKO/shTNFRI-Fc/HO-1 quadruple gene modified pigs.

PubMed

Kim, Geon A; Lee, Eun Mi; Jin, Jun-Xue; Lee, Sanghoon; Taweechaipaisankul, Anukul; Hwang, Jong Ik; Alam, Zahid; Ahn, Curie; Lee, Byeong Chun

2017-08-01

As an alternative source of organs for transplantation into humans, attention has been directed to pigs due to their similarities in biological features and organ size. However, severe immune rejection has prevented successful xenotransplantation using pig organs and tissues. To overcome immune rejection, recently developed genetic engineering systems such as TALEN coupled with somatic cell nuclear transfer (SCNT) to make embryos could be used to produce pigs compatible with xenotransplantation. We used the TALEN system to target the non-Gal antigen cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene in pigs that is naturally deleted in humans. Gal-deleted cells expressing both soluble human tumor necrosis factor receptor I IgG 1 -Fc (shTNFRI-Fc) and human hemagglutinin -tagged-human heme oxygenase-1 (hHO-1) were transfected with a TALEN target for CMAH. Cells lacking CMAH were negatively selected using N-glyconeuraminic acid (Neu5Gc)/magnetic beads and the level of Neu5Gc expression of isolated cells were analyzed by FACS and DNA sequencing. Cloned embryos using 3 different genetically modified cell clones were respectively transferred into 3 recipients, with 55.6% (5/9) becoming pregnant and three cloned pigs were produced. Successful genetic disruption of the CMAH gene was confirmed by sequencing, showing lack of expression of CMAH in tail-derived fibroblasts of the cloned piglets. Besides decreased expression of Neu5Gc in piglets produced by SCNT, antibody-mediated complement-dependent cytotoxicity assays and natural antibody binding for examining immuno-reactivity of the quadruple gene modified pigs derived from endothelial cells and fibroblasts were reduced significantly compared to those of wild type animals. We conclude that by combining the TALEN system and transgenic cells, targeting of multiple genes could be useful for generating organs for xenotransplantation. We produced miniature pigs with quadruple modified genes CMAHKO/GTKO/shTNFRI-Fc/hHO-1 that will be suitable for xenotransplantation by overcoming hyperacute, acute and anti-inflammatory rejection.

A Novel Strategy for Selection and Validation of Reference Genes in Dynamic Multidimensional Experimental Design in Yeast

PubMed Central

Cankorur-Cetinkaya, Ayca; Dereli, Elif; Eraslan, Serpil; Karabekmez, Erkan; Dikicioglu, Duygu; Kirdar, Betul

2012-01-01

Background Understanding the dynamic mechanism behind the transcriptional organization of genes in response to varying environmental conditions requires time-dependent data. The dynamic transcriptional response obtained by real-time RT-qPCR experiments could only be correctly interpreted if suitable reference genes are used in the analysis. The lack of available studies on the identification of candidate reference genes in dynamic gene expression studies necessitates the identification and the verification of a suitable gene set for the analysis of transient gene expression response. Principal Findings In this study, a candidate reference gene set for RT-qPCR analysis of dynamic transcriptional changes in Saccharomyces cerevisiae was determined using 31 different publicly available time series transcriptome datasets. Ten of the twelve candidates (TPI1, FBA1, CCW12, CDC19, ADH1, PGK1, GCN4, PDC1, RPS26A and ARF1) we identified were not previously reported as potential reference genes. Our method also identified the commonly used reference genes ACT1 and TDH3. The most stable reference genes from this pool were determined as TPI1, FBA1, CDC19 and ACT1 in response to a perturbation in the amount of available glucose and as FBA1, TDH3, CCW12 and ACT1 in response to a perturbation in the amount of available ammonium. The use of these newly proposed gene sets outperformed the use of common reference genes in the determination of dynamic transcriptional response of the target genes, HAP4 and MEP2, in response to relaxation from glucose and ammonium limitations, respectively. Conclusions A candidate reference gene set to be used in dynamic real-time RT-qPCR expression profiling in yeast was proposed for the first time in the present study. Suitable pools of stable reference genes to be used under different experimental conditions could be selected from this candidate set in order to successfully determine the expression profiles for the genes of interest. PMID:22675547

Difference in suitable mechanical properties of three-dimensional, synthetic scaffolds for self-renewing mouse embryonic stem cells of different genetic backgrounds.

PubMed

Lee, Myungook; Ahn, Jong Il; Ahn, Ji Yeon; Yang, Woo Sub; Hubbell, Jeffrey A; Lim, Jeong Mook; Lee, Seung Tae

2017-11-01

We evaluated whether the genetic background of embryonic stem cells (ESCs) affects the properties suitable for three-dimensional (3D) synthetic scaffolds for cell self-renewal. Inbred R1 and hybrid B6D2F1 mouse ESC lines were cultured for 7 days in hydrogel scaffolds with different properties derived from conjugating 7.5, 10, 12.5, or 15% (wt/vol) vinylsulfone-functionalized three-, four-, or eight-arm polyethylene glycol (PEG) with dicysteine-containing crosslinkers with an intervening matrix metalloproteinase-specific cleavage sites. Cell proliferation and expression of self-renewal-related genes and proteins by ESCs cultured in feeder-free or containing 2D culture plate or 3D hydrogel were monitored. As a preliminary experiment, the E14 ESC-customized synthetic 3D microenvironment did not maintain self-renewal of either the R1 or B6D2F1 ESCs. The best R1 cell proliferation (10.04 vs. 0.16-4.39; p < 0.0001) was observed in the four-arm 7.5% PEG-based hydrogels than those with other properties, whereas the F1 ESCs showed better proliferation when they were embedded in the three-arm 10% hydrogels. Self-renewal-related gene and protein expression by ESCs after feeder-free 3D culture was generally maintained compared with the feeder-containing 2D culture, but expression patterns and quantities differed. However, the feeder-free 3D culture yielded better expression than the feeder-free 2D culture. In conclusion, genetic background determined the suitability of hydrogel scaffolds for self-renewal of ESCs, which requires customization for the mechanical properties of each cell line. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2261-2268, 2017. © 2016 Wiley Periodicals, Inc.

Dynamic analysis and electronic circuit implementation of a novel 3D autonomous system without linear terms

NASA Astrophysics Data System (ADS)

Kengne, J.; Jafari, S.; Njitacke, Z. T.; Yousefi Azar Khanian, M.; Cheukem, A.

2017-11-01

Mathematical models (ODEs) describing the dynamics of almost all continuous time chaotic nonlinear systems (e.g. Lorenz, Rossler, Chua, or Chen system) involve at least a nonlinear term in addition to linear terms. In this contribution, a novel (and singular) 3D autonomous chaotic system without linear terms is introduced. This system has an especial feature of having two twin strange attractors: one ordinary and one symmetric strange attractor when the time is reversed. The complex behavior of the model is investigated in terms of equilibria and stability, bifurcation diagrams, Lyapunov exponent plots, time series and Poincaré sections. Some interesting phenomena are found including for instance, period-doubling bifurcation, antimonotonicity (i.e. the concurrent creation and annihilation of periodic orbits) and chaos while monitoring the system parameters. Compared to the (unique) case previously reported by Xu and Wang (2014) [31], the system considered in this work displays a more 'elegant' mathematical expression and experiences richer dynamical behaviors. A suitable electronic circuit (i.e. the analog simulator) is designed and used for the investigations. Pspice based simulation results show a very good agreement with the theoretical analysis.

Application of eGFP to label human periodontal ligament stem cells in periodontal tissue engineering.

PubMed

Wen, Yong; Lan, Jing; Huang, Haiyun; Yu, Meijiao; Cui, Jun; Liang, Jin; Jiang, Baoqi; Xu, Xin

2012-09-01

To establish human periodontal ligament stem cells (hPDLSC) with high and stable expression of enhanced green fluorescent protein (eGFP) and to obtain an ideal vector expression system that suitable for gene therapy in periodontal tissue engineering. hPDLSCs were transfected with eGFP for 48h via different MOI (25, 50, 100, 200 and 400) by lentiviral vector, the transfection efficiency was evaluated by fluorescent microscopy and flow cytometry, and transfected hPDLSCs proliferation was evaluated by MTT. Pluripotent, differentiation capacity and ALP expression status were determined further. Osteoblast-associated genes expressions for osteogenesis were evaluated by quantitative-PCR. In addition, rat molar periodontal fenestration defect model was used for evaluating periodontal tissue engineering. The transfection efficiency after 48h were 44.7%, 60.9%, 71.7%, 85.8%, and 86.9% respectively. There was no significant effect of transfection (at different MOI levels of 25, 50, 100, and 200) on the proliferation of hPDLSCs (designated as eGFP-hPDLSCs) compared with hPDLSCs (P>0.05). However, proliferation of eGFP hPDLSCs at MOI 400 became slower (P<0.05). Both eGFP hPDLSCs and hPDLSCs were able to differentiate into osteocytes and adipocytes under certain conditioned media. At 7 days, expression levels of COL-1, RUNX2 in hPDLSCS were higher than those in eGFP hPDLSCs (P<0.05); expression levels of ALP and OPN in eGFP hPDLSCs were similar to those in hPDLSCs (P>0.05). Newly regenerated bone formation was observed in the defect model used. Among the transfection conditions, 48h transfection at MOI 200 is optimal for labelling hPDLSCs with eGFP in a lentiviral vector. There is no change in capability of the eGFP hPDLSCs osteogenesis. The lentiviral vector with eGFP is an appropriate expression vector system and hPDLSCs are ideal seeding cells for gene therapy in periodontal tissue engineering. Copyright © 2012 Elsevier Ltd. All rights reserved.

Targeting Rapamycin to Podocytes Using a Vascular Cell Adhesion Molecule-1 (VCAM-1)-Harnessed SAINT-Based Lipid Carrier System

PubMed Central

Visweswaran, Ganesh Ram R.; Gholizadeh, Shima; Ruiters, Marcel H. J.; Molema, Grietje; Kok, Robbert J.; Kamps, Jan. A. A. M.

2015-01-01

Together with mesangial cells, glomerular endothelial cells and the basement membrane, podocytes constitute the glomerular filtration barrier (GFB) of the kidney. Podocytes play a pivotal role in the progression of various kidney-related diseases such as glomerular sclerosis and glomerulonephritis that finally lead to chronic end-stage renal disease. During podocytopathies, the slit-diaphragm connecting the adjacent podocytes are detached leading to severe loss of proteins in the urine. The pathophysiology of podocytopathies makes podocytes a potential and challenging target for nanomedicine development, though there is a lack of known molecular targets for cell selective drug delivery. To identify VCAM-1 as a cell-surface receptor that is suitable for binding and internalization of nanomedicine carrier systems by podocytes, we investigated its expression in the immortalized podocyte cell lines AB8/13 and MPC-5, and in primary podocytes. Gene and protein expression analyses revealed that VCAM-1 expression is increased by podocytes upon TNFα-activation for up to 24 h. This was paralleled by anti-VCAM-1 antibody binding to the TNFα-activated cells, which can be employed as a ligand to facilitate the uptake of nanocarriers under inflammatory conditions. Hence, we next explored the possibilities of using VCAM-1 as a cell-surface receptor to deliver the potent immunosuppressant rapamycin to TNFα-activated podocytes using the lipid-based nanocarrier system Saint-O-Somes. Anti-VCAM-1-rapamycin-SAINT-O-Somes more effectively inhibited the cell migration of AB8/13 cells than free rapamycin and non-targeted rapamycin-SAINT-O-Somes indicating the potential of VCAM-1 targeted drug delivery to podocytes. PMID:26407295

Development of a proteomic approach to monitor protein synthesis in mycotoxin producing moulds.

PubMed

Milles, J; Krämer, J; Prange, A

2007-12-01

In general, proteome studies compare different states of metabolism to investigate external or internal influences on protein expression. In the context of mycotoxin production the method could open another view on this complex and could be helpful to gain knowledge about proteins which are involved in metabolism (enzymes, transporters). In this short technical report, we describe a new protocol suitable for protein preparation for whole proteome analysis ofFusarium graminearum. Cell lysis was performed by grinding the mycelium with liquid nitrogen. Proteins were extracted with TCA/acetone and then cleaned; the isolated proteins were separated in a 2D-gel electrophoresis system (BioRad) using different pH gradients. The protocol established seems also generally applicable for other mycotoxin producing fungi.

Identification of novel loci for the generation of reporter mice

PubMed Central

Rebecchi, Monica; Levandis, Giovanna

2017-01-01

Abstract Deciphering the etiology of complex pathologies at molecular level requires longitudinal studies encompassing multiple biochemical pathways (apoptosis, proliferation, inflammation, oxidative stress). In vivo imaging of current reporter animals enabled the spatio-temporal analysis of specific molecular events, however, the lack of a multiplicity of loci for the generalized and regulated expression of the integrated transgenes hampers the creation of systems for the simultaneous analysis of more than a biochemical pathways at the time. We here developed and tested an in vivo-based methodology for the identification of multiple insertional loci suitable for the generation of reliable reporter mice. The validity of the methodology was tested with the generation of novel mice useful to report on inflammation and oxidative stress. PMID:27899606

Generalized look-ahead number conversion from signed digit to complement representation with optical logic operations

NASA Astrophysics Data System (ADS)

Qian, Feng; Li, Guoqiang

2001-12-01

In this paper a generalized look-ahead logic algorithm for number conversion from signed-digit to its complement representation is developed. By properly encoding the signed digits, all the operations are performed by binary logic, and unified logical expressions can be obtained for conversion from modified-signed-digit (MSD) to 2's complement, trinary signed-digit (TSD) to 3's complement, and quaternary signed-digit (QSD) to 4's complement. For optical implementation, a parallel logical array module using electron-trapping device is employed, which is suitable for realizing complex logic functions in the form of sum-of-product. The proposed algorithm and architecture are compatible with a general-purpose optoelectronic computing system.

Identifying and engineering promoters for high level and sustainable therapeutic recombinant protein production in cultured mammalian cells.

PubMed

Ho, Steven C L; Yang, Yuansheng

2014-08-01

Promoters are essential on plasmid vectors to initiate transcription of the transgenes when generating therapeutic recombinant proteins expressing mammalian cell lines. High and sustained levels of gene expression are desired during therapeutic protein production while gene expression is useful for cell engineering. As many finely controlled promoters exhibit cell and product specificity, new promoters need to be identified, optimized and carefully evaluated before use. Suitable promoters can be identified using techniques ranging from simple molecular biology methods to modern high-throughput omics screenings. Promoter engineering is often required after identification to either obtain high and sustained expression or to provide a wider range of gene expression. This review discusses some of the available methods to identify and engineer promoters for therapeutic recombinant protein expression in mammalian cells.

Generation and testing anti-influenza human monoclonal antibodies in a new humanized mouse model (DRAGA: HLA-A2. HLA-DR4. Rag1 KO. IL-2Rγc KO. NOD).

PubMed

Mendoza, Mirian; Ballesteros, Angela; Qiu, Qi; Pow Sang, Luis; Shashikumar, Soumya; Casares, Sofia; Brumeanu, Teodor-D

2018-02-01

Pandemic outbreaks of influenza type A viruses have resulted in numerous fatalities around the globe. Since the conventional influenza vaccines (CIV) provide less than 20% protection for individuals with weak immune system, it has been considered that broadly cross-neutralizing antibodies may provide a better protection. Herein, we showed that a recently generated humanized mouse (DRAGA mouse; HLA-A2. HLA-DR4. Rag1KO. IL-2Rgc KO. NOD) that lacks the murine immune system and expresses a functional human immune system can be used to generate cross-reactive, human anti-influenza monoclonal antibodies (hu-mAb). DRAGA mouse was also found to be suitable for influenza virus infection, as it can clear a sub-lethal infection and sustain a lethal infection with PR8/A/34 influenza virus. The hu-mAbs were designed for targeting a human B-cell epitope ( 180 WGIHHPPNSKEQ QNLY 195 ) of hemagglutinin (HA) envelope protein of PR8/A/34 (H1N1) virus with high homology among seven influenza type A viruses. A single administration of HA 180-195 specific hu-mAb in PR8-infected DRAGA mice significantly delayed the lethality by reducing the lung damage. The results demonstrated that DRAGA mouse is a suitable tool to (i) generate heterotype cross-reactive, anti-influenza human monoclonal antibodies, (ii) serve as a humanized mouse model for influenza infection, and (iii) assess the efficacy of anti-influenza antibody-based therapeutics for human use.

Children as Storytellers: Teaching the Basic Tools.

ERIC Educational Resources Information Center

Hamilton, Martha; Weiss, Mitch

1993-01-01

Explains how to teach children to be storytellers, including retelling stories, choosing the story, learning the story, voice factors, facial expressions, audience participation, and stage fright. A sidebar lists sources for stories that are suitable for young children to retell. (LRW)

Linear precoding based on polynomial expansion: reducing complexity in massive MIMO.

PubMed

Mueller, Axel; Kammoun, Abla; Björnson, Emil; Debbah, Mérouane

Massive multiple-input multiple-output (MIMO) techniques have the potential to bring tremendous improvements in spectral efficiency to future communication systems. Counterintuitively, the practical issues of having uncertain channel knowledge, high propagation losses, and implementing optimal non-linear precoding are solved more or less automatically by enlarging system dimensions. However, the computational precoding complexity grows with the system dimensions. For example, the close-to-optimal and relatively "antenna-efficient" regularized zero-forcing (RZF) precoding is very complicated to implement in practice, since it requires fast inversions of large matrices in every coherence period. Motivated by the high performance of RZF, we propose to replace the matrix inversion and multiplication by a truncated polynomial expansion (TPE), thereby obtaining the new TPE precoding scheme which is more suitable for real-time hardware implementation and significantly reduces the delay to the first transmitted symbol. The degree of the matrix polynomial can be adapted to the available hardware resources and enables smooth transition between simple maximum ratio transmission and more advanced RZF. By deriving new random matrix results, we obtain a deterministic expression for the asymptotic signal-to-interference-and-noise ratio (SINR) achieved by TPE precoding in massive MIMO systems. Furthermore, we provide a closed-form expression for the polynomial coefficients that maximizes this SINR. To maintain a fixed per-user rate loss as compared to RZF, the polynomial degree does not need to scale with the system, but it should be increased with the quality of the channel knowledge and the signal-to-noise ratio.

Preliminary results of the search for possible Martian landing sites to be considered for future European exploration missions

NASA Astrophysics Data System (ADS)

Martin, P.

2007-08-01

The recently adopted European Space Policy aims at expanding and coordinating the role and activities of Europe's space actors with the purpose of increasing both scientific knowledge in selected space domains and the European presence in the Solar System, as well as optimising the relevant societal benefits. With our Moon and in particular Mars as primary targets of exploration goals for the Solar System, and following a number of very successful orbital missions performing detailed remote sensing and mapping of these planetary bodies, probe landings on the surface of the Moon and Mars represent the next stepping stone of the exploration of our close planetary environment. Along with developing the hardware capabilities required for Europe to reach such ambitious goals, it therefore becomes increasingly important to pinpoint with precision a number of landing sites well suited for the safety and scientific success of future robotic missions. Focusing on Mars, and although a number of candidate landing sites and associated catalogs with available scientific justification already exist, the results being obtained by orbiters such as Mars Express and Mars Reconnaissance Orbiter are fundamentally transforming our knowledge of the planet's surface, which in turns highlights the need to review, update and revise the candidate sites for future landing missions on Mars. Detailed investigations of possible future Martian landing sites for European missions are ongoing, based on the wealth of scientific data and high-resolution mapping products available. In order to support the identification of suitable sites, various mapping products (geological, hyperspectral and compositional) can be consolidated, and various areas of Mars identified in the recent scientific literature as primary targets for landing can be taken into account for further, refined assessment of their suitability for landing. Seasonal and climatic effects potentially influencing landing shall also be considered, as well as lessons learned from past landing experiences. Finally, nolanding zones shall be identified based on a number of available criteria. A preliminary investigation and classification of potential landing sites for future European Mars exploration missions is summarised here, with the assumed following general requirements: • Moderate latitudes (e.g., 15ºS to 45ºN). Such a latitude range would be suitable for Exomars. • Low-to-moderate elevation (e.g., below 2000 m) • Relatively flat surface in the landing ellipse (e.g., slopes < 15º) • Low-to-moderate rock abundance (e.g., < 20%) • Moderate thermal inertia (rock/dust abundance) • Suitability to overriding scientific goals and targets of interest (e.g., niches of extant life, olivine sites, or sites where phyllosilicates or hydrated sulfates were identified) • Other constraints that shall be taken into account in a more detailed study: - Atmospheric and aeolian activity - Power and communications systems requirements - Illumination and temperature requirements - Biological potential and planetary protection Possible landing regions on Mars resulting from this preliminary investigation can be categorised into two classes, depending on the level of risk assessed for the landing in terms of, e.g., roughness and rock abundance: • Low-risk regions: Amazonis Planitia, Utopia Planitia, and Elysium Planitia. One of their potential drawbacks is that most areas of these regions exhibit a relatively high dust index which could be detrimental to the scientific interest of the in-situ mission. • Moderate-risk regions: - Syrtis Major / Nili Fossae, where phyllosilicates and hydrated minerals can be found based on recent evidence from orbit (Mars Express/OMEGA). - Isidis Planitia, in particular because this region presents a low vertical roughness. - Chryse/Acidalia Planitia, where phyllosilicates, hydrated minerals and sulfates can be found. - The region that spans the terrains from Sinus Meridiani to Syrtis Major, between 15ºS and 45ºN. This region exhibits a high dust index, and is represented by rougher, heavily cratered terrains in many areas. Within these regions, a more detailed identification of landing sites can be performed by refining the study (top-down approach) using higher-resolution geological and compositional maps (e.g., Mars Express/HRSC-OMEGA and/or MRO/HiRISECRISM) coupled with other parameters and constraints. Such detail work will be reported and the resulting suitable landing sites will be made available to the sciencedriven and success-oriented selection process for future Mars missions such as Exomars. This shall then be confronted with bottom-up approaches consisting in the pre-selection of sites purely based on scientific goals prior to the assessment of their suitability for landing.

Gene Expression Measurement Module (GEMM) - A Fully Automated, Miniaturized Instrument for Measuring Gene Expression in Space

NASA Technical Reports Server (NTRS)

Pohorille, Andrew; Peyvan, Kia; Karouia, Fathi; Ricco, Antonio

2012-01-01

The capability to measure gene expression on board spacecraft opens the door to a large number of high-value experiments on the influence of the space environment on biological systems. For example, measurements of gene expression will help us to understand adaptation of terrestrial life to conditions beyond the planet of origin, identify deleterious effects of the space environment on a wide range of organisms from microbes to humans, develop effective countermeasures against these effects, and determine the metabolic bases of microbial pathogenicity and drug resistance. These and other applications hold significant potential for discoveries in space biology, biotechnology, and medicine. Supported by funding from the NASA Astrobiology Science and Technology Instrument Development Program, we are developing a fully automated, miniaturized, integrated fluidic system for small spacecraft capable of in-situ measurement of expression of several hundreds of microbial genes from multiple samples. The instrument will be capable of (1) lysing cell walls of bacteria sampled from cultures grown in space, (2) extracting and purifying RNA released from cells, (3) hybridizing the RNA on a microarray and (4) providing readout of the microarray signal, all in a single microfluidics cartridge. The device is suitable for deployment on nanosatellite platforms developed by NASA Ames' Small Spacecraft Division. To meet space and other technical constraints imposed by these platforms, a number of technical innovations are being implemented. The integration and end-to-end technological and biological validation of the instrument are carried out using as a model the photosynthetic bacterium Synechococcus elongatus, known for its remarkable metabolic diversity and resilience to adverse conditions. Each step in the measurement process-lysis, nucleic acid extraction, purification, and hybridization to an array-is assessed through comparison of the results obtained using the instrument with those from standard laboratory protocols. Once developed, the system can be used with minor modifications for multiple experiments on different platforms in space, including extension to higher organisms and microbial monitoring. A proposed version of GEMM that is capable of handling both microbial and tissue samples on the International Space Station will be briefly summarized.

Expression profiling of marker genes responsive to the defence-associated phytohormones salicylic acid, jasmonic acid and ethylene in Brachypodium distachyon.

PubMed

Kouzai, Yusuke; Kimura, Mamiko; Yamanaka, Yurie; Watanabe, Megumi; Matsui, Hidenori; Yamamoto, Mikihiro; Ichinose, Yuki; Toyoda, Kazuhiro; Onda, Yoshihiko; Mochida, Keiichi; Noutoshi, Yoshiteru

2016-03-02

Brachypodium distachyon is a promising model plants for grasses. Infections of Brachypodium by various pathogens that severely impair crop production have been reported, and the species accordingly provides an alternative platform for investigating molecular mechanisms of pathogen virulence and plant disease resistance. To date, we have a broad picture of plant immunity only in Arabidopsis and rice; therefore, Brachypodium may constitute a counterpart that displays the commonality and uniqueness of defence systems among plant species. Phytohormones play key roles in plant biotic stress responses, and hormone-responsive genes are used to qualitatively and quantitatively evaluate disease resistance responses during pathogen infection. For these purposes, defence-related phytohormone marker genes expressed at time points suitable for defence-response monitoring are needed. Information about their expression profiles over time as well as their response specificity is also helpful. However, useful marker genes are still rare in Brachypodium. We selected 34 candidates for Brachypodium marker genes on the basis of protein-sequence similarity to known marker genes used in Arabidopsis and rice. Brachypodium plants were treated with the defence-related phytohormones salicylic acid, jasmonic acid and ethylene, and their transcription levels were measured 24 and 48 h after treatment. Two genes for salicylic acid, 7 for jasmonic acid and 2 for ethylene were significantly induced at either or both time points. We then focused on 11 genes encoding pathogenesis-related (PR) 1 protein and compared their expression patterns with those of Arabidopsis and rice. Phylogenetic analysis suggested that Brachypodium contains several PR1-family genes similar to rice genes. Our expression profiling revealed that regulation patterns of some PR1 genes as well as of markers identified for defence-related phytohormones are closely related to those in rice. We propose that the Brachypodium immune hormone marker genes identified in this study will be useful to plant pathologists who use Brachypodium as a model pathosystem, because the timing of their transcriptional activation matches that of the disease resistance response. Our results using Brachypodium also suggest that monocots share a characteristic immune system, defined as the common defence system, that is different from that of dicots.

Recombinant hosts suitable for simultaneous saccharification and fermentation

DOEpatents

Ingram, Lonnie O'Neal; Zhou, Shengde

2007-06-05

The invention provides recombinant host cells containing at least one heterologous polynucleotide encoding a polysaccharase under the transcriptional control of a surrogate promoter capable of increasing the expression of the polysaccharase. In addition, the invention further provides such hosts with genes encoding secretory protein/s to facilitate the secretion of the expressed polysaccharase. Preferred hosts of the invention are ethanologenic and capable of carrying out simultaneous saccharification fermentation resulting in the production of ethanol from complex cellulose substrates.

Identification of novel and robust internal control genes from Volvariella volvacea that are suitable for RT-qPCR in filamentous fungi.

PubMed

Tao, Yongxin; van Peer, Arend Frans; Huang, Qianhui; Shao, Yanping; Zhang, Lei; Xie, Bin; Jiang, Yuji; Zhu, Jian; Xie, Baogui

2016-07-12

The selection of appropriate internal control genes (ICGs) is a crucial step in the normalization of real-time quantitative PCR (RT-qPCR) data. Housekeeping genes are habitually selected for this purpose, despite accumulating evidence on their instability. We screened for novel, robust ICGs in the mushroom forming fungus Volvariella volvacea. Nine commonly used and five newly selected ICGs were evaluated for expression stability using RT-qPCR data in eight different stages of the life cycle of V. volvacea. Three different algorithms consistently determined that three novel ICGs (SPRYp, Ras and Vps26) exhibited the highest expression stability in V. volvacea. Subsequent analysis of ICGs in twenty-four expression profiles from nine filamentous fungi revealed that Ras was the most stable ICG amongst the Basidiomycetous samples, followed by SPRYp, Vps26 and ACTB. Vps26 was expressed most stably within the analyzed data of Ascomycetes, followed by HH3 and β-TUB. No ICG was universally stable for all fungal species, or for all experimental conditions within a species. Ultimately, the choice of an ICG will depend on a specific set of experiments. This study provides novel, robust ICGs for Basidiomycetes and Ascomycetes. Together with the presented guiding principles, this enables the efficient selection of suitable ICGs for RT-qPCR.

Assessment of reference gene stability in Rice stripe virus and Rice black streaked dwarf virus infection rice by quantitative Real-time PCR.

PubMed

Fang, Peng; Lu, Rongfei; Sun, Feng; Lan, Ying; Shen, Wenbiao; Du, Linlin; Zhou, Yijun; Zhou, Tong

2015-10-24

Stably expressed reference gene(s) normalization is important for the understanding of gene expression patterns by quantitative Real-time PCR (RT-qPCR), particularly for Rice stripe virus (RSV) and Rice black streaked dwarf virus (RBSDV) that caused seriously damage on rice plants in China and Southeast Asia. The expression of fourteen common used reference genes of Oryza sativa L. were evaluated by RT-qPCR in RSV and RBSDV infected rice plants. Suitable normalization reference gene(s) were identified by geNorm and NormFinder algorithms. UBQ 10 + GAPDH and UBC + Actin1 were identified as suitable reference genes for RT-qPCR normalization under RSV and RBSDV infection, respectively. When using multiple reference genes, the expression patterns of OsPRIb and OsWRKY, two virus resistance genes, were approximately similar with that reported previously. Comparatively, by using single reference gene (TIP41-Like), a weaker inducible response was observed. We proposed that the combination of two reference genes could obtain more accurate and reliable normalization of RT-qPCR results in RSV- and RBSDV-infected plants. This work therefore sheds light on establishing a standardized RT-qPCR procedure in RSV- and RBSDV-infected rice plants, and might serve as an important point for discovering complex regulatory networks and identifying genes relevant to biological processes or implicated in virus.

Inducing mutations through γ-irradiation in seeds of Mucuna pruriens for developing high L-DOPA-yielding genotypes.

PubMed

Singh, Susheel Kumar; Yadav, Deepti; Lal, Raj Kishori; Gupta, Madan M; Dhawan, Sunita Singh

2017-04-01

To develop elite genotypes in Mucuna pruriens (L.) DC with high L-DOPA (L-3, 4 dihydroxyphenylalanine) yields, with non-itching characteristics and better adaptability by applying γ-irradiation. Molecular and chemical analysis was performed for screening based on specific characteristics desired for developing suitable genotypes. Developed, mutant populations were analyzed for L-DOPA % in seeds through TLC (thin layer chromatography), and the results obtained were validated with the HPLC (High performance liquid chromatography). The DNA (Deoxyribonucleic acid) was isolated from the leaf at the initial stage and used for DNA polymorphism. RNA (Ribonucleic acid) was isolated from the leaf during maturity and used for expression analysis. The selected mutant T-I-7 showed 5.7% L-DOPA content compared to 3.18% of parent CIM-Ajar. The total polymorphism obtained was 57% with the molecular marker analysis. The gene expression analysis showed higher fold change expression of the dopadecarboxylase gene (DDC) in control compared to selected mutants (T-I-7, T-II-23, T-IV-9, T-VI-1). DNA polymorphism was used for the screening of mutants for efficient screening at an early stage. TLC was found suitable for the large-scale comparative chemical analysis of L-DOPA. The expression profile of DDC clearly demonstrated the higher yields of L-DOPA in selected mutants developed by γ-irradiation in the seeds of the control.

Identification of suitable reference genes in bone marrow stromal cells from osteoarthritic donors.

PubMed

Schildberg, Theresa; Rauh, Juliane; Bretschneider, Henriette; Stiehler, Maik

2013-11-01

Bone marrow stromal cells (BMSCs) are key cellular components for musculoskeletal tissue engineering strategies. Furthermore, recent data suggest that BMSCs are involved in the development of Osteoarthritis (OA) being a frequently occurring degenerative joint disease. Reliable reference genes for the molecular evaluation of BMSCs derived from donors exhibiting OA as a primary co-morbidity have not been reported on yet. Hence, the aim of the study was to identify reference genes suitable for comparative gene expression analyses using OA-BMSCs. Passage 1 bone marrow derived BMSCs were isolated from n=13 patients with advanced stage idiopathic hip osteoarthritis and n=15 age-matched healthy donors. The expression of 31 putative reference genes was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using a commercially available TaqMan(®) assay. Calculating the coefficient of variation (CV), mRNA expression stability was determined and afterwards validated using geNorm and NormFinder algorithms. Importin 8 (IPO8), TATA box binding protein (TBP), and cancer susceptibility candidate 3 (CASC3) were identified as the most stable reference genes. Notably, commonly used reference genes, e.g. beta-actin (ACTB) and beta-2-microglobulin (B2M) were among the most unstable genes. For normalization of gene expression data of OA-BMSCs the combined use of IPO8, TBP, and CASC3 gene is recommended. © 2013.

Combining stable insect cell lines with baculovirus-mediated expression for multi-HA influenza VLP production.

PubMed

Sequeira, Daniela P; Correia, Ricardo; Carrondo, Manuel J T; Roldão, António; Teixeira, Ana P; Alves, Paula M

2018-05-24

Safer and broadly protective vaccines are needed to cope with the continuous evolution of circulating influenza virus strains and promising approaches based on the expression of multiple hemagglutinins (HA) in a virus-like particle (VLP) have been proposed. However, expression of multiple genes in the same vector can lead to its instability due to tandem repetition of similar sequences. By combining stable with transient expression systems we can rationally distribute the number of genes to be expressed per platform and thus mitigate this risk. In this work, we developed a modular system comprising stable and baculovirus-mediated expression in insect cells for production of multi-HA influenza enveloped VLPs. First, a stable insect High Five cell population expressing two different HA proteins from subtype H3 was established. Infection of this cell population with a baculovirus vector encoding three other HA proteins from H3 subtype proved to be as competitive as traditional co-infection approaches in producing a pentavalent H3 VLP. Aiming at increasing HA expression, the stable insect cell population was infected at increasingly higher cell concentrations (CCI). However, cultures infected at CCI of 3×10 6 cells/mL showed lower HA titers per cell in comparison to standard CCI of 2×10 6 cells/mL, a phenomenon named "cell density effect". To lessen the negative impact of this phenomenon, a tailor-made refeed strategy was designed based on the exhaustion of key nutrients during cell growth. Noteworthy, cultures supplemented and infected at a CCI of 4×10 6 cells/mL showed comparable HA titers per cell to those of CCI of 2×10 6 cells/mL, thus leading to an increase of up to 4-fold in HA titers per mL. Scalability of the modular strategy herein proposed was successfully demonstrated in 2L stirred tank bioreactors with comparable HA protein levels observed between bioreactor and shake flasks cultures. Overall, this work demonstrates the suitability of combining stable with baculovirus-mediated expression in insect cells as an efficient platform for production of multi-HA influenza VLPs, surpassing the drawbacks of traditional co-infection strategies and/or the use of larger, unstable vectors. Copyright © 2017 Elsevier Ltd. All rights reserved.

Hypersonic three-dimensional nonequilibrium boundary-layer equations in generalized curvilinear coordinates

NASA Technical Reports Server (NTRS)

Lee, Jong-Hun

1993-01-01

The basic governing equations for the second-order three-dimensional hypersonic thermal and chemical nonequilibrium boundary layer are derived by means of an order-of-magnitude analysis. A two-temperature concept is implemented into the system of boundary-layer equations by simplifying the rather complicated general three-temperature thermal gas model. The equations are written in a surface-oriented non-orthogonal curvilinear coordinate system, where two curvilinear coordinates are non-orthogonial and a third coordinate is normal to the surface. The equations are described with minimum use of tensor expressions arising from the coordinate transformation, to avoid unnecessary confusion for readers. The set of equations obtained will be suitable for the development of a three-dimensional nonequilibrium boundary-layer code. Such a code could be used to determine economically the aerodynamic/aerothermodynamic loads to the surfaces of hypersonic vehicles with general configurations. In addition, the basic equations for three-dimensional stagnation flow, of which solution is required as an initial value for space-marching integration of the boundary-layer equations, are given along with the boundary conditions, the boundary-layer parameters, and the inner-outer layer matching procedure. Expressions for the chemical reaction rates and the thermodynamic and transport properties in the thermal nonequilibrium environment are explicitly given.

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

PubMed Central

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

2004-01-01

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

Imaging Subcellular Structures in the Living Zebrafish Embryo.

PubMed

Engerer, Peter; Plucinska, Gabriela; Thong, Rachel; Trovò, Laura; Paquet, Dominik; Godinho, Leanne

2016-04-02

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Rational Design of Mini-Cas9 for Transcriptional Activation.

PubMed

Ma, Dacheng; Peng, Shuguang; Huang, Weiren; Cai, Zhiming; Xie, Zhen

2018-04-20

Nuclease dead Cas9 (dCas9) has been widely used for modulating gene expression by fusing with different activation or repression domains. However, delivery of the CRISPR/Cas system fused with various effector domains in a single adeno-associated virus (AAV) remains challenging due to the payload limit. Here, we engineered a set of downsized variants of Cas9 including Staphylococcus aureus Cas9 (SaCas9) that retained DNA binding activity by deleting conserved functional domains. We demonstrated that fusing FokI nuclease domain to the N-terminal of the minimal SaCas9 (mini-SaCas9) or to the middle of the split mini-SaCas9 can trigger efficient DNA cleavage. In addition, we constructed a set of compact transactivation domains based on the tripartite VPR activation domain and self-assembled arrays of split SpyTag:SpyCatch peptides, which are suitable for fusing to the mini-SaCas9. Lastly, we produced a single AAV containing the mini-SaCas9 fused with a downsized transactivation domain along with an optimized gRNA expression cassette, which showed efficient transactivation activity. Our results highlighted a practical approach to generate down-sized CRISPR/Cas9 and gene activation systems for in vivo applications.

Cross-domain expression recognition based on sparse coding and transfer learning

NASA Astrophysics Data System (ADS)

Yang, Yong; Zhang, Weiyi; Huang, Yong

2017-05-01

Traditional facial expression recognition methods usually assume that the training set and the test set are independent and identically distributed. However, in actual expression recognition applications, the conditions of independent and identical distribution are hardly satisfied for the training set and test set because of the difference of light, shade, race and so on. In order to solve this problem and improve the performance of expression recognition in the actual applications, a novel method based on transfer learning and sparse coding is applied to facial expression recognition. First of all, a common primitive model, that is, the dictionary is learnt. Then, based on the idea of transfer learning, the learned primitive pattern is transferred to facial expression and the corresponding feature representation is obtained by sparse coding. The experimental results in CK +, JAFFE and NVIE database shows that the transfer learning based on sparse coding method can effectively improve the expression recognition rate in the cross-domain expression recognition task and is suitable for the practical facial expression recognition applications.

A practical application of the geometrical theory on fibered manifolds to an autonomous bicycle motion in mechanical system with nonholonomic constraints

NASA Astrophysics Data System (ADS)

Haddout, Soufiane

2018-01-01

The equations of motion of a bicycle are highly nonlinear and rolling of wheels without slipping can only be expressed by nonholonomic constraint equations. A geometrical theory of general nonholonomic constrained systems on fibered manifolds and their jet prolongations, based on so-called Chetaev-type constraint forces, was proposed and developed in the last decade by O. Krupková (Rossi) in 1990's. Her approach is suitable for study of all kinds of mechanical systems-without restricting to Lagrangian, time-independent, or regular ones, and is applicable to arbitrary constraints (holonomic, semiholonomic, linear, nonlinear or general nonholonomic). The goal of this paper is to apply Krupková's geometric theory of nonholonomic mechanical systems to study a concrete problem in nonlinear nonholonomic dynamics, i.e., autonomous bicycle. The dynamical model is preserved in simulations in its original nonlinear form without any simplifying. The results of numerical solutions of constrained equations of motion, derived within the theory, are in good agreement with measurements and thus they open the possibility of direct application of the theory to practical situations.

Quantum integrable systems from conformal blocks

NASA Astrophysics Data System (ADS)

Chen, Heng-Yu; Qualls, Joshua D.

2017-05-01

In this note, we extend the striking connections between quantum integrable systems and conformal blocks recently found in [M. Isachenkov and V. Schomerus, Phys. Rev. Lett. 117, 071602 (2016), 10.1103/PhysRevLett.117.071602] in several directions. First, we explicitly demonstrate that the action of the quartic conformal Casimir operator on general d-dimensional scalar conformal blocks can be expressed in terms of certain combinations of commuting integrals of motions of the two particle hyperbolic BC2 Calogero-Sutherland system. The permutation and reflection properties of the underlying Dunkl operators play crucial roles in establishing such a connection. Next, we show that the scalar superconformal blocks in superconformal field theories (SCFTs) with four and eight supercharges and suitable chirality constraints can also be identified with the eigenfunctions of the same Calogero-Sutherland system; this demonstrates the universality of such a connection. Finally, we observe that the so-called "seed" conformal blocks for constructing four point functions for operators with arbitrary space-time spins in four-dimensional CFTs can also be linearly expanded in terms of Calogero-Sutherland eigenfunctions.

A Dissipative Systems Theory for FDTD With Application to Stability Analysis and Subgridding

NASA Astrophysics Data System (ADS)

Bekmambetova, Fadime; Zhang, Xinyue; Triverio, Piero

2017-02-01

This paper establishes a far-reaching connection between the Finite-Difference Time-Domain method (FDTD) and the theory of dissipative systems. The FDTD equations for a rectangular region are written as a dynamical system having the magnetic and electric fields on the boundary as inputs and outputs. Suitable expressions for the energy stored in the region and the energy absorbed from the boundaries are introduced, and used to show that the FDTD system is dissipative under a generalized Courant-Friedrichs-Lewy condition. Based on the concept of dissipation, a powerful theoretical framework to investigate the stability of FDTD methods is devised. The new method makes FDTD stability proofs simpler, more intuitive, and modular. Stability conditions can indeed be given on the individual components (e.g. boundary conditions, meshes, embedded models) instead of the whole coupled setup. As an example of application, we derive a new subgridding method with material traverse, arbitrary grid refinement, and guaranteed stability. The method is easy to implement and has a straightforward stability proof. Numerical results confirm its stability, low reflections, and ability to handle material traverse.

Selection and validation of reference genes for RT-qPCR indicates that juice of sugarcane varieties modulate the expression of C metabolism genes in the endophytic diazotrophic Herbaspirillum rubrisubalbicans strain HCC103.

PubMed

Polese, Valéria; de Paula Soares, Cleiton; da Silva, Paula Renata Alves; Simões-Araújo, Jean Luiz; Baldani, José Ivo; Vidal, Marcia Soares

2017-12-01

Quantitative reverse transcription PCR (RT-qPCR) is an important tool for evaluating gene expression. However, this technique requires that specific internal normalizing genes be identified for different experimental conditions. To date, no internal normalizing genes are available for validation of data analyses for Herbaspirillum rubrisubalbicans strain HCC103, an endophyte that is part of the sugarcane consortium inoculant. This work seeks to identify and evaluate suitable reference genes for gene expression studies in HCC103 grown until middle log phase in sugarcane juice obtained from four sugarcane varieties or media with three different carbon sources. The mRNA levels of five candidate genes (rpoA, gyrA, dnaG, recA and gmK) and seven target genes involved in carbon metabolism (acnA, fbp, galE, suhB, wcaA, ORF_0127.0101 and _0127.0123) were quantified by RT-qPCR. Analysis of expression stability of these genes was carried out using geNorm and Normfinder software. The results indicated that the HCC103 dnaG and gyrA genes are the most stable and showed adequate relative expression level changes among the different sugarcane juices. The highest expression level was seen for ORF_0127.0101, which encodes a sugar transporter, in juice from sugarcane variety RB867515 and glucose as the carbon source. The suhB gene, encoding SuhB inositol monophosphatase, had a higher relative expression level on 0.5% glucose, 100% sugarcane juice from variety RB867515 and 0.5% aconitate. Together the results suggest that dnaG and gyrA genes are suitable as reference genes for RT-qPCR analysis of strain HCC103 and that juice from different sugarcane varieties modulates the expression of key genes involved in carbon metabolism.

Modeling gene expression measurement error: a quasi-likelihood approach

PubMed Central

Strimmer, Korbinian

2003-01-01

Background Using suitable error models for gene expression measurements is essential in the statistical analysis of microarray data. However, the true probabilistic model underlying gene expression intensity readings is generally not known. Instead, in currently used approaches some simple parametric model is assumed (usually a transformed normal distribution) or the empirical distribution is estimated. However, both these strategies may not be optimal for gene expression data, as the non-parametric approach ignores known structural information whereas the fully parametric models run the risk of misspecification. A further related problem is the choice of a suitable scale for the model (e.g. observed vs. log-scale). Results Here a simple semi-parametric model for gene expression measurement error is presented. In this approach inference is based an approximate likelihood function (the extended quasi-likelihood). Only partial knowledge about the unknown true distribution is required to construct this function. In case of gene expression this information is available in the form of the postulated (e.g. quadratic) variance structure of the data. As the quasi-likelihood behaves (almost) like a proper likelihood, it allows for the estimation of calibration and variance parameters, and it is also straightforward to obtain corresponding approximate confidence intervals. Unlike most other frameworks, it also allows analysis on any preferred scale, i.e. both on the original linear scale as well as on a transformed scale. It can also be employed in regression approaches to model systematic (e.g. array or dye) effects. Conclusions The quasi-likelihood framework provides a simple and versatile approach to analyze gene expression data that does not make any strong distributional assumptions about the underlying error model. For several simulated as well as real data sets it provides a better fit to the data than competing models. In an example it also improved the power of tests to identify differential expression. PMID:12659637

[Functional expression of an omega-3 fatty acid desaturase gene from Glycine max in Saccharomyces cerevisiae].

PubMed

Zhang, Hong-Tao; Yang, Jia-Sen; Shan, Lei; Bi, Yu-Ping

2006-01-01

Alpha-linolenic acid(ALA, C18:3delta9,12,15 ) is an essential fatty acid which has many sanitary functions to human. However, its contents in diets are often not enough. In plants, omega-3 fatty acid desaturases(FAD) catalyze linoleic acid(LA, C18:2delta9,12) into ALA. The seed oil of Glycine max contains high level of ALA. To investigate the functions of Glycine max omega-3FAD, the cDNA of GmFAD3 C was amplified by RT-PCR from immature seeds, then cloned into the shuttle expression vector p416 to generate the recombinant vector p4GFAD3C. The resulting vector was transformed into Saccharomyces cerevisiae K601 throuth LiAc method. The positive clones were screened on the CM(Ura-) medium and identified by PCR, and then cultured in CM (Ura-) liquid medium with exogenous LA in 20 degrees C for three days. The intracellular fatty acid composition of the engineering strain Kp416 and Kp4GFAD3C was analyzed by gas chromatography (GC). A novel peak in strain Kp4GFAD3C was detected,which was not detectable in control, Comparison of the retention times of the newly yielded peak with that of authentic standard indicated that the fatty acid is ALA. The content of ALA reached to 3.1% of the total fatty acid in recombinant strain, the content of LA correspondingly decreased from 22% to 16.2% by contrast. It was suggested that the protein encoded by GmFAD3 C can specifically catalyze 18 carbon PUFA substrate of LA into ALA by taking off hydrogen atoms at delta15 location. In this study, we expressed a Glycine max omega-3 fatty acid desaturase gene in S. cerevisiae; An efficient and economical yeast expressing system(K601-p416 system) which is suitable for the expression of FAD was built.

Identification and validation of reference genes for quantitative real-time PCR normalization and its applications in lycium.

PubMed

Zeng, Shaohua; Liu, Yongliang; Wu, Min; Liu, Xiaomin; Shen, Xiaofei; Liu, Chunzhao; Wang, Ying

2014-01-01

Lycium barbarum and L. ruthenicum are extensively used as traditional Chinese medicinal plants. Next generation sequencing technology provides a powerful tool for analyzing transcriptomic profiles of gene expression in non-model species. Such gene expression can then be confirmed with quantitative real-time polymerase chain reaction (qRT-PCR). Therefore, use of systematically identified suitable reference genes is a prerequisite for obtaining reliable gene expression data. Here, we calculated the expression stability of 18 candidate reference genes across samples from different tissues and grown under salt stress using geNorm and NormFinder procedures. The geNorm-determined rank of reference genes was similar to those defined by NormFinder with some differences. Both procedures confirmed that the single most stable reference gene was ACNTIN1 for L. barbarum fruits, H2B1 for L. barbarum roots, and EF1α for L. ruthenicum fruits. PGK3, H2B2, and PGK3 were identified as the best stable reference genes for salt-treated L. ruthenicum leaves, roots, and stems, respectively. H2B1 and GAPDH1+PGK1 for L. ruthenicum and SAMDC2+H2B1 for L. barbarum were the best single and/or combined reference genes across all samples. Finally, expression of salt-responsive gene NAC, fruit ripening candidate gene LrPG, and anthocyanin genes were investigated to confirm the validity of the selected reference genes. Suitable reference genes identified in this study provide a foundation for accurately assessing gene expression and further better understanding of novel gene function to elucidate molecular mechanisms behind particular biological/physiological processes in Lycium.

In vivo imaging of endothelial cell adhesion molecule expression after radiosurgery in an animal model of arteriovenous malformation.

PubMed

Raoufi-Rad, Newsha; McRobb, Lucinda S; Lee, Vivienne S; Bervini, David; Grace, Michael; Ukath, Jaysree; Mchattan, Joshua; Sreenivasan, Varun K A; Duong, T T Hong; Zhao, Zhenjun; Stoodley, Marcus A

2017-01-01

Focussed radiosurgery may provide a means of inducing molecular changes on the luminal surface of diseased endothelium to allow targeted delivery of novel therapeutic compounds. We investigated the potential of ionizing radiation to induce surface expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells (EC) in vitro and in vivo, to assess their suitability as vascular targets in irradiated arteriovenous malformations (AVMs). Cultured brain microvascular EC were irradiated by linear accelerator at single doses of 0, 5, 15 or 25 Gy and expression of ICAM-1 and VCAM-1 measured by qRT-PCR, Western, ELISA and immunocytochemistry. In vivo, near-infrared (NIR) fluorescence optical imaging using Xenolight 750-conjugated ICAM-1 or VCAM-1 antibodies examined luminal biodistribution over 84 days in a rat AVM model after Gamma Knife surgery at a single 15 Gy dose. ICAM-1 and VCAM-1 were minimally expressed on untreated EC in vitro. Doses of 15 and 25 Gy stimulated expression equally; 5 Gy was not different from the unirradiated. In vivo, normal vessels did not bind or retain the fluorescent probes, however binding was significant in AVM vessels. No additive increases in probe binding were found in response to radiosurgery at a dose of 15 Gy. In summary, radiation induces adhesion molecule expression in vitro but elevated baseline levels in AVM vessels precludes further induction in vivo. These molecules may be suitable targets in irradiated vessels without hemodynamic derangement, but not AVMs. These findings demonstrate the importance of using flow-modulated, pre-clinical animal models for validating candidate proteins for vascular targeting in irradiated AVMs.

Analytical tools for identifying bicycle route suitability, coverage, and continuity.

DOT National Transportation Integrated Search

2012-05-01

This report presents new tools created to assess bicycle suitability using geographic information systems (GIS). Bicycle suitability is a rating of how appropriate a roadway is for bicycle travel based on attributes of the roadway, such as vehi...

Polarization-based compensation of astigmatism.

PubMed

Chowdhury, Dola Roy; Bhattacharya, Kallol; Chakraborty, Ajay K; Ghosh, Raja

2004-02-01

One approach to aberration compensation of an imaging system is to introduce a suitable phase mask at the aperture plane of an imaging system. We utilize this principle for the compensation of astigmatism. A suitable polarization mask used on the aperture plane together with a polarizer-retarder combination at the input of the imaging system provides the compensating polarization-induced phase steps at different quadrants of the apertures masked by different polarizers. The aberrant phase can be considerably compensated by the proper choice of a polarization mask and suitable selection of the polarization parameters involved. The results presented here bear out our theoretical expectation.

Identification of suitable fundus images using automated quality assessment methods.

PubMed

Şevik, Uğur; Köse, Cemal; Berber, Tolga; Erdöl, Hidayet

2014-04-01

Retinal image quality assessment (IQA) is a crucial process for automated retinal image analysis systems to obtain an accurate and successful diagnosis of retinal diseases. Consequently, the first step in a good retinal image analysis system is measuring the quality of the input image. We present an approach for finding medically suitable retinal images for retinal diagnosis. We used a three-class grading system that consists of good, bad, and outlier classes. We created a retinal image quality dataset with a total of 216 consecutive images called the Diabetic Retinopathy Image Database. We identified the suitable images within the good images for automatic retinal image analysis systems using a novel method. Subsequently, we evaluated our retinal image suitability approach using the Digital Retinal Images for Vessel Extraction and Standard Diabetic Retinopathy Database Calibration level 1 public datasets. The results were measured through the F1 metric, which is a harmonic mean of precision and recall metrics. The highest F1 scores of the IQA tests were 99.60%, 96.50%, and 85.00% for good, bad, and outlier classes, respectively. Additionally, the accuracy of our suitable image detection approach was 98.08%. Our approach can be integrated into any automatic retinal analysis system with sufficient performance scores.

The chemiluminescence based Ziplex automated workstation focus array reproduces ovarian cancer Affymetrix GeneChip expression profiles.

PubMed

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.

Suitability and perspectives on using recombinant insect cells for the production of virus-like particles.

PubMed

Yamaji, Hideki

2014-03-01

Virus-like particles (VLPs) can be produced in recombinant protein production systems by expressing viral surface proteins that spontaneously assemble into particulate structures similar to authentic viral or subviral particles. VLPs serve as excellent platforms for the development of safe and effective vaccines and diagnostic antigens. Among various recombinant protein production systems, the baculovirus-insect cell system has been used extensively for the production of a wide variety of VLPs. This system is already employed for the manufacture of a licensed human papillomavirus-like particle vaccine. However, the baculovirus-insect cell system has several inherent limitations including contamination of VLPs with progeny baculovirus particles. Stably transformed insect cells have emerged as attractive alternatives to the baculovirus-insect cell system. Different types of VLPs, with or without an envelope and composed of either single or multiple structural proteins, have been produced in stably transformed insect cells. VLPs produced by stably transformed insect cells have successfully elicited immune responses in vivo. In some cases, the yield of VLPs attained with recombinant insect cells was comparable to, or higher than, that obtained by baculovirus-infected insect cells. Recombinant insect cells offer a promising approach to the development and production of VLPs.

MECHANISMS OF NANODIAMOND PARTICLE INDUCED IL-8 EXPRESSION IN HUMAN AIRWAY EPITHELIAL CELLS

EPA Science Inventory

Nanodiamond particles (NDP) prepared by detonation under confined conditions have a number of industrial and analytical applications. Previous in vitro studies have reported NDP to be biologically inert with negligible cytotoxicity, implying that they are potentially suitable for...

Assessing the habitat suitability of agricultural landscapes for characteristic breeding bird guilds using landscape metrics.

PubMed

Borges, Friederike; Glemnitz, Michael; Schultz, Alfred; Stachow, Ulrich

2017-04-01

Many of the processes behind the decline of farmland birds can be related to modifications in landscape structure (composition and configuration), which can partly be expressed quantitatively with measurable or computable indices, i.e. landscape metrics. This paper aims to identify statistical relationships between the occurrence of birds and the landscape structure. We present a method that combines two comprehensive procedures: the "landscape-centred approach" and "guild classification". Our study is based on more than 20,000 individual bird observations based on a 4-year bird monitoring approach in a typical agricultural area in the north-eastern German lowlands. Five characteristic bird guilds, each with three characteristic species, are defined for the typical habitat types of that area: farmland, grassland, hedgerow, forest and settlement. The suitability of each sample plot for each guild is indicated by the level of persistence (LOP) of occurrence of three respective species. Thus, the sample plots can be classified as "preferred" or "less preferred" depending on the lower and upper quartiles of the LOP values. The landscape structure is characterized by 16 different landscape metrics expressing various aspects of landscape composition and configuration. For each guild, the three landscape metrics with the strongest rank correlation with the LOP values and that are not mutually dependent were identified. For four of the bird guilds, the classification success was better than 80%, compared with only 66% for the grassland bird guild. A subset of six landscape metrics proved to be the most meaningful and sufficiently classified the sample areas with respect to bird guild suitability. In addition, derived logistic functions allowed the production of guild-specific habitat suitability maps for the whole landscape. The analytical results show that the proposed approach is appropriate to assess the habitat suitability of agricultural landscapes for characteristic bird guilds.

An Efficient Electroporation Protocol for the Genetic Modification of Mammalian Cells

PubMed Central

Chicaybam, Leonardo; Barcelos, Camila; Peixoto, Barbara; Carneiro, Mayra; Limia, Cintia Gomez; Redondo, Patrícia; Lira, Carla; Paraguassú-Braga, Flávio; Vasconcelos, Zilton Farias Meira De; Barros, Luciana; Bonamino, Martin Hernán

2017-01-01

Genetic modification of cell lines and primary cells is an expensive and cumbersome approach, often involving the use of viral vectors. Electroporation using square-wave generating devices, like Lonza’s Nucleofector, is a widely used option, but the costs associated with the acquisition of electroporation kits and the transient transgene expression might hamper the utility of this methodology. In the present work, we show that our in-house developed buffers, termed Chicabuffers, can be efficiently used to electroporate cell lines and primary cells from murine and human origin. Using the Nucleofector II device, we electroporated 14 different cell lines and also primary cells, like mesenchymal stem cells and cord blood CD34+, providing optimized protocols for each of them. Moreover, when combined with sleeping beauty-based transposon system, long-term transgene expression could be achieved in all types of cells tested. Transgene expression was stable and did not interfere with CD34+ differentiation to committed progenitors. We also show that these buffers can be used in CRISPR-mediated editing of PDCD1 gene locus in 293T and human peripheral blood mononuclear cells. The optimized protocols reported in this study provide a suitable and cost-effective platform for the genetic modification of cells, facilitating the widespread adoption of this technology. PMID:28168187

Cancer-targeting siRNA delivery from porous silicon nanoparticles.

PubMed

Wan, Yuan; Apostolou, Sinoula; Dronov, Roman; Kuss, Bryone; Voelcker, Nicolas H

2014-10-01

Porous silicon nanoparticles (pSiNPs) with tunable pore size are biocompatible and biodegradable, suggesting that they are suitable biomaterials as vehicles for drug delivery. Loading of small interfering RNA (siRNA) into the pores of pSiNPs can protect siRNA from degradation as well as improve the cellular uptake. We aimed to deliver MRP1 siRNA loaded into pSiNPs to glioblastoma cells, and to demonstrate downregulation of MRP1 at the mRNA and protein levels. 50-220 nm pSiNPs with an average pore size of 26 nm were prepared, followed by electrostatic adsorption of siRNA into pores. Oligonucleotide loading and release profiles were investigated; MRP1 mRNA and protein expression, cell viability and cell apoptosis were studied. Approximately 7.7 µg of siRNA was loaded per mg of pSiNPs. Cells readily took up nanoparticles after 30 min incubation. siRNA-loaded pSiNPs were able to effectively downregulate target mRNA (~40%) and protein expression (31%), and induced cell apoptosis and necrosis (33%). siRNA loaded pSiNPs downregulated mRNA and protein expression and induced cell death. This novel siRNA delivery system may pave the way towards developing more effective tumor therapies.

Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors.

PubMed

Wang, Hai-Chao; An, Huai-Jie; Yu, Yun-Zhou; Xu, Qing

2015-02-01

DNA vaccines require improvement for human use because they are generally weak stimulators of the immune system in humans. The efficacy of DNA vaccines can be improved using a viral replicon as vector to administer antigen of pathogen. In this study, we comprehensively evaluated the conventional non-viral DNA, viral replicon DNA or viral replicon particles (VRP) vaccines encoding different forms of anthrax protective antigen (PA) for specific immunity and protective potency against anthrax. Our current results clearly suggested that these viral replicon DNA or VRP vaccines derived from Semliki Forest virus (SFV) induced stronger PA-specific immune responses than the conventional non-viral DNA vaccines when encoding the same antigen forms, which resulted in potent protection against challenge with the Bacillus anthracis strain A16R. Additionally, the naked PA-expressing SFV replicon DNA or VRP vaccines without the need for high doses or demanding particular delivery regimens elicited robust immune responses and afforded completely protective potencies, which indicated the potential of the SFV replicon as vector of anthrax vaccines for use in clinical application. Therefore, our results suggest that these PA-expressing SFV replicon DNA or VRP vaccines may be suitable as candidate vaccines against anthrax. Copyright © 2015 Elsevier B.V. All rights reserved.

HRT-UML: a design method for hard real-time systems based on the UML notation

NASA Astrophysics Data System (ADS)

D'Alessandro, Massimo; Mazzini, Silvia; di Natale, Marco; Lipari, Giuseppe

2002-07-01

The Hard Real-Time-Unified Modelling Language (HRT-UML) method aims at providing a comprehensive solution to the modeling of Hard Real Time systems. The experience shows that the design of Hard Real-Time systems needs methodologies suitable for the modeling and analysis of aspects related to time, schedulability and performance. In the context of the European Aerospace community a reference method for design is Hierarchical Object Oriented Design (HOOD) and in particular its extension for the modeling of hard real time systems, Hard Real-Time-Hierarchical Object Oriented Design (HRT-HOOD), recommended by the European Space Agency (ESA) for the development of on-board systems. On the other hand in recent years the Unified Modelling Language (UML) has been gaining a very large acceptance in a wide range of domains, all over the world, becoming a de-facto international standard. Tool vendors are very active in this potentially big market. In the Aerospace domain the common opinion is that UML, as a general notation, is not suitable for Hard Real Time systems, even if its importance is recognized as a standard and as a technological trend in the near future. These considerations suggest the possibility of replacing the HRT-HOOD method with a customized version of UML, that incorporates the advantages of both standards and complements the weak points. This approach has the clear advantage of making HRT-HOOD converge on a more powerful and expressive modeling notation. The paper identifies a mapping of the HRT-HOOD semantics into the UML one, and proposes a UML extension profile, that we call HRT-UML, based on the UML standard extension mechanisms, to fully represent HRT-HOOD design concepts. Finally it discusses the relationships between our profile and the UML profile for schedulability, performance and time, adopted by OMG in November 2001.

Evaluation of New Reference Genes in Papaya for Accurate Transcript Normalization under Different Experimental Conditions

PubMed Central

Chen, Weixin; Chen, Jianye; Lu, Wangjin; Chen, Lei; Fu, Danwen

2012-01-01

Real-time reverse transcription PCR (RT-qPCR) is a preferred method for rapid and accurate quantification of gene expression studies. Appropriate application of RT-qPCR requires accurate normalization though the use of reference genes. As no single reference gene is universally suitable for all experiments, thus reference gene(s) validation under different experimental conditions is crucial for RT-qPCR analysis. To date, only a few studies on reference genes have been done in other plants but none in papaya. In the present work, we selected 21 candidate reference genes, and evaluated their expression stability in 246 papaya fruit samples using three algorithms, geNorm, NormFinder and RefFinder. The samples consisted of 13 sets collected under different experimental conditions, including various tissues, different storage temperatures, different cultivars, developmental stages, postharvest ripening, modified atmosphere packaging, 1-methylcyclopropene (1-MCP) treatment, hot water treatment, biotic stress and hormone treatment. Our results demonstrated that expression stability varied greatly between reference genes and that different suitable reference gene(s) or combination of reference genes for normalization should be validated according to the experimental conditions. In general, the internal reference genes EIF (Eukaryotic initiation factor 4A), TBP1 (TATA binding protein 1) and TBP2 (TATA binding protein 2) genes had a good performance under most experimental conditions, whereas the most widely present used reference genes, ACTIN (Actin 2), 18S rRNA (18S ribosomal RNA) and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) were not suitable in many experimental conditions. In addition, two commonly used programs, geNorm and Normfinder, were proved sufficient for the validation. This work provides the first systematic analysis for the selection of superior reference genes for accurate transcript normalization in papaya under different experimental conditions. PMID:22952972

Modelling white-water rafting suitability in a hydropower regulated Alpine River.

PubMed

Carolli, Mauro; Zolezzi, Guido; Geneletti, Davide; Siviglia, Annunziato; Carolli, Fabiano; Cainelli, Oscar

2017-02-01

Cultural and recreational river ecosystem services and their relations with the flow regime are still poorly investigated. We develop a modelling-based approach to assess recreational flow requirements and the spatially distributed river suitability for white-water rafting, a typical service offered by mountain streams, with potential conflicts of interest with hydropower regulation. The approach is based on the principles of habitat suitability modelling using water depth as the main attribute, with preference curves defined through interviews with local rafting guides. The methodology allows to compute streamflow thresholds for conditions of suitability and optimality of a river reach in relation to rafting. Rafting suitability response to past, present and future flow management scenarios can be predicted on the basis of a hydrological model, which is incorporated in the methodology and is able to account for anthropic effects. Rafting suitability is expressed through a novel metric, the "Rafting hydro-suitability index" (RHSI) which quantifies the cumulative duration of suitable and optimal conditions for rafting. The approach is applied on the Noce River (NE Italy), an Alpine River regulated by hydropower production and affected by hydropeaking, which influences suitability at a sub-daily scale. A dedicated algorithm is developed within the hydrological model to resemble hydropeaking conditions with daily flow data. In the Noce River, peak flows associated with hydropeaking support rafting activities in late summer, highlighting the dual nature of hydropeaking in regulated rivers. Rafting suitability is slightly reduced under present, hydropower-regulated flow conditions compared to an idealized flow regime characterised by no water abstractions. Localized water abstractions for small, run-of-the-river hydropower plants are predicted to negatively affect rafting suitability. The proposed methodology can be extended to support decision making for flow management in hydropower regulated streams, as it has the potential to quantify the response of different ecosystem services to flow regulation. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparative Analysis.

DTIC Science & Technology

1987-11-01

differential qualita- tive (DQ) analysis, which solves the task, providing explanations suitable for use by design systems, automated diagnosis, intelligent...solves the task, providing explanations suitable for use by design systems, automated diagnosis, intelligent tutoring systems, and explanation based...comparative analysis as an important component; the explanation is used in many different ways. * One way method of automated design is the principlvd

Linking Health Concepts in the Assessment and Evaluation of Water Distribution Systems

ERIC Educational Resources Information Center

Karney, Bryan W.; Filion, Yves R.

2005-01-01

The concept of health is not only a specific criterion for evaluation of water quality delivered by a distribution system but also a suitable paradigm for overall functioning of the hydraulic and structural components of the system. This article views health, despite its complexities, as the only criterion with suitable depth and breadth to allow…

76 FR 50197 - Notice Inviting Publishers To Submit Tests for a Determination of Suitability for Use in the...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-08-12

... Suitability for Use in the National Reporting System for Adult Education AGENCY: Office of Vocational and... review and approval for use in the National Reporting System for Adult Education (NRS). FOR FURTHER... National Reporting System for Adult Education regulations, 34 CFR part 462 (NRS regulations), include the...

Matrix elements of N-particle explicitly correlated Gaussian basis functions with complex exponential parameters

NASA Astrophysics Data System (ADS)

Bubin, Sergiy; Adamowicz, Ludwik

2006-06-01

In this work we present analytical expressions for Hamiltonian matrix elements with spherically symmetric, explicitly correlated Gaussian basis functions with complex exponential parameters for an arbitrary number of particles. The expressions are derived using the formalism of matrix differential calculus. In addition, we present expressions for the energy gradient that includes derivatives of the Hamiltonian integrals with respect to the exponential parameters. The gradient is used in the variational optimization of the parameters. All the expressions are presented in the matrix form suitable for both numerical implementation and theoretical analysis. The energy and gradient formulas have been programed and used to calculate ground and excited states of the He atom using an approach that does not involve the Born-Oppenheimer approximation.

Matrix elements of N-particle explicitly correlated Gaussian basis functions with complex exponential parameters.

PubMed

Bubin, Sergiy; Adamowicz, Ludwik

2006-06-14

In this work we present analytical expressions for Hamiltonian matrix elements with spherically symmetric, explicitly correlated Gaussian basis functions with complex exponential parameters for an arbitrary number of particles. The expressions are derived using the formalism of matrix differential calculus. In addition, we present expressions for the energy gradient that includes derivatives of the Hamiltonian integrals with respect to the exponential parameters. The gradient is used in the variational optimization of the parameters. All the expressions are presented in the matrix form suitable for both numerical implementation and theoretical analysis. The energy and gradient formulas have been programmed and used to calculate ground and excited states of the He atom using an approach that does not involve the Born-Oppenheimer approximation.

Immunodiagnostic Properties of Wucheraria bancrofti SXP-1, a Potential Filarial Diagnostic Candidate Expressed in Tobacco Plant, Nicotiana tabacum.

PubMed

Ganapathy, Mathangi; Chakravarthi, M; Charles, S Jason; Harunipriya, P; Jaiganesh, S; Subramonian, N; Kaliraj, P

2015-08-01

Transgenic tobacco plants were developed expressing WbSXP-1, a diagnostic antigen isolated from the cDNA library of L3 stage larvae of Wucheraria bancrofti. This antigen produced by recombinant Escherichia coli has been demonstrated by to be successful as potential diagnostic candidate against lymphatic filariasis. A rapid format simple and qualitative flow through immune-filtration diagnostic kit has been developed for the identification of IgG antibodies to the recombinant WbSXP-1 and is being marketed by M/S Span Diagnostics Ltd in India and Africa. Here, we present the results of experiments on the transformation and expression of the same filarial antigen, WbSXP-1, in tobacco plant, Nicotiana tabacum, to produce plant-based diagnostic antigen. It was possible to successfully transform the tobacco plant with WbSXP-1, the integration of the parasite-specific gene in plants was confirmed by PCR amplification and the expression of the filarial protein by Western blotting. The immunoreactivity of the plant-produced WbSXP-1 was assessed based on its reaction with the monoclonal antibodies developed against the E. coli-produced protein. Immunological screening using clinical sera from patients indicates that the plant-produced protein is comparable to E. coli-produced diagnostic antigen. The result demonstrated that plants can be used as suitable expression systems for the production of diagnostic proteins against lymphatic filariasis, a neglected tropical infectious disease which has a negative impact on socioeconomic development. This is the first report of the integration, expression and efficacy of a diagnostic candidate of lymphatic filariasis in plants.Key MessageTransgenic tobacco plants with WbSXP-1, a filarial diagnostic candidate, were developed. The plant-produced protein showed immunoreactivity on par with the E. coli product.

Validation of internal controls for gene expression analysis in the intestine of rats infected with Hymenolepis diminuta.

PubMed

Hoque, Tafazzal; Bhogal, Meetu; Boghal, Meetu; Webb, Rodney A

2007-12-01

The non-invasive parasitic cestode Hymenolepis diminuta induces hypertrophy, hyperplasia and other changes in cell activity in the intestine of rats which are indicated in the expression of mRNA. We have investigated various house-keeping genes (GAPDH, beta-actin, 18S and HPRT) and other internal controls (total RNA/unit biomass, total RNA/unit length of intestine) to validate gene expression in the rat intestine after cestode infection and drug-induced neuromodulation. Variation in GAPDH, beta-actin, 18S and HPRT expression was observed in rat jejunal tissue according to treatment. Total RNA/unit length of intestine was found to be the most suitable internal control for normalizing target gene mRNA expression in both infected and/or drug-induced rat intestine. This normalization method may be applied to studies of gene expression levels in intestinal tissue where hypertrophy, hyperplasia, rapid growth and cell differentiation generally occur.

Blastema induction in aneurogenic state and Prrx-1 regulation by MMPs and FGFs in Ambystoma mexicanum limb regeneration.

PubMed

Satoh, Akira; makanae, Aki; Hirata, Ayako; Satou, Yutaka

2011-07-15

Urodele amphibians can regenerate amputated limbs. It has been considered that differentiated dermal tissues generate multipotent and undifferentiated cells called blastema cells during limb regeneration. In early phases of limb regeneration, blastema cells are induced by nerves and the apical epithelial cap (AEC). We had previously investigated the role of neurotrophic factors in blastema or blastema-like formation consisting of Prrx-1 positive cells. A new system suitable for investigating early phases of limb regeneration, called the accessory limb model (ALM), was recently developed. In this study, we performed a comparative transcriptome analysis between a blastema and wound using ALM. Matrix metalloproteinase (MMP) and fibroblast growth factor (FGF) signaling components were observed to be predominantly expressed in ALM blastema cells. Furthermore, we found that MMP activity induced a blastema marker gene, Prrx-1, in vitro, and FGF signaling pathways worked in coordination to maintain Prrx-1 expression and ALM blastema formation. Furthermore, we demonstrated that these two activities were sufficient to induce an ALM blastema in the absence of a nerve in vivo. Copyright © 2011 Elsevier Inc. All rights reserved.

Caenorhabditis elegans as a model system to study post-translational modifications of human transthyretin

NASA Astrophysics Data System (ADS)

Henze, Andrea; Homann, Thomas; Rohn, Isabelle; Aschner, Michael; Link, Christopher D.; Kleuser, Burkhard; Schweigert, Florian J.; Schwerdtle, Tanja; Bornhorst, Julia

2016-11-01

The visceral protein transthyretin (TTR) is frequently affected by oxidative post-translational protein modifications (PTPMs) in various diseases. Thus, better insight into structure-function relationships due to oxidative PTPMs of TTR should contribute to the understanding of pathophysiologic mechanisms. While the in vivo analysis of TTR in mammalian models is complex, time- and resource-consuming, transgenic Caenorhabditis elegans expressing hTTR provide an optimal model for the in vivo identification and characterization of drug-mediated oxidative PTPMs of hTTR by means of matrix assisted laser desorption/ionization - time of flight - mass spectrometry (MALDI-TOF-MS). Herein, we demonstrated that hTTR is expressed in all developmental stages of Caenorhabditis elegans, enabling the analysis of hTTR metabolism during the whole life-cycle. The suitability of the applied model was verified by exposing worms to D-penicillamine and menadione. Both drugs induced substantial changes in the oxidative PTPM pattern of hTTR. Additionally, for the first time a covalent binding of both drugs with hTTR was identified and verified by molecular modelling.

Biocompatibility evaluation of HDPE-UHMWPE reinforced β-TCP nanocomposites using highly purified human osteoblast cells.

PubMed

Shokrgozar, M A; Farokhi, M; Rajaei, F; Bagheri, M H A; Azari, Sh; Ghasemi, I; Mottaghitalab, F; Azadmanesh, K; Radfar, J

2010-12-15

Biocompatibility of β-TCP/HDPE-UHMWPE nanocomposite as a new bone substitute material was evaluated by using highly purified human osteoblast cells. Human osteoblast cells were isolated from bone tissue and characterized by immunofluorescence Staining before and after purification using magnetic bead system. Moreover, proliferation, alkaline phosphatase production, cell attachment, calcium deposition, gene expression, and morphology of osteoblast cells on β-TCP/HDPE-UHMWPE nanocomposites were evaluated. The results have shown that the human osteoblast cells were successfully purified and were suitable for subsequent cell culturing process. The high proliferation rate of osteoblast cells on β-TCP/HDPE-UHMWPE nanocomposite confirmed the great biocompatibility of the scaffold. Expression of bone-specific genes was taken place after the cells were incubated in composite extract solutions. Furthermore, osteoblast cells were able to mineralize the matrix next to composite samples. Scanning electron microscopy demonstrated that cells had normal morphology on the scaffold. Thus, these results indicated that the nanosized β-TCP/HDPE-UHMWPE blend composites could be potential scaffold, which is used in bone tissue engineering. Copyright © 2010 Wiley Periodicals, Inc.

Primer sets for cloning the human repertoire of T cell Receptor Variable regions

PubMed Central

Boria, Ilenia; Cotella, Diego; Dianzani, Irma; Santoro, Claudio; Sblattero, Daniele

2008-01-01

Background Amplification and cloning of naïve T cell Receptor (TR) repertoires or antigen-specific TR is crucial to shape immune response and to develop immuno-based therapies. TR variable (V) regions are encoded by several genes that recombine during T cell development. The cloning of expressed genes as large diverse libraries from natural sources relies upon the availability of primers able to amplify as many V genes as possible. Results Here, we present a list of primers computationally designed on all functional TR V and J genes listed in the IMGT®, the ImMunoGeneTics information system®. The list consists of unambiguous or degenerate primers suitable to theoretically amplify and clone the entire TR repertoire. We show that it is possible to selectively amplify and clone expressed TR V genes in one single RT-PCR step and from as little as 1000 cells. Conclusion This new primer set will facilitate the creation of more diverse TR libraries than has been possible using currently available primer sets. PMID:18759974

Kinetics of Eucalyptus globulus delignification in a methanol-water medium

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

Gilarranz, M.A.; Rodriguez, F.; Santos, A.

1999-09-01

The kinetics of Eucalyptus Globulus delignification in methanol-water pulping has been studied. A total of 17 isothermal runs at a liquor-to-wood ratio of 50 L/kg were carried out to develop the kinetic model describing the system. In a first series of experiments, eight models were considered to study the influence of temperature on the delignification rate. The most suitable model, which was discriminated according to statistical criteria, describes delignification as the consecutive dissolution of three lignin species: initial, bulk, and residual lignin, their content in wood being 10, 69, and 21%, respectively. Initial and residual delignification were considered as irreversiblemore » reactions and bulk delignification as reversible. The influence of hydrogen ion concentration was taken into account by means of a general power-law expression. The model proposed was taken into account by means of a general power-law expression. The model proposed was validated by reproducing the experimental data from four runs carried out under nonisothermal conditions and a liquor-to-wood ratio of 7 L/kg, which are closer to industrial operating conditions.« less

Transgenic elite indica rice plants expressing CryIAc delta-endotoxin of Bacillus thuringiensis are resistant against yellow stem borer (Scirpophaga incertulas).

PubMed

Nayak, P; Basu, D; Das, S; Basu, A; Ghosh, D; Ramakrishnan, N A; Ghosh, M; Sen, S K

1997-03-18

Generation of insect-resistant, transgenic crop plants by expression of the insecticidal crystal protein (ICP) gene of Bacillus thuringiensis (Bt) is a standard crop improvement approach. In such cases, adequate expression of the most appropriate ICP against the target insect pest of the crop species is desirable. It is also considered advantageous to generate Bt-transgenics with multiple toxin systems to control rapid development of pest resistance to the ICP. Larvae of yellow stem borer (YSB), Scirpophaga incertulas, a major lepidopteran insect pest of rice, cause massive losses of rice yield. Studies on insect feeding and on the binding properties of ICP to brush border membrane receptors in the midgut of YSB larvae revealed that cryIAb and cryIAc are two individually suitable candidate genes for developing YSB-resistant rice. Programs were undertaken to develop Bt-transgenic rice with these ICP genes independently in a single cultivar. A cryIAc gene was reconstructed and placed under control of the maize ubiquitin 1 promoter, along with the first intron of the maize ubiquitin 1 gene, and the nos terminator. The gene construct was delivered to embryogenic calli of IR64, an elite indica rice cultivar, using the particle bombardment method. Six highly expressive independent transgenic ICP lines were identified. Molecular analyses and insect-feeding assays of two such lines revealed that the transferred synthetic cryIAc gene was expressed stably in the T2 generation of these lines and that the transgenic rice plants were highly toxic to YSB larvae and lessened the damage caused by their feeding.

Effect of cadmium exposure on hepatopancreas and gills of the estuary mud crab (Scylla paramamosain): Histopathological changes and expression characterization of stress response genes.

PubMed

Zhu, Qi-Hui; Zhou, Zhong-Kai; Tu, Dan-Dan; Zhou, Yi-Lian; Wang, Cong; Liu, Ze-Peng; Gu, Wen-Bin; Chen, Yu-Yin; Shu, Miao-An

2018-02-01

Cadmium (Cd) is a heavy metal that accumulates easily in organisms and causes several detrimental effects, including tissue damage. Cd contamination from anthropogenic terrestrial sources flows into rivers, and through estuaries to the ocean. To evaluate the toxic effects of Cd on estuary crustaceans, we exposed the mud crab Scylla paramamosain to various Cd concentrations (0, 10.0, 20.0, and 40.0mg/L) for 24h. We also exposed mud crabs to a fixed Cd concentration (20.0mg/L) for various periods of time (0, 6, 12, 24, 48, and 72h). We observed that after exposure to Cd, the surfaces of the gill lamellae were wrinkled, and the morphologies of the nuclei and mitochondria in the hepatopancreas were altered. We analyzed the expression profiles of 36 stress-related genes after Cd exposure, including those encoding metallothioneins, heat shock proteins, apoptosis-related proteins, and antioxidant proteins, with quantitative reverse transcription PCR. We found that exposure to Cd altered gene expression, and that some genes might be suitable bioindicators of Cd stress. Gene expression profiles were organ-, duration-, and concentration-dependent, suggesting that stress-response genes might be involved in an innate defense system for handling heavy metal exposure. To the best of our knowledge, this study is the first one of histopathology and stress-response gene expression pattern of Scylla paramamosain after Cd exposure. Our work could increase our understanding of the effect of environmental toxins on estuary crustaceans. Copyright © 2017 Elsevier B.V. All rights reserved.

Bacterially produced Pt-GFP as ratiometric dual-excitation sensor for in planta mapping of leaf apoplastic pH in intact Avena sativa and Vicia faba.

PubMed

Geilfus, Christoph-Martin; Mühling, Karl H; Kaiser, Hartmut; Plieth, Christoph

2014-01-01

Ratiometric analysis with H(+)-sensitive fluorescent sensors is a suitable approach for monitoring apoplastic pH dynamics. For the acidic range, the acidotropic dual-excitation dye Oregon Green 488 is an excellent pH sensor. Long lasting (hours) recordings of apoplastic pH in the near neutral range, however, are more problematic because suitable pH indicators that combine a good pH responsiveness at a near neutral pH with a high photostability are lacking. The fluorescent pH reporter protein from Ptilosarcus gurneyi (Pt-GFP) comprises both properties. But, as a genetically encoded indicator and expressed by the plant itself, it can be used almost exclusively in readily transformed plants. In this study we present a novel approach and use purified recombinant indicators for measuring ion concentrations in the apoplast of crop plants such as Vicia faba L. and Avena sativa L. Pt-GFP was purified using a bacterial expression system and subsequently loaded through stomata into the leaf apoplast of intact plants. Imaging verified the apoplastic localization of Pt-GFP and excluded its presence in the symplast. The pH-dependent emission signal stood out clearly from the background. PtGFP is highly photostable, allowing ratiometric measurements over hours. By using this approach, a chloride-induced alkalinizations of the apoplast was demonstrated for the first in oat. Pt-GFP appears to be an excellent sensor for the quantification of leaf apoplastic pH in the neutral range. The presented approach encourages to also use other genetically encoded biosensors for spatiotemporal mapping of apoplastic ion dynamics.

Kuiper Belt Objects Along the Pluto-Express Path

NASA Technical Reports Server (NTRS)

Jewitt, David (Principal Investigator)

1997-01-01

The science objective of this work is to identify objects in the Kuiper Belt which will, in the 5 years following Pluto encounter, be close to the flight path of NASA's Pluto Express. Our hope is that we will find a Kuiper Belt object or objects close enough that a spacecraft flyby will be possible. If we find a suitable object, the science yield of Pluto Express will be substantially enhanced. The density of objects in the Kuiper Belt is such that we are reasonably likely to find an object close enough to the flight path that on-board gas thrusters can effect a close encounter.